Received: 13 February 2020 Revised: 6 May 2020 Accepted: 6 May 2020

DOI: 10.1111/1541-4337.12579

COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY

Comparative review and the recent progress in detection

technologies of meat product adulteration

Yun-Cheng Li1,2 Shu-Yan Liu1 Fan-Bing Meng1,2 Da-Yu Liu1,2

Yin Zhang1,2 Wei Wang2 Jia-Min Zhang2

1 Collegeof Pharmacy and Biological

Engineering, Chengdu University, Chengdu,
China
2 KeyLaboratory of Meat Processing of
Sichuan Province, Chengdu University,
Chengdu, China
Correspondence
Dr Fan-Bing Meng, College of Pharmacy and
Biological Engineering, Chengdu University,
No. 2025 Chengluo Avenue, Chengdu, Sichuan
610106, China.
Email: mfb1020@163.com
Funding information
Provincial Key Research and Development
Program of Sichuan, Grant/Award Numbers:
2019, YFS0525; Sichuan Science and Technol-
ogy Program, Grant/Award Numbers: 2019,
YFN0174, 20ZDYF0467
Abstract
Meat adulteration, mainly for the purpose of economic pursuit, is widespread and
leads to serious public health risks, religious violations, and moral loss. Rapid, effec-
tive, accurate, and reliable detection technologies are keys to effectively supervis-
ing meat adulteration. Considering the importance and rapid advances in meat adul-
teration detection technologies, a comprehensive review to summarize the recent
progress in this area and to suggest directions for future progress is beneficial. In
this review, destructive meat adulteration technologies based on DNA, protein, and
metabolite analyses and nondestructive technologies based on spectroscopy were
comparatively analyzed. The advantages and disadvantages, application situations of
these technologies were discussed. In the future, determining suitable indicators or
markers is particularly important for destructive methods. To improve sensitivity and
save time, new interdisciplinary technologies, such as biochips and biosensors, are
promising for application in the future. For nondestructive techniques, convenient and
effective chemometric models are crucial, and the development of portable devices
based on these technologies for onsite monitoring is a future trend. Moreover, omics
technologies, especially proteomics, are important methods in laboratory detection
because they enable multispecies detection and unknown target screening by using
mass spectrometry databases.

KEYWORDS
adulteration, analytical methods, destructive technologies, meat, nondestructive technologies

1 I N T RO D U C T I O N cally motivated, such as the low-cost addition of duck meat to

Meat and meat products are an important food source for
humans in both developed and developing countries (Daniel,
Cross, Koebnick, & Sinha, 2011). Many important nutrients,
such as proteins, fats, minerals, and vitamins, can be supplied
by meats (Cascella et al., 2018; Kamruzzaman, Makino, &
Oshita 2015b; Zheng, Li, Wei, & Peng, 2019). Although there
are various national and international laws for supervising the
quality and safety of meat and meat products, meat adulter-
ation is still widespread. Most meat adulteration is economi-
mutton (Zheng et al., 2019), which causes consumers to suf-
fer economic losses. A small amount of adulteration is due to
adventitious contamination during processing (Naaum et al.,
2018). Either way, meat adulteration may lead to serious pub-
lic health risks, such as the exposure to toxins, pathogens, or
allergens in these products (Magiati, Myridaki, Christopou-
los, & Kalogianni, 2019; Spink & Moyer, 2011). For exam-
ple, coronaviruses, such as Middle East respiratory syndrome
(MERS-CoV) and severe acute respiratory syndrome coron-
avirus (SARS-CoV), might be transmitted to humans through

Compr Rev Food Sci Food Saf. 2020;1–41. wileyonlinelibrary.com/journal/crf3 © 2020 Institute of Food Technologists® 1
2 DETECTION METHODS OF MEAT ADULTERATION…
F I G U R E 1 Commonly applied technologies for meat product adulteration detection
the consumption of adulterated wild animal meat (Amin,
Hamid, & Ali, 2016; Kingsley, 2016). In addition, meat adul-
teration could also violate religious concerns; for example,
pork or pork-associated products are not acceptable in Kosher
and Halal food laws (Lim & Ahmed, 2016). Furthermore,
meat adulteration has become a problematic concern for all
meat industry chains at all levels of the production and distri-
bution process, from farmers to regulators and from producers
to consumers.
Although many strategies have been adopted to assure the
authenticity of meat and meat products, such as protected des-
ignation of origin, protected geographical indication, certifi-
cate of specific characteristics, and so on (Abbas et al., 2018),
the coverage is too small, and it is unrealistic to certify all meat
products for protection from adulteration. Therefore, effective
supervision is very important for ensuring the suitable devel-
opment of the meat industry, and rapid, effective, accurate,
and reliable detection technologies are fundamental technical
support for this goal.
In the last two decades, authenticity detection technolo-
gies for meat and meat products have been established or
optimized for different markers, such as polymerase chain
reactions (PCRs) based on deoxyribonucleic acids (DNAs),
immunological technologies based on proteins, spectroscopic
technologies based on specific metabolites, and so on (Fig-
ure 1). These technologies have their own characteristics and
have some disadvantages regarding adulteration detection for
meat and meat products. In this review, recent advances of the
detection technologies were comprehensively discussed, and
the merits and demerits between technologies were compared.
Finally, the future perspectives about the detection technolo-
gies of meat adulteration are also discussed. Although many
older literatures were included, this review mainly focused the
references published in near 5 years.
2 TECHNOLOGIES BA SED ON D NA
DNA is the main material for storing, replicating, and trans-
mitting genetic information. DNA exists in all tissues of indi-
vidual animals and is more conserved than proteins (Kumar
et al., 2015; Xiang et al., 2017). More importantly, DNA
fragments have shown better thermal stability than that of
proteins in processed meat, so they could be chosen as
markers for authenticity determination in processed meat
(Kaltenbrunner, Hochegger, & Cichna-Markl, 2018a; Kang
& Tanaka, 2018; Kumar et al., 2015; Ruiz-Valdepeñas Mon-
tiel et al., 2017; Xu et al., 2018). Although the variation
of DNA content in meat tissues and species could impact
the meat quantification, the PCR and its derived technolo-
gies based on DNA are the most commonly used tech-
niques in the detection of meat and meat product adul-
teration due to their sensitivity, simplicity, and reliability
(Table 1).
The target genes and DNA fragments used as mark-
ers for identifying meat product adulteration have mainly
been derived from mitochondrial DNA (mtDNA) (Figure 2)
(Dai et al., 2015), such as the mitochondrial D-loop region,
cytochrome b (CytB) genes, cytochrome c oxidase subunit I,
II, and III (COI, COII, and COIII) genes, ATPase subunit
6 and 8 (ATPase6 and ATPase8), 12SrRNA and 16SrRNA
(Table 2), because mtDNA possesses several advantages
over genomic DNA, such as being more efficiently arranged
and easily accessible, not undergoing recombination (Kumar
et al., 2015), and so on. In addition, new genomic DNA
has also been identified as a stable marker for the detec-
tion of meat product adulteration, such as replication pro-
tein A1 (RPA1) (Ren, Deng, Huang, Chen, & Ge, 2017),
prion protein PrP (Prnp) (Kaltenbrunner et al., 2018a), and
soon.
TABLE 1 Comparative analysis of the common applied meat adulteration techniques

Multispecies Operator Detection Commercial Application

Techniques Specificity Sample preparation Detection time detection requirements costs availability locations
Direct PCR High but vulnerable Sampling→smashing or Time-consuming Yes Professional High Commercial kits Lab
ground→DNA extraction→ available
purification→ quantification
Real-time PCR High Sampling→smashing or Time-consuming Yes Professional High Commercial kits Lab
ground→DNA extraction→ available
purification→ quantification
PCR-RFLP High Sampling→smashing or Time-consuming Yes Professional High Commercial kits Lab
ground→DNA extraction→ available
purification→ quantification
LAMP High Sampling→smashing or Less time- Yes Professional High Commercial kits Lab or onsite
DETECTION METHODS OF MEAT ADULTERATION…

ground→DNA extraction→ consuming available

purification→ quantification
ddPCR High Sampling→smashing or Less time- Yes Professional High No Lab
ground→DNA extraction→ consuming
purification→ quantification
DNA barcoding High Sampling→smashing or Less time- Yes Professional High Public databases Lab
ground→DNA extraction→ consuming available (BOLD)
purification→ quantification
ELISA High Sample ground→protein Less time- No Simple training Low Commercial kits Lab or onsite
extraction→ quantification consuming available
Protein High Sample ground→protein Less time- No Simple training Low No Lab or onsite
immunosensor extraction→ quantification consuming
Protein mass High Sample ground→ protein Time-consuming Yes Professional High No Lab
extraction→ purification→
digestion
Metabolite Low and vulnerable Metabolomics: sample Time-consuming Yes Professional High No Lab
profiling ground→ metabolites
extraction→ purification;
electronic nose: no or little
IRS Low and vulnerable No or little Time-saving Yes Simple training Low Commercial Lab or onsite
portable device
available
RS Low and vulnerable No or little Time-saving Yes Simple training Low Commercial Lab or onsite
portable device
available
HSI Low and vulnerable No or little Time-saving Yes Professional Low No Lab or onsite
LIBS High No or little Time-saving Yes Professional Low No Lab or onsite
3
4 DETECTION METHODS OF MEAT ADULTERATION…
F I G U R E 2 Flowchart of meat adulteration detection technologies based on DNA
2.1 Direct PCR
The direct PCR method has the characteristics of high sen-
sitivity, high resolution, and specificity, so it is commonly
used in meat authenticity and origin traceability (Bhat et al.,
2016; Ha et al., 2017). Ha et al. (2017) developed species-
specific PCR methods of the mitochondrial D-loop to detect
pork adulteration in commercial beef and/or chicken prod-
ucts, and the methods were able to detect as little as 1%
pork in heat-treated pork-beef-chicken mixtures. However,
the conventional single-species PCR method could only detect
one specific species of adulterant in products (Kumar, 2015),
which is of low commercial value because there might be
many other adulterants in the products.
Multiplex PCR assays with multiple species-specific
primers have been greatly developed since they offer mul-
tiple target detection in a single reaction (Ali et al., 2015;
Böhme, Calo-Mata, Barros-Velázquez, & Ortea, 2019; Dai
et al., 2015; Hou et al., 2015). Ali et al. (2015) designed
five pairs of species-specific primers targeting mitochondrial
ND5, ATPase 6 gene, and CytB to simultaneously detect
cat, dog, pig, monkey, and rat meats in Islamic foods. The
detection limits were 0.01 to 0.02 ng DNA in the raw prod-
uct and 1% suspected meats in meatballs, which showed
potential value in the Halal food industry and Halal regula-
tory bodies. In addition, the authors successfully applied this
method in commercial sample identification. Multiplex PCR
technologies for other meat species, such as chicken, lamb,
ostrich, horse, rat, buffalo, fox species, and so on, have also
been developed (Hossain et al., 2016; Kitpipit, Sittichan, &
Thanakiatkrai, 2014; Li, Li et al., 2019; Liu, Wang et al., 2019;
Qin et al., 2019; Wang, Hang, & Geng, 2019).
However, the multiplex PCR method usually applies com-
paratively longer and variable-length DNA templates (Köp-
pel, Ruf, Zimmerli, & Breitenmoser, 2008; Sakai et al.,
2011), which are not stable under the harsh conditions of
food processing, such as high-temperature autoclaving and
baking (Al Amin, Abd Hamid, & Ali, 2016). In addition,
some closely related species may not be differentiated by
direct PCR technologies (Doosti et al., 2014). Moreover, the
direct PCR method is still time-consuming, requires com-
plex operation and gel electrophoresis, and cannot be accu-
rately quantified (Li, Jalbani et al., 2019; Shabani et al., 2015)
through spectrophotometric methods because the PCR prod-
ucts easily undergo interference with single-stranded DNA,
RNA, proteins, and so on (Kang & Tanaka, 2018), which
restricts its application in industry and commercial settings
(Ali, Hashim, Mustafa, & Man, 2012; Ali et al., 2012). There-
fore, an upgraded technology has been developed in recent
years, that is PCR associated with biochips (Cottenet, Son-
nard, Blancpain, Ho, Leong, & Chuah, 2016; Iwobi, Huber,
Hauner, Miller, & Busch, 2011; Li, Li et al., 2019). The DNA
biochip technologies have the advantages of the high sensitive
and simultaneous detection of multispecies compared to sim-
plex PCR methods (Cottenet et al., 2016; Iwobi et al., 2011).
Recently, Li, Li et al. (2019) developed two independent mul-
tiplex PCR methods based on 12S rRNA, 16S rRNA, ND2,
and COI. Using microchip electrophoresis to replace tradi-
tional gel electrophoresis, 14 animal species could be simulta-
neously detected with detection limits as low as 0.02 ng DNA.
This study provides a good reference for improving the tradi-
tional PCR methods for meat adulteration detection (Table 2).
2.2 Real-time PCR
Compared to direct PCR, real-time PCR showed higher speci-
ficity, higher sensitivity, and scope for automation, and it can
effectively mitigate PCR contamination (Al-Kahtani, Ismail,
& Asif Ahmed, 2017). More importantly, real-time PCR
methods can achieve quantitative analysis through the linear
relationship between the amount of DNA and the Ct value
(Fang & Zhang, 2016; Kumar et al., 2015). Real-time PCR
technology has been applied for the identification of pork,
beef, turkey, chicken, and lamb in quantities less than 0.1%,
even in heat-processed meat products (Kumar et al., 2015).
Real-time PCR is performed by monitoring the fluores-
cence signal, which allows for deducing the initial quantity
of the target genes without additional steps (Xu et al., 2018).
SYBR Green and TaqMan technology are commonly used
in quantitative methods (the working principle is outlined
in the review of Kumar et al., 2015). SYBR Green technol-
ogy can only detect a single species, but the detection cost
was lower than that of TaqMan technology. Li, Jalbani et al.
(2019) developed a novel reference primer-based mitochon-
drial 12S rRNA for quantitative determination of goat meat
adulterated with pork by using real-time PCR. The method
showed high specificity and sensitivity for goat meat mixed
TABLE 2 Recent 5 years representative studies of DNA-based technologies for meat adulteration identification
Detection
Detection items technology Target marker (primer, 5′ → 3′ ) Limits of detection References
Pork adulteration in Simplex PCR MItochondrial D-loop region for pork 1% pork in the Ha et al. (2017)
beef, chicken, and (GGTTCTTACTTCAGGACCATC/GTGTACGCACGTGTATGTAC) heat-treated meat
mutton samples
Beef adulteration in Simplex PCR Mitochondrial D-loop region for beef 0.1% beef adulteration Karabasanavar et al.
other meat (TATCAAAAATCCCAATAACTCAACACA/GGCCCGGAGCGAGAAG) in raw, cooked, (2017)
autoclaved, and
micro-oven
processed samples
Pork adulteration in Multiplex PCR CytB for beef (CATCGCACGAATATAATACA-C3- 0.5% to 1% w/w pork Skouridou et al.
beef or chicken TGGAGTAATCCTTCTGCTCACAGT/TGTAAAACGACGGCCAGT-C3- could be detected (2019)
DETECTION METHODS OF MEAT ADULTERATION…

products GGATTGCTGATAAGAGGTTGGTG) when mixed with

CytB for chicken beef or chicken
(ATTACGACGAACTCAATGAA-C3-ATTCCCTACATTGGACACA/TGTAAAACGACGGCCAGT-
C3-TGATAGTAATACCTGCGATTGCA)
CytB for pork (ATAGGCTGGTTCGTAATCGG-C3-
CATCCCAATTATAATATCCAACTC/TGTAAAACGACGGCCAGT-C3-
ATTTCTTGGCCTGTGTGT)
Fox, raccoon, or Multiplex PCR COXI gene for fox (ACCGCACATGCCTTCGTAA/CGTGAAGTATGCTCGTGTATCC) 1% (w/w) level Li and Guan (2019)
mink in COXI gene for raccon (TTAGTAGCTCATAACGCCTTG/CGTGAGTATGTTCTTGGTTAG)
commercial beef 12S rRNA gene for mink (TTGACTGGTTCCACTGATG/CCGTTAGGAGTATTGTGATTC)
and mutton meat
Cattle, pig, duck, Multiplex PCR COI for buffalo (CTGTGTTCGCCATTATAGGA/GTGGTTAGATCTACGGTTGAG) 1 pg pure DNA and Wang, Hang et al.
and buffalo meat COI for cattle (GAACTCTGCTCGGAGACGAC/GGTACACGGTTCAGCCTGTT) 0.1% (w/w) (2019)
COI for pig (GGAGCAGTGTTCGCCATTAT/TTCTCGTTTTGATGCGAATG) adulterated meat
COI for duck (TAATTGGCACAGCACTCAGC/TTATCAGGGGGACCAATCAG)
Chicken, duck, pork, Multiplex PCR Cytb for chicken (AATCGCAGGTATTACTATCATCCAC/GGTGAGTATGAGAGTTAAGCCCAG) 0.05% (w/w) for each Qin et al. (2019)
and beef COIII for duck (GCCATCCTACTACTCCTCACCA/CCAGATTTTAGAGATTGGAAGTCA) species
ATPase subunit 8/6 for pork
(TACCCAGCAAGCCCAGAATC/GAGATTGTGCGGTTATTAATGAGTC)
Cytb for beef (CAACAGGAATCTCCTCAGACGTAGA/GCTAGAATTAGTAAGAGGGCCCCTAA)
Rat, fox, duck, and Multiplex PCR ATPase 6 for rat (ATCATCAGAACGCCTTATTAGC/GGTTCGTCCTTTTGGTGTATG) 0.1 ng/𝜇L mtDNA for Liu, Wang et al.
mutton COX2 for fox (GTCAAATCATAGGTGAAACCCC/TAGAGAAGGAAGAGCAATCAGG) rat, duck, and goat (2019)
tRNA, ND1 for duck (CATCAACAAAGAGTGCGTCAAA/GTTTAACCTAGGTCACTGGGCA) meat; 0.05 ng/𝜇L
Cytb for goat mtDNA for fox
(GACCTCCCAGCTCCATCAAACATCTCATCTTGATGAAA/AGGTTTGTGCCAATATATGGAATT) meat
(Continues)
5
TABLE 2 (Continued)
6

Detection
Detection items technology Target marker (primer, 5′ → 3′ ) Limits of detection References
Dog, donkey, fox, Multiplex PCR ATPase 6 for dog 0.1 ng/𝜇L mtDNA for Liu, Tao et al. (2019)
chicken, beef, (TGGCTCTAGCCGTTCGATTA/CCTTCCTTCCTTCCCCCAAGGCAACAGCAAATTCTAGG) chicken, horse, and
pork, horse, and tRNA phe 12S rRNA for donkey fox meat; 0.05
rabbit meat (CTCTTCCCCAGTTAATGTAGCTT/CCTTCCTTCCTTCCCCCCTATCGTGTGGTCAGAGATATT) ng/𝜇L mtDNA for
16S rRNA for fox (AACTTAGACCGAACCATATTG- dog, cattle, pig,
CATC/CCTTCCTTCCTTCCCCCGGGGTTTGAAGTTCATAAGTTTGG) donkey, and rabbit
Cytb for chicken (GACCTCCCAGCTCCATCAAACATCTCATCTTGAT- meat
GAAA/CCTTCCTTCCTTCCCCCCAGATGAAGAAGAATGAGGCG)
Cytb for cattle (GACCTCCCAGCTCCATCAAACATCTCATCTTGAT-
GAAA/CCTTCCTTCCTTCCCCCCTAGAAAAGTGTAAGACCGTAAT)
Cytb for pig (GACCTCCCAGCTCCATCAAACATCTCATCTTGAT-
GAAA/CCTTCCTTCCTTCCCCCTGATAGTAGATTTGTGATGACCG)
Cytb for horse (GACCTCCCAGCTCCATCAAACATCTCATCTTGAT-
GAAA/CCTTCCTTCCTTCCCCCCAGATTCACTCGACGAGGGT)
Cytb for rabbit (GACCTCCCAGCTCCATCAAACATCTCATCTTGAT-
GAAA/CCTTCCTTCCTTCCCCCGAGGAGAAGAATGGCTACAAGGAA)
Group I: cattle, Multiplex PCR COI for cattle (GTTCTTCACGACACATACTACGTT/GCAAATACAGCTCCTATTGATAAA) 0.02 ng DNA for Li, Li et al. (2019)
donkey, dog, fox, ND2 for donkey (ATTCTCCCCACCCTAATGGCT/ACTAACCTATTCCACCTCCCTAACT) group I and 0.2 ng
raccoon-dog, deer, 16S rRNA for fox, dog, and raccoon-dog DNA for group II
and horse (AAGGGAATGATGAAAGACAT/GAGTTGATCCTTTTAGATTGTT)
Group II: pig, sheep, 12S rRNA for deer (GCTCACGACACCTTGCACAG/GCTTTAACACACTTTACGCCGTATG)
goat, chicken, 16S rRNA for horse (CAACCCAAACTAACTCCT/ATAGATGCATGCCTGTGTT)
duck, cat, and COI for cat (TCATGTTAATAGTTTTCATAGTG/TGTGGTTAGTTCTACTATGGC)
mouse COI for pig (TACTTCTACTATCCCTGCCAGTTC/TGATAAAGGATAGGGTCTCCACCA)
16S rRNA for mouse
(GACATCCCAATGGTGTAGAAGCTATT/GTCCTTTCGTACTGGGAGAAATCGTA)
12S rRNA for sheep and goat
(AAAATAAATGACGAAAGTAACCCTAC/GCCAAGTCCTTTGAGTTTCGG)
12S rRNA for duck and chicken
(GAGAACTACGAGCACAAACGCTT/CCCATAGGCTATACCTTGACCTGT)
Cattle and buffalo Multiplex PCR 12S rRNA for cattle 2.23 ng/𝜇L DNA for Dantas et al. (2019)
(CTAGAGGAGCCTGTTCTATAATCGATA/AAATAGGGTTAGATGCACTGAATCCAT) cattle (B. taurus)
12S rRNA for buffalo and 2.31 ng/𝜇L
(CTAGAGGAGCCTGTTCTATAATCGATA/TTCATAATAACTTTCGTGTTGTTGGGTGT) DNA for buffalo (B.
bubalis)
Chicken, mutton Multiplex PCR PRLR for sheep and goat (ATCTTCCACATGGCCTTTCC/CAGCAATGTTGTGGTAAGAA) 16 pg DNA per Balakrishna et al.
(sheep and goat), ND5 gene for cow (GGTTTCATTTTAGCAATAGCATGG/GTCCAATCAAGGGTATGTTTGAG) reaction and 0.01% (2019)
beef, and pork CytB for chicken (TCACACTCATAGCCACCGC/GATAGTAATACCTGCGATTGC) (w/w) of each
adulteration in ActB for pork (GCTGGAGTCTTTCTCGTGT/TGGATGGCCACGTACATG) sample
buffalo
DETECTION METHODS OF MEAT ADULTERATION…

(Continues)
TABLE 2 (Continued)
Detection
Detection items technology Target marker (primer, 5′ → 3′ ) Limits of detection References
Mice adulteration in Multiplex PCR ND1 for mice (CGGCATCCTACAACCATTTGC/CGGCTCGTAAAGC-TCCGAA) 5% (w/w) of the mice Raharjo et al. (2018)
meatball (beef, contamination
chicken, pork,
horse, and goat)
Fox, dog, mink, and Mitochondrial genomic DNA for fox Unspecified Wu et al. (2018)
rabbit (ACAGGGGAGAAAACGCTGTTC/GGCCTCCCCGAGATGAATC)
Mitochondrial genomic DNA for dog
(GCGGCAAAGTTAACGGCAGT/GGATGGGAAGCAAACTCCGA)
Mitochondrial genomic DNA for mink
(TTCGGCGCTGCCTTAATTGTC/AAGACCTGGGACCCGAGAGTT)
Mitochondrial genomic DNA for rabbit
(TAGCAGTAGGGATGACAGGGTTT/GCCTTAGAGTAGGGTCTTTTTGG)
DETECTION METHODS OF MEAT ADULTERATION…

Chicken, turkey, and Multiplex PCR 12S rRNA gene for turkey (GAAACCCAAATCAATAGCCATC/CCCTGGCTTTCGTGGCTTTA) 1 pg template for each Kim et al. (2018)
duck CytB for chicken (AGCAATTCCCTACATTGGACACA/GATGATAGTAATACCTGCGATTGCA) species
CytB for duck (CCGTCCTAATCCTATTCCTGGTC/GGAATAGGAGGATGGTGAAGTAAGTA)
Turkey, mutton, Multiplex PCR Mitochondrial ATPase subunit 8 gene for turkey 30 pg DNA per Prusakova et al.,
chicken, beef, (CCATAAACAAACCTTCAACAACAAAACCAAT/GGAGGGCCGGGAGAAGTAGAGATG) reaction 2018
pork, cat, dog, Mitochondrial ATPase subunit 8 gene for mutton
mouse, human, (CTCAAAACACAACTTCTACCACAACCCAG/AACAATGAGGGTAACGAGGGGGAGA)
and rat Mitochondrial ATPase subunit 8 gene for chicken
(CAATTAAACCCAAACCCATGATTCTCCA/GATTCCTAGTAGGCAGGGGCTTGAGAAT)
Mitochondrial ATPase subunit 8 gene for beef (AACATGACTGACAATGATCTTAT-
CAATATTCTTGA/ATAGTAGGCTTGGGAATAGTACGATAAGGGTT)
Mitochondrial ATPase subunit 8 gene for pork
(GCCACAACTAGATACATCCACATGATTCATTAC/TTGTTGGATCGAGATTGTGCGGTT)
Mitochondrial ATPase subunit 8 gene for cat (CACTAAAACAACGGAATCCCTGA-
GAAAAA/TAGGTGAAGGGAATAAAATGCTTGGAAATATA)
Mitochondrial ATPase subunit 8 gene for dog
(CGATAACCAAATCTGCTAAAATTGCTGG/AATGGAGATTAACCGATTATTGATTAGGCG)
Mitochondrial ATPase subunit 8 gene for mouse
(ACTAAAAGTCTCATCACAAACATTCCCACTG/GTGCTCAGGTTCGTCCTTTTGGTGT)
Mitochondrial ATPase subunit 8 gene for rat
(AAAATTGTAGCCACAGGAAAAACAGACAAT/GTGCTCAGGTTCGTCCTTTTGGTGT)
Mitochondrial ATPase subunit 8 gene for human
(AAAGCCCATAAAAATAAAAAATTATAACAAACCC/TTGGAGGTGGGGATCAATAGAGGG)
Beef and buffalo Multiplex PCR DNA for cattle 10% buffalo meat in de Oliveira et al.
meat (CTAGAGGAGCCTGTTCTATAATCGATA/AAATAGGGTTAGATGCACTGAATCCAT) the ground beef (2018)
DNA for buffalo samples and 0.1%
(CTAGAGGAGCCTGTTCTATAATCGATA/TTCATAATAACTTTCGTGTTGTTGGGTGT) beef in the ground
buffalo meat
samples
7

(Continues)
 8

TABLE 2 (Continued)
Detection
Detection items technology Target marker (primer, 5′ → 3′ ) Limits of detection References
Beef, lamb, and pork Multiplex PCR CytB for beef (GACCTCCCAGCTCCATCAAACATCTCATCTTGAT- 1% (w/w) of binary Song et al. (2017)
GAAA/CTAGAAAAGTGTAAGACCCGTAATATAAG) mixtures
CytB for lamb (GACCTCCCAGCTCCATCAAACATCTCATCTTGAT-
GAAA/AAACATAGCCTATGAATGCTGTGGCTATTGTC)
CytB for pork (GACCTCCCAGCTCCATCAAACATCTCATCTTGAT-
GAAA/CTGTTCCGATATAAGGGATAGCTGATAGTAGA)
Beef, pork, and Multiplex PCR Targeting double genes for all species 0.1% (w/w) of binary Hossain, Ali, Hamid,
buffalo meat CytB for beef (CGGCACAAATTTAGTCGAAT/TGGACTATGGCAATTGCTATG) mixtures and Asing et al. (2017)
ND5 for beef (GGTTTCATTTTAGCAATAGCATGG/GTCCAATCAAGGGTATGTTTGAG) meatball products
CytB for buffalo (GGGTTCTAGCCCTAGTTCTCTCT/ATGGCCGGAACATCATACTT)
ND5 for beef (TCGCCTAGCTTCTTACACAAAC/TGGTTTGTGACTGTGATGGAT)
CytB for pork (TATCCCTTATATCGGAACAGACCTC/GCAGGAATAGGAGATGTACGG)
ND5 for beef (GATTCCTAACCCACTCAAACG/GGTATGTTTGGGCATTCATTG)
Beef and buffalo in Multiplex PCR Targeting double genes 1% (w/w) meat in Hossain, Ali, Hamid,
meat curry and CytB for beef (GGAGTACTAGCCCTAGCCTTCTC/CTACTAGGGCTCAGAATAGGCATT) admixed and burger Hossain et al.
burger products ND5 for beef (GGTTTCATTTTAGCAATAGCATGG/GTCCAATCAAGGGTATGTTTGAG) products (2017)
CytB for buffalo (GGGTTCTAGCCCTAGTTCTCTCT/ATGGCCGGAACATCATACTT)
ND5 for buffalo (TCGCCTAGCTTCTTACACAAAC/TGGTTTGTGACTGTGATGGAT)
Pork, duck, chicken, Multiplex PCR 16S rRNA gene for goat (GCATCCTTTATGGACTAGC/GGGCGATAATTTGATTTGAC) 9.1% (w/w) of the Xu et al. (2016)
horse, and cat 16S rRNA gene for mouse (CAGCTTTTAACCATTGTAG/CAGAGAAGTTATAGGTGG) adulteration in
meats adulteration 16S rRNA gene for pork (CAACAATACACCAAAATG/TGTTGGTGAATTTGTTGAGC) mutton samples
in mutton 16S rRNA gene for duck (CCAGACATATACAAAAACTC/CTGATTATCCGTGAGAGAC)
16S rRNA gene for chicken (CCACACTAACAAGCAATAC/GGTTTATGTGTGGGTGGAC)
16S rRNA gene for horse (CTAGTCATCTATTTAAACC/GTGTTGTTTTTTGTGAATC)
16S rRNA gene for cat (GTCAAAAATAATGCAACC/CCTAATTTGCATACCTGTG)
Duck, beef, and Multiplex PCR CytB for duck (CCGTCCTAATCCTATTCCTGGTC/GGAATAGGAGGATGGTGAAGTAAGTA) 1% (w/w) of target Qin et al. (2016)
lamb meats in CytB for beef (ATCCTTCCATTTATCATCATAGCAA/GAGCTAGAATTAGTAAGAGGGCC) species in raw and
meat mixtures CytB for lamb (GAGTAATCCTCCTATTTTGCGACA/AGGTTTGTGCCAATATATGGAATT) heat-treated (100,
121, and 200 ◦ C for
20 min) meat
mixtures
Soybean and chicken Multiplex PCR 12S rRNA for chicken (TGAGAACTACGAGCACAAAC/GGGCTATTGAGCTCACTGTT) 0.05 ng DNA for each Tafvizi and
meat in beef CytB for beef (GACCTCCCAGCTCCATCAAACATCTCATCTTGAT- species. Hashemzadegan
hamburgers GAAA/CTAGAAAAGTGTAAGACCCGTAATATAAG) (2016)
(Continues)
DETECTION METHODS OF MEAT ADULTERATION…
TABLE 2 (Continued)
Detection
Detection items technology Target marker (primer, 5′ → 3′ ) Limits of detection References
Cat, dog, pig, Multiplex PCR CytB for cat (GGAATAATGTTTCGACCACTAAGC/TGCCTGAGATGGGTATTAGGAT) 0.01 to 0.02 ng DNA Ali et al. (2015)
monkey, and rat ATPase 6 for dog (TGGCTCTAGCCGTTCGATTA/AAGGCAACAGCAAATTCTAGG) under raw states and
meats in Islamic ND5 for pig (CCATCCCAATTATAATATCCAACTC/TGATTATTTCTTGGCCTGTGTGT) 1% suspected meats
foods ND5 for monkey (TGAGACCTCCAACAAATACTAGC/CTCTATGGCAGAAGGTAGTCAG) in meatball
ATPase 6 for rat (ATCATCAGAACGCCTTATTAGC/AGGTTCGTCCTTTTGGTGTATG) formulation
Chicken, duck, and Multiplex PCR 12S rRNA for chicken (CTATAATCGATAATCCACGATTCA/CTTGACCTGTCTTATTAGCGAGG) 1% target meat content Hou et al. (2015)
goose in meat CytB for duck (CGAGGCTTCTACTACGGC/GGGTGATTCCTGCGATTA) of total meat weight
products D-loop for goose (ACAGGACATACCCTAACA/GTCCAGGCTTAGATTGTG)
Bovine and porcine Multiplex PCR CytB for bovine (GCCATATACTCTCCTTGGTGACA/GTAGGCTTGGGAATAGTACGA) 0.1% w/w of both Shabani et al. (2015)
sources gelatin CytB for porcine (GCCTAAATCTCCCCTCAATGGTA/ATGAAAGAGGCAAATAGATTTTCG) porcine and bovine
gelatin
DETECTION METHODS OF MEAT ADULTERATION…

Goat meat SYBR Green 12S rRNA for goat 10% (w/w) of pork Li, Jalbani et al.
adulterated with real-time PCR (CTAGAGGAGCCTGTTCTATAATCGATAA/TGACCTAACGTCTTTATGTGTGGTG) adulteration in goat (2019)
pork 16S rRNA for reference meat
(AAGACGAGAAGACCCTTGGACTTTA/GATTGCGCTGTTATCCCTAGGGTA)
Cattle and horse TaqMan Nuclear DNA for cattle (C/AAGACAAAGGTCAGGAAGTAATC/AGATGAGGGAAGAGCAGGTCTG, 5% of limit of Wang, Liu, Zhang,
real-time PCR probe:FAM-CCTTCAGAGAGGGTCCCACTGGTCC-BHQ1) quantification Zhou, and Liu
SCPEP1 for horse (TGGCTCTGAAGATGATGAGGCTG/GAGGCAATGATTTTGTTCTGCGT, (2019)
probe:Cy5-CGCTTCCGCTCCCGTGCCAAGG-BHQ3)
LcoR for internal reference (CCAGCCAGCCCAATAGCACAA/GAGGTGAGTCTTGGTCAGCCAT,
probe:HEX-TGCYGAAAGCATCTC-MGB)
Pork, chicken, and TaqMan D-loop for pork (AGCTGGACTTCATGGAACTC/GCACGTTATGTCCTGTAACC, 0.5% (w/w) of each Kim and Kim (2019)
beef in real-time PCR probe:FAM-GATCCGGCACGACAATCCAA-MGBNFQ) species in meat
commercial CytB for beef (CCTTCTCTATCCTAATTCTTGCTC/AGTAGGTCTGCTACTAGGGC, mixtures, 0.1 pg of
processed meat probe:NED-TTCCGACCACTCAGCCAATG-MGBNFQ) DNA for all three
products CytB for chicken (AGCAATTCCCTACATTGGACACA/GATGATAGTAATACCTGCGATTGCA, target species
probe:VIC-ACAACCCAACCCTTACCCGATTCTTC-MGBNFQ)
Cat, rabbit, rat, and TaqMan CytB for cat (GGAATAATGTTTCGACCACTAAGC/TGCCTGAGATGGGTATTAGGAT, 0.1% (w/w) under Ahamad et al. (2019)
squirrel meat in real-time PCR probe:TAMRA/TCTGACTCCT/TAO/AGTAGCGGATCTCCTAACCC/3IAbRQ) admixed samples
food products CytB for rabbit (TCCGATACCTCCACGCTAAC/GGAGGATGATGCCAATGTTTC,
probe:Cy5/GTAGGCCGC/TAO/GGAATCTACTATGGATCATAC/3IAbRQ)
CytB for squirrel (TCCGACCTCTAAGCCAATG/ACTAACAGCTGGCATAAATAGAAGG,
probe:ROXN/GCCTGTAGA/TAO/ATACCCCTTTATCACAATCGG/3IAbRQ)
ATP6 for rat (CATCATCAGAACGCCTTATTAGC/AGGTTCGTCCTTTTGGTGTATG,
probe:HEX/CGCCTCCAC/ZEN/ACATTTCAACACTGACTAAT/3IABkFQ)
18S rRNA for internal reference
(GGTAGTGACGAAAAATAACAATACAGGAC/ATACGCTATTGGAGCTGGAATTACC,
probe: FAM-AAGTGGACTCATTCCAATTACAGGGCCT-ZEN/IOWA BLACK FQ)
(Continues)
9
 10

TABLE 2 (Continued)
Detection
Detection items technology Target marker (primer, 5′ → 3′ ) Limits of detection References
Donkey-hide gelatin TaqMan ND5 for donkey (TGCTAGCCTCATTATCAGTAT/GTGATGAGGATACGTGCT, 1 ng/𝜇L DNA for each Zhang, Cui, Cheng,
adulterated with real-time PCR probe:FAM-TCTACCAATCATATCATCAATCCTCAAC-BQ1) species Wei, and Ma
horse, ox, and pig CytB for pig (CAAAGCAACCCTCACACGATT/TGCGAGGGCGGTAATGAT, (2019)
gelatin probe:FAM-TTCGCCTTCCACTTTATCCTGCCATTC-BQ1)
COI for horse (GTAACCGCCCATGCATTC/CAGGTGCTCCAATTATCAGG,
probe:FAM-TATGGTCATACCCATTATAATCGGAGGATTCGGA-BQ1)
COI for ox (GGAACTCTGCTCGGAGAC/GTTACCGAACCCTCCAATTATG,
probe:FAM-TCTACAACGTAGTTGTAACCGCACACGC-BQ1)
Goat meat Real-time PCR RPA1 for goat (CTGACACACGGGACACATCTCC/AAGCTAAACATGGACCCACATG,probe:FAM- Suspected adulteration Wang, Wang et al.
adulteration with TAAGCCAGCCTTGTGCGTGTGGTCC-BHQ1) could be deduced (2019)
pork RPA1 for pig (ACCCAGACGAACTGCTCAA/TGGCGTCACTGATAGGTAAAT, by a good linear
probe:FAM-TCACAGGCGTGGGCTTTCTGC-BHQ1) correlation (R2 =
0.9868) in a range
from 5% to 80%
Pork adulteration in TaqMan ATPase 6 for pig (CACACCCACACAACTAT/GGTAGAAAGTGGGCTAGTGAT, 5 pg of pork total Raharjo et al. (2019)
meat and real-time PCR probe:FAM/TCAGCAACC/ZEN/GTATTCACAGGATTCCG/3IABkFQ) DNA and 1% (w/w)
meatballs pork contamination
in beef meatball
Pork, duck, and beef TaqMan ATP8 for pig (ATCTACATGATTCATTACAATTAC/CTATGTTTTTGAGTTTTGAGTTCA, Unspecified Song, Chen, Zhao,
in Sichuan real-time PCR probe:FAM-ATCTCAAACTACTCATACCCA-TAMARA) Ouyang, and Song
sausage products 16S rRNA for duck (AAGCCTTCCTCTAGCTCAGC/AGAAAATGCTTTAGTTAAGTC, (2019)
of China probe:FAM-CTCAGCCGCTTAAACAACGC-TAMRA)
BGH for beef (CCGATGGATGTGTTCAGAGCT/GCCAAATGTCTGGGTGTAGATACC,
probe:FAM-TCGGCTTTAGGGCTTCCGAATGTGAA-TAMRA)
18S rRNA for internal reference
(TCTGCCCTATCAACTTTCGATGGTA/AATTTGCGCGCCTGCTGCCTTCCTT,
probe:FAM-ATCTCAAACTACTCATACCCA-TAMARA)
Crocodiles meat in TaqMan CytB for crocodiles (CTCATAGCCACCATCCTCAC/CAAAAGACGCCATAG, 0.1% (w/w) target Nizar et al. (2019)
food chain real-time PCR probe:Cy5-CCTCATCTT/TAO/CCTCCACGAACGCGG/IOWA BLACK RQ) meat in model
18S rRNA for internal reference chicken meatball
(GGTAGTGACGAAAAATAACAATACAGGAC/ATACGCTATTGGAGCTGGAATTACC,
probe:FAM-AAGTGGACTCATTCCAATTACAGGGCCT- ZEN/IOWA BLACK FQ)
Wild boar meat in SYBR Green Mitochondrial D-loop for wild boar (ACTAATCAGCCCATGCTCAC/TGACTGTGTTAGGGCCTTTG) 4.68 ng DNA of wild Arini et al. (2018)
meatball real-time PCR boar meat and 2.34
formulation ng of wild boar in
meatball product
(Continues)
DETECTION METHODS OF MEAT ADULTERATION…
TABLE 2 (Continued)
Detection
Detection items technology Target marker (primer, 5′ → 3′ ) Limits of detection References
Pork adulteration in SYBR Green CytB for pork (CTGCCCTGAGGACAAATATCATTC/AAGCCCCCTCAGATTCATTCTACG) 0.01% (w/w) of pork Kang and Tanaka
processed beef real-time PCR CytB for beef (CTGCCGAGACGTGAACTACG/AAGCCTCGTCCTACGTGCATAT) in both raw and (2018)
products 18S rRNA for internal reference heat-treated samples
(CTGCCCTATCAACTTTCGATGGTA/TTGGATGTGGTAGCCGTTTCTCA)
Duck, pork, beef, TaqMan Mitochondrial DNA for pig (CAGCAACCGTATTCACAGGAT/GGCTACTGGTTGAATAAATAGGC, 0.01% (w/w) template Xu et al. (2018)
and chicken real-time PCR probe:Cy3-TTTCTACCACAA+GGAACACCCGCC-BHQ2) DNA of each
Mitochondrial DNA for cow (TTGAATTAGGCCATGAAGCA/CGACTTGTCTCCTCTCATGTAGC, species
probe:FAM-CGCCCGTCA+CCCTCCTCAAATA-BHQ1)
Mitochondrial DNA for chicken
(TAGCCCTAAATCTAGATACCTCCC/ACCGCCAAGTCCTTAGAGTTT,
probe:HEX-CACATGTATCCGC+CTGAGAACTACGA-BHQ1)
Mitochondrial DNA for duck (ACCTTCCCGCACCCTCTAAT/GGCAGATGGCGAGCAGAGAT,
DETECTION METHODS OF MEAT ADULTERATION…

probe:Cy5-ATCTCYG+CYTGATGAAACTTCGG-BHQ1)
Pork and chicken in TaqMan ACTB gene for pork (CGTAGGTGCACAGTAGGTCTGAC/GGCCAGACTGGGGACATG, 5 copies per reaction Wang, Cai, He,
meat products real-time PCR probe:FAM-CCAGGTCGGGGAGTC-NFQ-MGB) Yang, and Pan
TGF-𝛽3 gene for chicken (CAGCTGGCCTGCCGGC/GCCCAGTGGAATGTGGTATTCA, (2018)
probe:FAM-TGCCACTCCTCTGCACCCAGTGC-TAMRA)
Chicken and pigeon TaqMan CytB for chicken (CGGCTTACTACTCGCCGCA/GAGGTTTCGGATTAGCCGGC, 0.01% of each species Kim and Kim (2018)
in raw and real-time PCR probe:FAM-ACACATGCCGAAACGTACAG-BHQ1) DNA was detectible
heat-treated meats CytB for pigeon (AGCAATTCCCTACATTGGACACA/GATGATAGTAATACCTGCGATTGCA, in chicken–pigeon
probe:HEX-ACAACCCAACCCTTACCCGATTCTTC-BHQ1) mixtures
Fox, dog, mink, and SYBR Green Mitochondrial genomic DNA for fox The results showed a Wu et al. (2018)
rabbit real-time PCR (ACAGGGGAGAAAACGCTGTTC/GGCCTCCCCGAGATGAATC) good linear
Mitochondrial genomic DNA for dog relationship at 0.1
(GCGGCAAAGTTAACGGCAGT/GGATGGGAAGCAAACTCCGA) ng/𝜇L pure DNA
MItochondrial genomic DNA for mink
(TTCGGCGCTGCCTTAATTGTC/AAGACCTGGGACCCGAGAGTT)
Mitochondrial genomic DNA for rabbit
(TAGCAGTAGGGATGACAGGGTTT/GCCTTAGAGTAGGGTCTTTTTGG)
Fallow deer TaqMan Nuclear MC1-R gene for fallow deer 0.1% of isolated DNA Kaltenbrunner,
adulterated in real-time PCR (GACACCATGGAGCCACAGATAA/AGGCAGCTGTGGTGCTAC, from the samples Hochegger, and
game meat probe:VIC-CGTCGATGACATTGTCCAG-MGBNFQ) Cichna-Markl
(2018b)
Pork adulterated in Eva Green CytB for pork (CGGAACAGACCTCGTAGAATG/GGTAATGATGAATGGCAGGATAAAG) 0.00001 ng/𝜇L of Lubis et al. (2018)
food samples real-time PCR porcine DNA and as
low as 0.001% pork
adulteration in raw
pork–chicken
binary mixture
11

(Continues)
 12

TABLE 2 (Continued)
Detection
Detection items technology Target marker (primer, 5′ → 3′ ) Limits of detection References
Chicken adulterated TaqMan Chromosomal Actb gene for chicken 10 pg of DNA Xiang et al. (2017)
in meat products real-time PCR (TGACCTAGGTACCTCAATCATC/CCTAGAGTGACTTAGAGACTGG, template
probe:FAM-CAGACCTGCTAAGGGCCAGCAATA-TAMRA)
Horse, beef, sheep, TaqMan Cyclic-GMP-phosphodiesterase gene for beef 0.01 ng DNA for each Iwobi et al. (2017)
and pork fractions real-time PCR (ACTCCTACCCATCATGCAGAT/TTTTTAAATATTTCAGCTAAGAAAAAAG, species
probe:ROX-AACATCAGGATTTTTGCTGCATTTGC-TAMRA)
Growth hormone receptor gene for horse
(CCAACTTCATCATGGACAACGC/CCAACTTCATCATGGACAACGC,
probe:Cy5-AAGTGCATCCCCGTGGCCCCTCA-BHQ2)
Prolactin receptor gene for sheep
(CCAACATGCCTTTAAACCCTCAA/GGAACTGTAGCCTTCTGACTCG,
probe:ATTO425-TGCCTTTCCTTCCCCGCCAGTCTC-BHQ1)
Beta-actin gene for pork (CGAGAGGCTGCCGTAAAGG/TGCAAGGAACACGGCTAAGTG,
probe:HEX-TCTGACGTGACTCCCCGACCTGG-BHQ1)
Cattle, buffalo, and TaqMan ND5 for cattle (GGTTTCATTTTAGCAATAGCATGG/GTCCAATCAAGGGTATGTTTGAG, 0.003 ng of target Hossain, Ali, Sultana
porcine meat in real-time PCR probe:Hex-ACAAATCTCAATACCTGAGACCTCCAACAGA-ZEN/IOWA BLACK FQ) DNA in a 20 𝜇L of et al. (2017)
food chain CytB for buffalo (GGGTTCTAGCCCTAGTTCTCTCT/ATGGCCGGAACATCATACTT, reaction mixture
probe:TAMRA-AATCCTCATTCTCATGCCCCTGCTACA-TAO-IOWA BLACK RQ)
CytB for pork (TATCCCTTATATCGGAACAGACCTC/GCAGGAATAGGAGATGTACGG,
probe:ROX-CCTGCCATTCATCATTACCGCCC- TAO-IOWA BLACK RQ)
18S rRNA for internal reference
(GGTAGTGACGAAAAATAACAATACAGGAC/ATACGCTATTGGAGCTGGAATTACC,
probe:FAM-AAGTGGACTCATTCCAATTACAGGGCCT-ZEN/IOWA BLACK FQ)
Mutton adulteration TaqMan CytB for murine (CCGCCCAATCACCCAAA/GCCTCCGATT CATGTT, 1 pg of DNA per Fang and Zhang
with murine meat real-time PCR probe:FAM-TACTGAATYCTAGTAGCCAAC-BHQ) reaction and 0.1% (2016)
Internal positive control probe: CY5-TGGTCTTCTTAGCGAGAGTG-BHQ murine
contamination in
meat mixtures
Horse adulteration in TaqMan 12S rRNA for horse (GCCCTTGGGATGGAGAGA/GAGACTTTCGTCCGGGTTAAAGT, 1 pg of horse DNA Pegels et al. (2015)
food and feed real-time PCR probe:6FAM-CTTGTTCTTAGGGTAGAAA-BBQ)
materials
Rabbit, rat, and PCR-RFLP CytB for squirrel (ATCTCCCCACTCCTTCCAAT/CGCGGCCTACATGTAAGAAT) 0.1% (w/w) meat in Ali et al. (2018)
squirrel meat in CytB for rabbit (TCCGATACCTCCACGCTAAC/GGAGGATGATGCCAATGTTTC) frankfurter
frankfurter ATP6 for rat (CATCATCAGAACGCCTTATTAGC/AGGTTCGTCCTTTTGGTGTATG) formulation
products 18S rRNA for endogenous control
(GTAGTGACGAAAAATAACAATACAGGAC/ATACGCTATTGGAGCTGGAATTACC)
Digestion enzyme: BtsI, MutI, and BtsCI
(Continues)
DETECTION METHODS OF MEAT ADULTERATION…
TABLE 2 (Continued)
Detection
Detection items technology Target marker (primer, 5′ → 3′ ) Limits of detection References
Sea snake meat PCR-RFLP CytB (GCCTGAAAAACCACCGTTGT/CCGTCTTTGGTTTACAAGAAC) Unspecified Suntrarachun et al.
adulteration in 12S rRNA (CAAGGTCTTGGTCTTR(A/G)AACCT/CCATGTTACGACTTGCCCT) (2018)
meat products 16S rRNA (GCAATGAAGTGCGCACACACCGCC/CGGTCTGAACTCAGATCACGT)
Digestion enzyme: AluI and HinfI
Chicken, beef, and PCR-RFLP Genomic DNA for chicken 1% (w/w) of each Kušec et al. (2017)
sheep meat in (GGGACACCCTCCCCCTTAATGACA/GGAGGGCTGGAAGAAGGAGTG) species
pork sausages Genomic DNA for beef (GCCATATACTCTCCTTGGTGACA/GTAGGCTTGGGAATAGTACGA)
Genomic DNA for sheep (ATGCTGTGGCTATTGTC/CCTAGGCATTTGCTTAATTTTA)
Digestion enzyme: HaeIII, RsaI, and HinfI
Beef, buffalo, and PCR-RFLP CytB for beef (CGGCACAAATTTAGTCGAAT/TGGACTATGGCAATTGCTATG) 0.1% of adulterated Hossain et al. (2016)
DETECTION METHODS OF MEAT ADULTERATION…

pork in in food ND5 for beef (GGTTTCATTTTAGCAATAGCATGG/GTCCAATCAAGGGTATGTTTGAG) species could be

chain CytB for buffalo (GGGTTCTAGCCCTAGTTCTCTCT/ATGGCCGGAACATCATACTT) detected in
ND5 for buffalo (TCGCCTAGCTTCTTACACAAAC/TGGTTTGTGACTGTGATGGAT) autoclaved
CytB for pork (TATCCCTTATATCGGAACAGACCTC/GCAGGAATAGGAGATGTACGG) frankfurters
ND5 for pork (GATTCCTAACCCACTCAAACG/GGTATGTTTGGGCATTCATTG)
Digestion enzyme: AluI, EciI, and FatI.
Canine meat in PCR-RFLP CytB for canine (CCTTACTAGGAGTATGCTTG/TGGGTGACTGATGAAAAAG) 0.01% (w/w) of canine Rahman et al. (2015)
burger 18S rRNA for internal control meat in chicken and
formulation (GGTAGTGACGAAAAATAACAA/ATACGCTATTGGAGCTGGAATTACCTACAGGAC) beef burger
Digestion enzyme: AluI formulation
Macaque monkey PCR-RFLP D-loop gene for macaque monkey (GAAATCAATATCCCGCACA/CTGGTTGTTATGGCCCTGAG) 0.1% of target in Rashid et al. (2015)
meat in food chain Digestion enzyme: AluI and CViKI-1 mixed matrices and
commercial
meatball products
Onsite authenticity LAMP LOC106782588 gene for horse (F3/B3: 15 copies in horses Zhang et al. (2019)
detection of horse TGGATCTGAGGAGATGAGG/TTGAGGAGCATAGTCTTGAA, FIP/BIP: and donkeys
and donkey meat BIO-ATAGCTCCATCAGATCCTGGGCCAGGGTGCAGTAAAAGC/FITC-
CAAGGGAAGGTAGAGTCAGAGGGGAGGATGTCAGTATGAGAG)
LOC106825524 gene for donkey (F3/B3:
TCCTTGCCCTCGATGTCAA/GGGGAAGAAATGGATTTAAGGTGT,FIP/BIP:
BIO-AGGGGGCAATACAGTCCCATGGATTTCACTGGGCGAGAG/Digoxin-
CCAGACTGGCACTACAAATGAAGATCCCCATCATTGTCCGG)
Gcg gene for endogenous reference (F3/B3:
CGTGCCCAAGATTTTGTG/CGGCCAAGTTCTTCAACAA,FIP/BIP:
BIO-GTCCCTTCAGCATGTCTCTCAAAGATGAATACCAAGAGGAACAGG/Cy5-
AGCTGCCAAGGAATTCATTGCTTCTGGGAAATCTCGCCT)
(Continues)
13
TABLE 2 (Continued)
14

Detection
Detection items technology Target marker (primer, 5′ → 3′ ) Limits of detection References
Horse meat in meat LAMP COI for horse 0.1% contamination in Wang, Wan et al.
products (F3/B3: TGAATAAGAAGATGAAGCCTAGA/GCTCACCACATGTTTACAGT,FIP/BIP: xenogeneic meat (2019)
GTAAAAGTATTCAGCTGACTAGCCA-CTCAGAGTATAGCTGGAGATCA/CCAGTAGGGATAG sources
CGATGATTATGG-AGGGATAGCGTTGACACA,LF/LB:CCCTGCAC
GGAGGAAATATTCA/TAGCTGATGTGAAGTATGCTCG)
Chicken in processed LAMP 16S rRNA for chicken (F3/B3: ACCCACCCCCTAAAGACA/GGAGCTTTGACGCACTCTT,FIP/BIP: 10 fg of chicken DNA Sul et al. (2019)
meat products TGGAGCTGTACCCCCATCGAAT-
CCCACCTTTGTCAACCTTGA/GAACACAACCTCCTCCAGCGG-
TGTTGGTGGCTGCTTGAA,LB:TAATCACCCCTCCCCGCACTG)
Pork in meat LAMP COI for horse (F3/B3: TCGCCTACGCTATTCTAC/GGAAGTATAAGATGGAGGCTA,FIP/BIP: ≥0.1 pg/𝜇L and Azam et al. (2018)
products GGATGTGTGTAGTATGGGCATTAACTAGGTGGAGTGTTGG/TTCGACCACTAAGTCAA 0.001% in pure meat
TGCCGGTTGTCCTCCAATTCATG,LF/LB:ATTAGGATTAGG and binary mixture,
ATGGAGGCTA/CTAGTAGCAGACCTCATTACAC) respectively
Screen and identify LAMP Gcg for horse (F3/B3: TTGCCAAACGTCACGATGA/TGTCTGCGGCCAAGTTCT,FIP/BIP: The LOD as low as 10 Xu et al. (2017)
mammals on site TGGCAGCTTGGCCTTCCAAATATTTTTGAGAGACATGCTGAAGGGA/TTGCTTGGCTGGTGAAAGGCCTTTTTCAA
pg, which was
CAATGGCACCTCTTC) equivalent to 3 to 5
copies in mammals
Cow components LAMP Mitochondrial D-loop for cow (F3/B3: CCTTCATAAAAATTTCCCCCTTA/GTTGGGAGACT 5 % (w/w) of cow Deb et al. (2016)
adulterated in CATCTAGG,FIP/BIP: GCTAAATTGAGTATTGAAGAGCGTG-TCTACCACCAC milk/meat mixed in
buffalo milk/meat TTTTAACAGA/CAAAGTCAATATATAAACGCAGGCC-GTGCCTTGCTTTGGGTTA) buffalo samples
Pork in Halal LAMP Mitochondrial DN1 for (F3/B3: 0.5 pg for pork DNA Ran et al. (2016)
products CCTCACCCTAGTAGAACGAA/GCTTGACATGGCTAGCAT,FIP/BIP:
GGTGAATAGTTTTAGGGCATCGGCTTTTACTACGAAAAGGACCCAACG/TTGCACCAA
TCCTAGCCTTATCCCTTTTGGGTAGGGTATTGGTAGTGGA)
Onsite detection LAMP Mitochondrial D-loop for pork (F3/B3: 1 pg for raw pork Lee et al. (2016)
pork in processed GCAGGTAAATTATTAGCTCATTCA/AAATCTAGGGGGGTAGGT,FIP/BIP: DNA and 0.1% of
meat products CTTTGTTTTTGGGGTTTGGCAAGTTACCCCCCATTAAACTTATGC/AACATTTAA pork in a beef–meat
CAACACAAACCACCAT TGCTTTCGTAGCACGTATT). 18S rRNA gene for endogenous reference mixture
(F3/B3: GACCCATTCGAACGTCTG/GTTATTTTTCGTCACTACCTCC, FIP/BIP:
GAACCCTGATTCCCCGTCAC
CCCTATCAACTTTCGATGGT/CGGAGAGGGAGCCTGAGAAAGGAGTGGGTAATTTCGCG)
Silver pomfret ddPCR COI for silver pomfret (TCTGACTTCTTCCCCCATCTTTC/CAGTTCCAGCGCCAGCTT, 0.1% (w/w) for meat Cao et al. (2020)
probe: FAM-CTGTTATTAGCTTCTTCCGGAGT-MGBNFQ) mixtures
Beef, pork, chicken, ddPCR COI for sheep (AGGAACCGCCTTAAGCCTA/GTCCGTTAGTAGTATTGTGATACCA) Unspecified Shehata et al. (2019)
turkey, and sheep Mitochondrial DNA for beef (CCATATACTCTCCTTGGTGAC/GTAGGCTTGGGAATAGTACGA)
in Canada sausage Mitochondrial DNA for pork (CTCAATGGTATGCCACAACTAG/CATTGTTGGATCGAGATTGTGC)
products CytB for chicken (TCTGGGCTTAACTCTCATACTCACC/GGTTACTAGTGGGTTTGCTGGG)
Mitochondrial DNA for turkey
(CCACCTAGAGGAGCCTGTTCTGTAAT/TTGAGCTCACTATTGATCTTTCATTTT)
DETECTION METHODS OF MEAT ADULTERATION…

(Continues)
TABLE 2 (Continued)
Detection
Detection items technology Target marker (primer, 5′ → 3′ ) Limits of detection References
Alaska pollock in ddPCR 16S rRNA for Alaska pollock (GTACCTTTTGCATCATGATT/TGGCTGCTTTTARGCCCA) 4.2 copy numbers for Noh et al. (2019)
seafood products Alaska pollock
Goat and sheep in ddPCR Chromosome 9 gene for goat 1 copies/𝜇L for each Wang, Cai, He,
commercial (GGAAGGAAAGAGAATGGGGATATGG/TCTCCACACACAGCCAAAACC, species Yang, Li et al.
products probe:FAM-ATCCATCTCTCCCTCCACTCCCTGCCTAA-TAMRA) (2018)
PRLR gene for sheep (CCAACATGCCTTTAAACCCTCAA/GGAACTGTAGCCTTCTGACTCG,
probe:FAM-TGCCTTTCCTTCCCCGCCAGTCTC-TAMRA)
Chicken in sheep or ddPCR RPA1 for chicken 0.1% (w/w) of each Ren et al. (2017)
goat meat (CAGAACCACACTCAACCTGTCTGA/TCGGGGAAATGTCTTACTGCAAG,probe:FAM- meat
DETECTION METHODS OF MEAT ADULTERATION…

products CTCCTAGCAGCCTGTGCCAAGGCCA-BHQ1)
RPA1 for sheep and goat (CTGACACACGGGACACMTCTCC/AAGCTAAACATGGACCCACATG,
probe:FAM-TAAGCCAGCCTTGTGCGTGTGGTCC-BHQ1)
Beef and pork in ddPCR ACTB gene for beef (GCGGCCTCGGAGTGTGTA/CCCCAGAATGAGGTTCA,probe:FAM- 0.1 ng/𝜇l DNA for Cai et al. (2017)
meat products TCAGTAGGTGCACAGTAC-MGB) beef and pork
ACTB gene for pork (CGTAGGTGCACAGTAGGTCTGAC/GGCCAGACTGGGGACATG,
probe:VIC-CCAGGTCGGGGAGTC-MGB)
Bovine, porcine, ddPCR CytB for chicken (TCTGGGCTTAACTCTCATACTCACC/GGTTACTAGTGGGTTTGCTGGG, 0.05% (w/w) of bovine Shehata et al. (2017)
chicken, and probe:FAM-CATTCCTAACACTAGCCCTA-BHQ1) and turkey, 0.01%
turkey species in 12S rRNA for turkey (w/w) of pork and
food and feed (CCACCTAGAGGAGCCTGTTCTGTAAT/TTGAGCTCACTATTGATCTTTCATTTT, chicken could be
probe:FAM-TCCACCCAACCACCTCTTGCCAACAC-BHQ1) detected for
ATPase for porcine (CTCAATGGTATGCCACAACTAG/CATTGTTGGATCGAGATTGTGC, heat-processed food
probe:FAM-ATCTCAAACTACTCATACCCAGCAAGCCCA-BHQ1)
ATP synthase for bovine (CCATATACTCTCCTTGGTGAC/GTAGGCTTGGGAATAGTACGA,
probe:FAM-TAGACACGTCAACATGACTGACAATGATC-BHQ1)
Internal control (AAGACATTGTGGATGCAGATGAGTA/TAGGCAAGTGCATCCTCCTC,
probe:CALFluorOrange-CTTGTCCCTCCTGTTGGTACTAGAGA-BHQ1)
Cattle, horse, and ddPCR Nuclear F2 gene for cattle 0.001% of each Floren et al. (2015)
pig in processed (CCTGTCTGCTGAGACGCCG/GTGGTAGAGTTGATTCTGGAATAGAAAGCAT, species.
meat products probe:HEX-CCCCGCCACCCGCAGTGTCT-BHQ1)
Nuclear F2 gene for horse
(GCCAGCAGGCTGAGAACG/GTGGTGCAGTTGATTCTGGAATAGGAAATTT,
probe:FAM-CCATGCCTCGCCCACCCTCA-BHQ1)
Nuclear F2 gene for pig (CTGCCAGCGGGCTGGGAATA/GGAGTTGACTCTGGAATAAGAAATTG,
probe:FAM-CGCCCCCGCCCCCAGGGTCT-BHQ1)
15
16
with pork within the 10% to 100% mixture-level range. Taq-
Man technology has higher specificity and sensitivity than
those of SYBR Green technology. More importantly, it can be
used for multispecies detection (Xu et al., 2018). Pegels et al.
(2015) selected the 12S rRNA of 73 base pairs of horse DNA
as the target gene to design primers and amplified them by
TaqMan real-time PCR. The primer had high specificity and
sensitivity and had no cross reaction with other species. The
detection limit was 1 pg/mg horse DNA. Through the specific
primers and TaqMan probe design based on the CytB gene,
less than 1 pg of DNA per reaction and 0.1% murine contam-
ination in mutton could be identified (Fang & Zhang, 2016).
During real-time PCR design, the quality of template DNA
and primers is the key. Reynisson et al. (2006) proved that
the combination of locked nucleic acids with TaqMan probes
could increase the fluorescence signal. Based on these results,
Xu et al. (2018) developed a multiplex TaqMan locked nucleic
acid real-time polymerase chain reaction assay to simulta-
neously detect multiple meat sources (duck, pork, beef, and
chicken). The detection limit reached 0.01% (w/w) for each
species, and the method showed 2% higher accuracy than
that of a conventional real-time PCR method. The purity
of the templates DNA has also a great impact on the real-
time PCR results. The single-stranded DNA, RNA, and DNA
polymerase could cause the results false positive or false
negatives. Besides, the quantitative results of real-time PCR
are largely affected by the template DNA concentrations.
Recently, Kang and Tanaka (2018) compared two common
DNA quantification methods of template DNA, spectropho-
tometric and spectrofluorometric methods, and the results
indicated that the spectrofluorometric DNA quantification
method was more appropriate for qPCR assays for processed
food products because the spectrofluorometric methods only
measured double-stranded DNA in the DNA extracts, which
eliminated the interference of single-stranded DNA, RNA,
proteins, and organic contaminants. However, the current
research application of real-time PCR is limited by the rela-
tively high cost of reagents and equipment compared to those
for conventional PCR (Liu, Wang et al., 2019).
2.3 Polymerase chain reaction-restriction
fragment length polymorphism
Polymerase chain reaction-restriction fragment length poly-
morphism (PCR-RFLP) is a technique for variation analy-
sis by using restriction endonuclease digestion to identify
specific sequences of conserved regions of DNA amplified
by using PCR. PCR-RFLP is a sensitive, accurate, and ver-
satile method for meat authenticity verification (Hsieh &
Hwang, 2004; Rashid et al., 2015), and more simple and
time-saving than real-time PCR (Ali et al., 2018). Rahman
et al. (2015) used PCR-RFLP coupled with a lab-on-a-chip
detection platform to detect dog meat in burger formula-
DETECTION METHODS OF MEAT ADULTERATION…
tions and designed primers for CytB with the ability to detect
0.01% (w/w) dog meat in chicken and beef burgers. Using the
variation sequence in a defined region of DNA, the differenti-
ation of even closely related species could be identified (Hos-
sain et al., 2016; Hsieh & Hwang, 2004). The PCR amplifi-
cation size with species-specific oligonucleotide primers and
the mtDNA segments of both donkey and horse are exactly the
same, so direct PCR could not be used to discriminate these
two species. Doosti et al. (2014) used PCR-RFLP coupled
with AluI restriction enzyme to successfully identify donkey
and horse species in Halal food. In addition, cattle-buffalo and
sheep-goat (Girish et al., 2005), cattle, yak, and buffalo (Chen,
Liu, & Yao, 2010), swine and wild boar (Mutalib et al., 2012),
and chicken, beef, and sheep meat (Kušec et al., 2017) have
also been successfully differentiated by using PCR-RFLP
technologies.
However, the PCR-RFLP technique is complex and
requires a suitably equipped laboratory and expensive
enzymes (Kumar et al., 2015), and the enzymatic process is
prone to incomplete digestion and leads to unreliable results.
In addition, some of the disadvantages of direct species-
specific PCR technologies could also affect PCR-RFLP, such
as not being able to be utilized for quantification (Hossain,
Ali, Hamid, Hossain et al., 2017). Therefore, some researchers
committed to improving this approach through combined
methods. Hossain et al. (2016) developed multiplex PCR cou-
pled with RFLP to simultaneously quantitatively and qual-
itatively differentiate cattle, buffalo, and porcine meat by
restricting the PCR products of ND5 and CytB through the
AluI, EciI, and FatI enzymes, and 0.1% (w/w) of adulter-
ated species could be detected in autoclaved frankfurters. This
technique not only ensured accuracy but also improved the
detection sensitivity and quantitation.
2.4 Loop-mediated isothermal amplification
Loop-mediated isothermal amplification (LAMP) is a newly
developed meat adulteration identification technology based
on DNA markers in recent years (Lee, Kim, Hong, & Kim,
2016; Zhang, Lowe, & Gooding, 2014). The LAMP assay
requires four to six different primers to recognize six to eight
precise gene sequences (Notomi et al., 2000), and DNA ampli-
fication is performed by a Bst or Gsp DNA polymerase with
strand-displacing activity (Cho, Dong, & Cho, 2014), which
endows the technology with high sensitivity and specificity. In
addition, amplification is carried out under isothermal con-
ditions (60 to 65 ◦ C) for 30 to 60 min and does not require
sophisticated and expensive instruments (Azam et al., 2018;
Cho et al., 2014; Deb et al., 2016; Wang, Wan et al., 2019).
More importantly, the reaction results of LAMP could be
directly monitored by naked eye observation because precipi-
tation of white pyrophosphate ions had been produced during
the reaction. Adding fluorescent dyes for color reactions could
DETECTION METHODS OF MEAT ADULTERATION…
improve the sensitivity and achieve real-time monitoring of
the reaction (Zhang et al., 2014; Zhang et al., 2019).
A number of studies have demonstrated that LAMP might
be a fast, efficient, and economical method for meat adul-
teration detection (Azam et al., 2018; Cho et al., 2014; Deb
et al., 2016; Ran et al., 2016; Sul et al., 2019; Wang, Wan
et al., 2019; Xu et al., 2017; Zhang et al., 2019). Using LAMP
combined with colorimetric detection technology for the COI
gene, 0.1% of horse meat could be detected from processed
meats (Wang, Wan et al., 2019), and this method had obvious
advantages in commercially processed meat products. How-
ever, LAMP technology has high requirements for primer
design. The primers cannot undergo dimer formation, comple-
mentation of the primer itself, and the formation of the hairpin
structure of the 3’ ends. Recently, Sul et al. (2019) designed
five primers of the chicken mitochondrial 16S rRNA gene,
including two outer primers (forward primer F3 and reverse
primer B3), two inner primers (forward inner primer FIP and
reverse inner primer BIP), and one loop primer (reverse loop
primer LB) to identify chicken in processed meat; by using
LAMP techniques, 0.1% chicken meat in a raw meat sample
and 1% chicken meat in a heat- and pressure-treated meat sam-
ple could be detected, the detection time was only approxi-
mately 30 min, and this method had been successfully applied
to commercial monitoring.
2.5 Droplet digital PCR
Droplet digital PCR (ddPCR) is a new method for nucleic
acid detection and quantification. The principle of this method
is to perform independent PCR on a large number of small
reactors in the form of droplets that contain or do not con-
tain one copy of the target molecule template in each reactor
(Figure 3), to achieve “single-molecule template PCR ampli-
fication” (Cai et al., 2017; Li, Bai et al., 2018; Pohl & Shih Ie,
2004). After amplification, the number of copies of the tar-
get sequence can be counted by the number of positive reac-
tors based on the fluorescence signal (Figure 3). The ddPCR
method achieved absolute DNA quantification, and no stan-
dard curve was required. In addition, because the droplet
distribution principle was adopted, each PCR system con-
tained only one template, which mitigated the interference of
foreign genes and improved the accuracy and sensitivity of
detection. Based on these advantages, the ddPCR method has
been used in the field of food quality safety control, such as
genetically modified foods (Demeke & Dobnik, 2018; Košir,
Demšar, Štebih, Žel, & Milavec, 2019), foodborne diseases
(Li, Zhang et al., 2019; Suresh, Harlow, & Nasheri, 2019), and
food adulteration (or food ingredient content) (Naaum et al.,
2018; Noh et al., 2019; Shehata et al., 2017; Shehata et al.,
2019; Xiang et al., 2017). By using ddPCR, direct relative
quantification could be achieved because low-concentration
templates could be detected in high numbers of nontarget
17
nucleic acids (Floren et al., 2015; Morisset, Štebih, Milavec,
Gruden, & Žel, 2013; Noh et al., 2019), which is very com-
mon in processed meat products. Shehata et al. (2017) devel-
oped a ddPCR method to detect adulterations in processed
meats; as low as 0.05% and 0.01% (w/w) of bovine and
turkey targets and pork and chicken targets could be identified,
respectively, and this method has been gradually introduced to
commercial applications (Naaum et al., 2018; Shehata et al.,
2019).
However, ddPCR still has many problems in practical
applications. For example, some of the existing ddPCR meth-
ods cannot be converted from the gene copy number to the
meat mass ratio, and some of the conversion steps are com-
plicated (Basu, 2017) because the cell density, genome size,
and copy numbers of target genes in the genomic DNA vary
among different animal species (Ballin, Vogensen, & Karls-
son, 2009; Ren et al., 2017). In addition, the ddPCR exper-
imental process requires highly precise operation because
the droplet partition and volumes may affect the detection
results (Basu, 2017; Demeke & Dobnik, 2018). These short-
comings limit the application of this method. Therefore, the
establishment of a simple, convenient, and accurate digital
PCR quantitative detection system to improve the stability
and commercial applicability of meat adulteration detection is
necessary.
2.6 DNA barcoding and next-generation
sequencing
The above reviewed DNA-based technologies are mainly tar-
geted detection methods, but in meat adulteration detections,
many unknown meat species should be identified (Cottenet,
Blancpain, Chuah, & Cavin, 2020). Following this need, an
untargeted detection technology named DNA barcoding had
been developed (Cavin, Cottenet, Cooper, & Zbinden, 2018;
Hebert, Cywinska, & Ball, 2003). Through PCR amplification
and sequencing of specific gene fragments, and then search
it in the Barcode of Life Data (BOLD) system and the U.S.
National Center for Biotechnology Information database, the
adulterated meat species could be identified (Fiorino et al.,
2018). Since the DNA barcoding method provides a rapid,
accurate, and unknown species identification, it is consid-
ered a promising meat adulteration detection technique and
is already used in animal meat identification (Ahmed et al.,
2018; Cottenet, et al., 2020; Shehata et al., 2019; Xing,
Hu, Han, Deng, & Chen, 2020; Xing et al., 2019) and fish
authentication (Fiorino et al., 2018; Giusti, & Armani, 2017;
Ha, Huong, Hung, & Guiguen, 2018; Xing, Zhang et al.,
2020).
The early DNA barcoding technology mainly relied on
Sanger DNA sequencing for an approximately 650 bp region
of COI and CtyB gene of the animal species (Böhme et al.,
2019; Xing, Zhang et al., 2020). However, when there are
18
F I G U R E 3 Detection principle of droplet digital PCR
multiple adulterated ingredients in meat products, the tradi-
tional Sanger sequencing will generate multiple or overlay-
ing sequencing peaks, resulting in false sequence information
(Yang, Ding et al., 2018). Therefore, a DNA metabarcoding
method had been constructed to implement multispecies iden-
tification in complex samples using next-generation sequenc-
ing (NGS) technology. Furthermore, for processed meat prod-
ucts, DNA can be degraded to small fragments (<200 bp)
depending on the treatment (Cavin et al., 2018). Thus, a mini-
barcoding method, which focuses on shorter DNA fragments
(100 to 200 bp), had been developed by using NGS technol-
ogy (Böhme et al., 2019; Xing, Hu et al., 2020). Compared
to the early DNA barcoding technology, mini-barcoding has
the advantages of a higher throughput and higher sensitivity
(Böhme et al., 2019; Xing, Hu et al., 2020). In addition, it
is applicable for meat identification even on highly processed
meat products when targeting small fragments (Cottenet et al.,
2020; Guisti & Armani, 2017). Recently, Cottenet et al. (2020)
successfully applied a commercial NGS Food Authenticity
Workflow to identify untargeted meat species, 46 pure and
mixture meat species were successfully tested, including some
close-related species, such as bison versus beef and red deer
versus reindeer. Furthermore, the method was also suitable for
processed (grounded, cooked, and canned) samples identifica-
tion. However, the DNA barcoding technology also has some
disadvantages, such as expensive sequencing costs, time-, and
sample-consuming (Fiorino et al., 2018). Meanwhile, devel-
oping more unique barcode candidates should also be focused
in the future research.
In addition to the technologies reviewed above, some other
DNA-based meat adulteration detection technologies, such as
DNA lateral flow (Ha, Thienes et al., 2018; Magiati et al.,
2019; Masiri et al., 2016), randomly amplified polymor-
phic DNA-polymerase chain reaction (Böhme et al., 2019;
Kumar et al., 2015), high-resolution melting (Fernandes,
Costa, Oliveira, & Mafra, 2018; Lopez-Oceja, Nuñez, Baeta,
Gamarra, & de Pancorbo, 2017), and so on, have also been
developed in recent years. Because these technologies are not
commonly used, this review did not focus on them.
DETECTION METHODS OF MEAT ADULTERATION…
3 PROTEIN-BA SED
TECHNOLOGIES
Meat adulteration detection by using PCR methods is usu-
ally affected by many factors, such as poor trace quantita-
tive analysis, sampling pollution, and DNA degradation in
meat processing (Di Pinto et al., 2015; Li, Bai et al., 2018;
Naveena et al., 2017). Moreover, DNA extraction is time-
consuming and must be optimized for each particular case to
ensure that enough DNA was obtained for the analysis (Song
et al., 2017). Protein is the main component of meat. The
specific protein composition and three-dimensional structure
of specific proteins have certain conservation and specificity
between species, which is suitable for meat adulteration detec-
tion. Moreover, some protein molecules are tissue specific and
can be used for the identification of less valuable additives,
such as connective tissue, blood plasma, or milk preparations
(Jiang, Fuller, Hsieh, & Rao, 2018; Montowska & Spychaj,
2018; Ofori & Hsieh, 2007, 2015).
Protein-based food adulteration identification technologies
mainly include the following three methods: electrophoretic
analysis based on protein band characteristics, immunoassays
based on antigen antibody-specific reactions, and mass spec-
trometric analysis based on proteins or short peptides. Tra-
ditional electrophoretic techniques have seldom been used
because their sensitivity is insufficient. Therefore, meat adul-
teration detection techniques based on proteins mainly include
immunoassays and mass spectrometric analysis methods
(Table 3).
3.1 Enzyme-linked immunosorbent assay
There are two kinds of immunoassay techniques used in
meat adulteration detection: enzyme-linked immunosorbent
assay (ELISA) and immunosensors. ELISA is the most widely
applied immunoassay method of meat adulteration detec-
tion (Ha, Thienes et al., 2018). During ELISA detection, an
enzyme conjugate is first prepared by using a known antigen
or antibody adsorbed on the surface of the solid phase, and
TABLE 3 Recent 5 years representative studies of protein-based technologies for meat adulteration identification
Detection items
Pork adulteration in beef
Pork adulteration in meat
Porcine hemoglobin in meat
products
Pork fat protein in other animal
meats
Fat adulteration in cooked and
noncooked of pork, beef, and
chicken
Cooked wild rat meat in pork,
beef, and chicken
Heated mammalian meats
adulterated in poultry meats
Cooked beef in the pork, horse,
chicken, goat, and sheep
meat
Cooked chicken/turkey in pork,
horse, goat, or sheep meat
Pork in cooked horse, beef,
chicken, goat, and lamb
meats
Wheat protein in ground chilled
pork and beef mixture
Soybean proteins in surimi
products
Mammalian muscle tissues in
raw meat and meat products
Pork adulteration in beef
meatballs
Pork adulteration in cooked
meatballs
Detection Method sensitivity(limit of
technology Immunogen and antibody detection) References
Direct ELISA Porcine immunoglobulins G (IgG) and polyclonal antibodies 0.01% (w/w) of pork in beef Seddaoui and Amine (2020)
Indirect competitive Porcine IgG and polyclonal antibodies 0.1% of pork adulteration Mandli et al. (2018)
ELISA
Indirect competitive Mammalian hemoglobin 13F7 and monoclonal antibodies 0.5 ppm of PHb Jiang, Fuller, Hsieh, and Rao
ELISA (MAbs 13F7) (2018)
Indirect ELISA Thermal stable-soluble protein (TSSP) and monoclonal 1% (w/w) of pork fat Kim et al. (2017)
antibodies (MAbs PF 2B8-31) adulteration
Indirect ELISA Skeletal muscle troponin I (smTnI) and monoclonal antibodies ND Park et al. (2015)
(commercial ab97427)
DETECTION METHODS OF MEAT ADULTERATION…
Sandwich ELISA Rat heat-resistant proteins and polyclonal antibodies 0.01 𝜇g/L based OD values Chen, Ran, Zeng, and Xin
(2020)
Sandwich ELISA Mammalian skeletal troponin and monoclonal antibodies 1% (g/g) of heated meats Jiang, Rao, Mittl, and Hsieh
(MAbs 6G1 and 8F10) adulterated in poultry meats (2020)
Sandwich ELISA ND 0.1% (w/w) of the cooked Thienes, Masiri, Benoit,
products Barrios-Lopez, Samuel,
Krebs et al. (2019)
Sandwich ELISA ND 0.1% (w/w) of the cooked Thienes, Masiri, Benoit,
products Barrios-Lopez, Samuel,
Meshgi et al. (2019)
Sandwich ELISA ND 0.1% (w/w) for cooked samples Thienes et al. (2018)
Sandwich ELISA Gliadin and monoclonal antibodies 1% (w/w) for spiked samples Petrášová et al. (2017)
Sandwich ELISA Soybean trypsin inhibitor (STI) and monoclonal antibodies 13.6 mg/kg samples Jiang et al. (2015)
Sandwich ELISA Skeletal muscle protein troponin I (TnI) and monoclonal 4.8 ng/mL of bovine TnI Zvereva et al. (2015)
antibodies
Electrochemical Porcine IgG and polyclonal antibodies 0.01% of pork adulteration Mandli et al. (2018)
immunosensor
Lateral flow Porcine IgG and polyclonal antibodies 0.1% (w/w) for pork in beef Kuswandi et al. (2017)
immunosensor meatballs
(Continues)
19

TABLE 3 (Continued)
Detection items
Horse meat adulteration in
meat products
Pork adulteration in raw meat
Bovine adipose tissue in meat
products
Duck, goose, and chicken in
processed meat products
Grain proteins adulteration into
meat products
Porcine blood plasma to
emulsion-type pork sausages
Shrimp species in seafood
Pork, beef, lamb, chicken,
duck, soy, peanut, and pea
adulteration in meat products
20
Detection Method sensitivity(limit of
technology Immunogen and antibody detection) References
Lateral flow Horse serum albumin (HSA) and polyclonal antibodies 0.01% and 1.0% adulteration Masiri et al. (2017)
immunosensor for raw and cooked horse
meat
Label-free Porcine serum albumin (PSA) and polyclonal antibodies 0.5 pg/mL PSA in buffer Lim and Ahmed (2016)
electrochemical solution
immunosensor
Label-free Ruminant-specific muscle protein and polyclonal antibodies 2% bovine fat-in-pork fat Hsieh and Gajewski (2015)
electrochemical 1% bovine fat-in-porcine
immunosensor meat-and-bone meal
0.5% bovine fat-in-soy meal
mixtures
LC–ESI–QTOF–MS Hemoglobin alpha for duck: FMCAVGAVLTAK ND Fornal and Montowska (2019)
LC–ESI–QQQ– Hemoglobin beta for goose: FFSSFGNLSSPTAILGNPMVR
MS/MS Myosin-binding protein C for chicken:
LDVPISGEPAPTVTWK
……a
HPLC-MS/MS Barley: IETPGPPYLAK, Oat: DFPITWPWK, Rice: Oats and rye: 5 mg/kg meat Jira and Munch (2019)
ELGAPDVGHPMSEVFR, Rye: TPFASTVAGIGGQ, product; barley and wheat:
Wheat: SVAVSQVAR 10 mg/kg meat product
……a
UHPLC-MS/MS Plasma peptide marker of: ISEPLATETVR 0.7% (w/w) meat substitution Stader, Judas, and Jira (2019)
GSLDEFFHR, ISPLPDITPADFK, DPFPDFFSPVLK by porcine plasma
SWATH-MS Myosin heavy chain type a for Marsupenaeus japonicas: ND Hu et al. (2018)
AAVELDDLHASAER
Arginine kinase for Fenneropenaeus chinensis:
GTYYPLTGMGK
Sarco/endoplasmic reticulum Ca2+ -ATPase for Litopenaeus
vannamei: IGVFGENEETAGK
……a
UPLC-MS/MS Conglutin/Ara h 6 for peanut: EIMNIPQQCNFR, Alpha 0.5% adulterations of any of the Li, Zhang et al. (2018)
subunit of beta conglycinin for soy: ESYFVDAQPK, P54 eight species
protein for pea: GIIGLVAEDR, Myoglobin for duck:
HGVTVLTQLGK, Creatine kinase M-type for chicken:
DLFDPVIQDR, Hemoglobin subunit beta for sheep:
VDEVGAEALGR, Carbonic anhydrase 3 for beef:
LVNELTEFAK, Hemoglobin subunit beta for pig:
VNVDEVGGEALGR
……a
(Continues)
DETECTION METHODS OF MEAT ADULTERATION…
TABLE 3 (Continued)
Detection items
Horse, pork, and beef meat in
smoked sausages
Duck, pig, cattle, chicken, and
sheep in cooked meats
Pork gelatin adulteration in
meat products
Buffalo, sheep, and goat meat
in minced meat and meat
products
Chicken blood in sheep whole
blood samples
Water buffalo and sheep meat
in raw and cooked ground
meat mixtures
Beef and pork meat in highly
processed food matrices
Chicken, duck, and goose meat
in processed meat products
Meat adulteration in
mammalian meat samples
ND, not determined.
a
Only one or two representative peptide markers are listed.
Detection Method sensitivity(limit of
technology Immunogen and antibody detection) References
Infusion MS Myosin-1 for pork: SALAHAVQSSR, Myoglobin for beef: 5% (w/w) for pork and beef in Montowska1 and Spychaj
HPSDFGADAQAAMSK, Myoglobin for horse: the three-component matrix (2018)
VEADIAGHGQEVLIR and 1% (w/w) for horse meat
……a
UPLC-TripleTOF- M-protein, striated muscle for chicken: FWIQAESLSPNSTYR, ND Wang, Zhou et al. (2018)
MS Alpha-enolase for duck: LMLDMDGSENK, Trifunctional
UPLC-MS/MS enzyme subunit alpha (mitochondrial) for pig:
FAGGNLDVLK, Stress-induced-phosphoprotein 1 for
bovine: ALDLDSNCK, Hemoglobin subunit beta for sheep:
FFEHFGDLSNADAVMNNPK
High-resolution MS Type I collagen: TGETGASGPPGFAGEK, 0.1% (w/w) of undesired pork Yang, Ghosh, and Beaudry
HGNRGEPGPAGSVGPAGAVGPR gelatin (2018)
DETECTION METHODS OF MEAT ADULTERATION…
……a
MALDI-TOF MS Myosin light chain 1 for sheep: EAFLLYDR, Myosin light 1.0% for raw meat and 0.1% Naveena et al. (2018)
chain 2 for buffalo: NMWAAFPPDVGGNVDYK, Myosin cooked samples
light chain 1 for goat: EAFLLYDR
……a
Internal extractive Hemoglobin for blood samples, peptide marker 2% chicken blood in sheep Song et al. (2017)
electrospray Not determined blood
ionization mass
spectrometry
(iEESI-MS)
MALDI-TOF MS Myosin light chain 1 for sheep: EAFLLYDRTGDGK, Myosin 0.5% (w/w) of buffalo meat in Naveena et al. (2017)
UPLC-QTOF light chain 2 for sheep: FSQEEIR; Myosin light chain 1 for sheep meat
sheep: EAFLLFDRTGECK, Myosin light chain 2 for sheep:
FSKEEIK
HPLC/ESI-MS/MS Collagen a2-chain for beef: IGQpGAVGPAGIR, Collagen 2% pork meat in Bolognese Prandi et al. (2017)
a2-chain for pork: TGQpGAVGPAGIR sauce
Nano-LC-QTOF- Pyruvate kinase for chicken: EPADAMAAGAVEASFK, 1% (w/w) of chicken or pork in Montowska and Fornal (2017)
MS/MS Alpha-enolase for duck: NYPVVSIEDPFDQDDWGAWK, chicken, duck, and goose
Hemoglobin alpha-A for goose: TYFPHFDLQHGSAQIK meat mixture, 0.8% (w/w)
……a beef proteins in commercial
poultry frankfurters
Q Exactive Orbitrap Myoglobin for pork: HPGDFGADAQGAMSK, Myosin-1 for 1% (w/w) of pork or horse meat Orduna et al. (2017)
LC-MS/MS horse: TLALLFSGPASADAEAGGK, Myosin-2 for beef: in a mixture before and after
TLAFLFSGTPTGDSEASGGTK, 𝛽-Hemoglobin for lamb: cooking
FFEHFGDLSNADAVMNNPK, 𝛽-Hemoglobin for chicken:
FFASFGNLSSPTAILGNPMVR
……a
21
22
F I G U R E 4 Schematic representation of ELISA. (a): Direct ELISA, (b): sandwich ELISA, and (c): indirect competitive ELISA
the enzyme conjugate can specifically bind to samples con-
taining antibodies or antigens. After washing and incubation,
the substrates are added and reacted a color, and the extent of
color development is positively correlated with the amount of
the antibody or antigen in the samples (Figure 4).
The commonly used ELISA methods for meat adulteration
detection are direct ELISA (Mandli, El Fatimi, Seddaoui, &
Amine, 2018; Seddaoui & Amine, 2020), sandwich ELISA
(Ayaz, Ayaz, & Erol, 2006; Hsieh & Ofori, 2014; Thienes
et al., 2018; Zvereva et al., 2015), and indirect competitive
ELISA (Hsieh & Ofori, 2014; Jiang et al., 2018; Mandli et al.,
2018) (Figure 4). Compared to DNA-based detection tech-
nologies, ELISA methods show simplicity of sample prepara-
tion, low cost, and less time consumption. In addition, ELISA
detection does not require complex equipment and is easily
feasible for onsite monitoring (Mandli et al., 2018; Thienes,
Masiri, Benoit, Barrios-Lopez, Samuel, Krebs et al., 2019).
Using direct ELISA methods developed for anti-pig IgG poly-
clonal antibody, as low as 0.01% (w/w) pork adulteration in
raw meat, could be determined in 14 hr and 15 min, but the
detection time decreased to 45 min when using a competi-
tive ELISA method that was developed by immobilizing an
IgG standard and competed with the IgG in samples (Mandli
et al., 2018). To implement fast and onsite quantitative deter-
mination, researchers were committed to developing species-
specific ELISA kits for meat adulteration, and these detection
kits have been applied in meat processing factories or food
regulatory agencies. For example, a sandwich ELISA method
was suggested as a reference method for animal meat adul-
DETECTION METHODS OF MEAT ADULTERATION…
teration identification in cooked and canned meat and poul-
try products by the USDA Food Safety and Inspection Ser-
vice (Perestam, Fujisaki, Nava, & Hellberg, 2017). Besides, a
sandwich ELISA kit developed with the monoclonal antibody
(MAb) technique had been successfully applied in meat adul-
teration of commercial meat products in Turkey, and 22.0% of
the samples were determined to not be in compliance with the
Turkish Food Codex (Ayaz et al., 2006). Recently, Microbi-
ologique, Inc. developed a series of sandwich ELISA kits for
the quantitative detection of pork, beef, and chicken/turkey
in cooked meat, and 0.1% (w/w) of the target species could
be detected in 70 min with no interference by common food
matrices, such as pizza, eggs, milk, and so on (Thienes et al.,
2018; Thienes, Masiri, Benoit, Barrios-Lopez, Samuel, Krebs
et al., 2019; Thienes, Masiri, Benoit, Barrios-Lopez, Samuel,
Meshgi et al., 2019).
Because ELISA methods depend on the specific reaction of
antigen and antibody, it can only detect meat species for which
specific antibodies have already been developed. Therefore,
developing proper protein markers for ELISA methods is
essential. The optimal protein marker should meet the follow-
ing criteria: (1) uniqueness between species, (2) high concen-
trations in raw meats or meat products, (3) fairly stable during
meat processing, especially during heat processing and pick-
ling, and (4) stable in the presence of food additives, such as
sodium nitrate or nitrite, sodium chloride, edible phosphates,
citrates, ascorbates, and so on (Zvereva et al., 2015). Skele-
tal troponin I (TnI) is a part of myofibrils and is present as a
constituent of the stable tropomyosin–troponin complex; it is
DETECTION METHODS OF MEAT ADULTERATION…
considered a protein specific to muscle cells. Therefore,
Zvereva et al. (2015) developed a sandwich ELISA method
based on TnI to detect beef, pork, lamb, and horse meat, but
this protein marker could not identify poultry chicken, turkey,
and duck meat. Animal porcine hemoglobin could retain
molecular integrity and stability after heat treatment and
remain stable under acidic and alkaline conditions. Therefore,
Jiang et al. (2018) proposed a MAb 13F7-based indirect com-
petitive ELISA to quantitatively detect porcine hemoglobin
in meat products, and the limit of detection (LOD) was as
low as l.5 mg/kg. More importantly, this method has poten-
tial application value in detecting diseased pork, a serious
food safety issue in developing countries, by determining the
residual porcine hemoglobin level in pork because the porcine
hemoglobin concentration is much higher in diseased pork
than in healthy pork due to ineffective bleeding. In addition,
the ELISA methods cannot implement multispecies detec-
tion, which is also one of the main disadvantages of this
technology.
3.2 Immunosensors
As mentioned above, the ELISA methods show some limi-
tations during meat adulteration detection due to false posi-
tives caused by cross-reactivity and proteolysis caused by heat
processing. Therefore, researchers are dedicated to develop-
ing more sensitive, time-saving, and low-cost protein-based
methods to identify meat adulteration. Recently, immunosen-
sors have been reported to identify food adulteration detec-
tion (Ruiz-Valdepeñas Montiel et al., 2019). The principle
of immunosensor methods is similar to that of ELISA meth-
ods, but the former uses a biosensor to transit and amplify
the optical, electrical, or other signal of immune response to
a detectable signal, so the sensitivity of the method is bet-
ter than that of ELISA. The immunosensor technique has
been widely used in food allergy, pesticide residue, and milk
adulteration analyses, among others. However, only a few
reports have utilized immunosensing for meat adulteration
detection (Kuswandi, Gani, & Ahmad, 2017; Lim & Ahmed,
2016; Mandli et al., 2018; Masiri et al., 2016). Using an
electrochemical competitive immunosensor based on an anti-
pig IgG polyclonal antibody, as low as 0.01% pork adul-
teration, could be identified within 20 min. Compared with
competitive ELISA methods (0.1% pork adulteration could
be identified in 45 min), the detection limit and detection
time were greatly improved (Mandli et al., 2018). By using
a lateral flow device, 0.01%, 0.1%, and 1% pork could be
identified from raw meat, beef meatballs, and cooked meat,
respectively (Kuswandi et al., 2017; Masiri et al., 2016).
Although the immunosensor method is not widely used, we
think it has good application prospects in the field of meat
adulteration identification, especially in manufactory onsite
monitoring.
23
3.3 Protein mass spectrometry analysis
Because DNA-based technologies and immunoassay tech-
nologies are limited by DNA and protein stability, respec-
tively, most of these methods exhibit false positives and high
detection limits when identifying heat processed meat. In
addition, for the detection of meat in complex food matri-
ces (such as emulsion-type processed meat) and distinc-
tion between similar meat species, such as sheep and goat,
chicken and turkey, and duck and goose (Table 3), DNA-
based technologies and immunoassay technologies are usu-
ally not suitable (Fornal & Montowska, 2019; Prandi et al.,
2017; Song et al., 2017; Stader, Judas, & Jira, 2019). There-
fore, researchers are dedicated to developing techniques that
are more accurate and have a wider range of applications for
meat adulteration identification. Recently, mass spectrome-
try technologies based on protein and peptide analysis have
rapidly evolved and have been increasingly applied for meat
species identification. Since the amino acid sequence of pep-
tides is more stable than DNA during meat processing, they
have an incomparable advantage in meat adulteration iden-
tification, especially for highly processed meat products and
similar meat species (Prandi et al., 2017). By using a UHPLC-
MS/MS method, 0.7% of porcine blood plasma could be iden-
tified from emulsion-type pork sausages (Stader et al., 2019).
Skeletal muscle proteins are processing stable and species
specific, so Montowska et al. (2015) developed a rapid LESA-
MS method with no fractionation steps before and after pro-
tein digestion to identify species-specific markers for meat
adulteration detection, and 25 species and heat stable peptide
markers have been identified in processed beef, pork, horse,
chicken, and turkey meat. This method has potential applica-
tion value in meat quality evaluation by assessing fiber-type
composition.
Generally, mass spectrometry technologies can identify
species by simultaneously monitoring multiple specific pep-
tides (Jira & Munch, 2019; Li, Zhang et al., 2018), which
reduces the probability of false positives. In addition to
specificity, a good peptide marker should be of well pro-
cess stability, suitable size (≥ 6 amino acids), and con-
taining no cysteine (Johnson et al., 2011). Through quan-
tification and evaluation of 11 species-specific proteins
and 14 unique peptides, Montowska and Spychaj (2018)
developed a label-free quantification method utilizing high-
resolution mass spectrometry to determine the authenticity
of cooked and smoked sausages. The LOD was 5% (w/w)
for pork and beef in a three-component matrix and 1%
(w/w) for horse meat. Because mass spectrometry technolo-
gies can achieve multimarker detection, it can be imple-
mented to identify similar species (Fornal & Montowska,
2019; Montowska & Fornal, 2017). Poultry species, espe-
cially duck versus goose and chicken versus turkey, are
more difficult to identify than mammalian species in meat
24
products. Recently, Fornal and Montowska (2019) developed
an LC-QQQ method for the detection of chicken, duck, and
goose meat in highly processed meat products, and the meth-
ods showed a high ability for quantification and qualification
and low matrix interference. More importantly, this method
successfully identified two adulterations of products with
undeclared species in commercial products subject to homog-
enization, smoking, cooking, semidrying, and sterilization
processing. Water buffalo, sheep, and goats have relatively
high homogeneity, and mixtures of their meat are difficult to
distinguish (Naveena et al., 2017; Naveena et al., 2018). Using
2DE-MS and OFFGEL-MS technologies based on the marker
of myosin light chain 1 and 2, Naveena et al. (2018) success-
fully determined sheep and goat meat from raw and cooked
water buffalo meat, and the detection limits were as low as
0.1% (w/w) in the OFFGEL-MS method.
In addition to the detection of adulterated species, mass
spectrometry also plays an important role in the detection
of inferior meat. Freezing is a commonly used method in
meat preservation, but long-term frozen meat, which is called
“zombie meat” in China, often causes nutrient loss and an
excess of pathogenic microorganisms and poses serious health
risks. However, some illegal manufacturers use zombie meat
instead of fresh meat during meat product processing, and
DNA-based and immunoassay techniques do not easily dis-
criminate between the same species of fresh and zombie meat.
Therefore, Kim et al. (2015) developed a 2DE-MS-based
proteomics method to detect fresh meat and freeze-thawed
pork. A total of 450 protein spots from meat exudates were
identified, and 22 proteins, mainly myofibrillar protein, myo-
globin, and so on, could be selected as markers to discrimi-
nate between fresh and freeze-thawed pork. However, the cost
of equipment and maintenance of mass spectrometric instru-
ments are high, and sample pretreatment is expensive. More-
over, the analysis of MS data is very complicated, especially
proteomics data analysis based on mass spectrometry, which
requires a strong mathematical statistics background; thus, the
technicians must be highly trained (Table 1), which restricts
its promotion and application.
4 TECHNOLOGIES BA SED ON
M E TA BO L I T E PROF I L I NG
Small-molecule metabolites, except proteins and nucleic
acids, vary in different meats. Therefore, meat adulteration
can be identified by comparative analysis of metabolite pro-
files in the samples. This technology can better reveal the
physiological and biochemical status of the processed sam-
ples and reflect the small differences in metabolites between
samples with excellent sensitivity and accuracy (Lim et al.,
2017). In meat adulteration identification, lipidomics and fla-
DETECTION METHODS OF MEAT ADULTERATION…
voromics are the main focus. For each meat species, the type
and quantity of fatty acids in tissues are specific, so the
lipidome could be used to distinguish the species of a meat
adulterant (Ballin, 2010). By using GC-MS and UHPLC-
MS in tandem with principal component analysis (PCA) and
partial least squares discriminant analysis (PLS-DA), Trivedi
et al. (2016) established a metabolomics and lipidomics
method to detect pork adulteration in beef. The results indi-
cated that 23 metabolites were significantly correlated with
pork adulteration in beef. Volatile compounds are another tar-
get marker for meat adulteration identification because the
flavor of meat products of different species has special char-
acteristics. By using an electronic nose and GC-MS detec-
tion, Nurjuliana et al. (2011), Zhang et al. (2015), and Haddi
et al. (2015) successfully developed a method using an elec-
tronic nose and multivariate analysis to identify meat adulter-
ation. These methods provide some new ideas for the iden-
tification of adulterated meats. However, because the animal
growth environment, meat storage, and processing conditions
have a great impact on the metabolite content, whether these
metabolites are species specific requires further confirmation.
In addition, from the current literatures report, the technolo-
gies based on metabolite profiling cannot achieve quantitative
analysis in the meat adulteration detection. Therefore, these
technologies are not commonly applied currently.
5 NON D E ST RU C T I V E
TECHNOLOGIES
DNA-, protein-, and metabolite-based meat adulteration iden-
tification techniques require sample pretreatment, such as tis-
sue disruption, target analyte extraction, and purification, and
these pretreatment processes are invasive, cumbersome, and
time-consuming (Wang, Peng, Sun, Zheng, & Wei, 2018).
Therefore, researchers are dedicated to developing new sim-
ple and noninvasive sample pretreatment techniques for the
detection of meat adulteration, of which spectroscopic tech-
niques are the most widely used. Spectroscopic analysis is
based on the principle that different components, such as
moisture, proteins, fatty acids, lipids, or elements, in meat
and meat products produce different spectra at different wave-
lengths (Rady & Adedeji, 2018; Wang, Peng et al., 2018).
Spectral technologies have been gradually applied to food
quality and safety control in recent years due to their time
savings, simple sample preparation, and lack of need for sam-
ple pretreatment (Table 1), which are called nondestructive
technologies (Wang, Peng et al., 2018; Zheng et al., 2019).
The current nondestructive techniques applied in meat adul-
teration mainly include infrared spectroscopy (IRS), Raman
spectroscopy (RS), hyperspectral imaging (HSI), and laser-
induced breakdown spectroscopy (LIBS) (Table 4).
TABLE 4 Recent 5 years representative studies of nondestructive analytical technologies for meat adulteration detection
Detection Spectral
Detection items technology Wavenumber range pretreatment Data analysis Performance summary References
Minced beef Vis/NIR 350 to 2,500 nm Standardization CARS, RF, and CARS-RF model provided optimal Weng et al.
adulteration and SG PLSR performance for beef adulterated with pork (2020)
(Rp 2 and RMSEP were 0.973 and 2.145).
CARS-PLSR showed the best prediction
for beef adulterated with beef heart (Rp 2
and RMSEP were 0.960 and 2.758)
Pork, chicken, dog, and NIRS 1,100 to 2,300 nm SNV, MSC, DT, PCA and PLS-DA Classification of validation sets between López-
horse meat and 1st/2nd 78.95% and 100%. The limit detection of Maestresalas
adulteration in derivative foal meat adulteration in minced beef as et al. (2019)
DETECTION METHODS OF MEAT ADULTERATION…

minced lamb and beef low as 1%, Lidia breed cattle in minced
beef as low as 2%
Classification of chicken NIRS 900 to 1,700 nm SG and KS LDA, RF, and SVM Differentiating breast samples from thighs Nolasco-Perez
parts and drumstick with 98.8% accuracy et al. (2018)
Plant and animal protein Vis/NIR 400 to 1,700 nm 1st/2nd LDA, decision trees, The highest correlation coefficient values of Rady and
adulterants in minced derivative, CART, KNN, the prediction models are 0.85 (1.77), 0.86 Adedeji (2018)
beef and pork NOR, SNV, PLS-DA, FFNN, (1.95), 0.86 (1.98), 0.86 (1.87), and 0.87
MSC, and MC SVM, NB, and (1.64) for assessing pork, TVP, chicken,
PLSR-IPLS WG, TVP, and prok +TVP in minced beef,
respectively, and 0.86 (1.79) for assessing
TVP in minced pork
Beef adulteration with UV/Vis/NIR 200 to 1,100 nm NOR, MSC, PW-AF and PLSR The best UV-Vis-NIR model was PW-AF Chen et al.
spoiled beef SNV, and model, which selected 29 wavelengths to (2018)
1st/2nd obtain RMSEs of 0.10 and 0.12 for cross
derivative validation and prediction, respectively
Beef and chicken in FT-IRS 1,700 to 1,071 cm−1 SNV, MSC, and PLSR and ANN ANN showed higher R2 of 0.999 and lower Keshavarzi,
meat products Min-Max training and testing performance errors Banadkoki,
NOR (RMSEcv = 0.32 and RMSEp = 0.73) in Faizi,
comparison with PLSR model (R2 = 0.889, Zolghadri, and
RMSEcv = 1.02, and RMSEp = 1.74) Shirazi (2020)
Pork adulterated in the FT-IRS 4,000 to 450 cm−1 1st/2nd PLS-DA and SVM PLS-DA showed better identification than the Yang, Wu et al.
beef and mutton derivative, SVM. The R2 of calibration and testing sets (2018)
MSC, SNV, in PLS-DA reached 0.99 and 0.99, RMSEC
SG, and NOR was 0.06, and both the RMSECV and
RMSEP were 0.08
(Continues)
25
 26

TABLE 4 (Continued)
Detection Spectral
Detection items technology Wavenumber range pretreatment Data analysis Performance summary References
Beef, chicken, mutton, FT-IRS 6,250 to 4,100 cm−1 SNV, 1st and 2nd PCA, SVM-C, and R2 of FT-NIR system was 0.85 to 0.94, SECV Wiedemair et al.
turkey, pork, and instrument derivations PLSR = 7.52% to 13.83%, RPD = 2.2 to 5.7. The (2018)
horse meat LOD was 1% for FT-NIR device
Beef adulterated with FT-IRS 4,000 to 850 cm−1 NOR, 1st and HCA, PCA, and PCA provided better classification than HCA Deniz et al.
chicken or turkey 2nd one-way ANOVA in both adulterated mixtures (2018)
meat derivations In PCA, 1,290 to 1,210 cm−1 , and 1,480 to
1,425 cm−1 was used in the identification
of turkey and chicken meats, and the 1,760
to 1,710 cm−1 showed significantly robust
in chicken meat identification
Pork and turkey hams FT-IRS 4,000 to 500 cm−1 – LOO and PNN The major protein-structure content of pork- Sinanoglou et al.
and turkey-ham samples is 𝛽-sheet. The (2018)
spectra of the turkey hams presented
significantly (P < 0.05) higher intensities
at 1,040 to 1,020 cm−1 compared to the
pork hams
Norwegian Salmon FT-IRS 4,000 to 450 cm−1 SNV, MSC, and PLS-DA When the subwaveband range covering 450 to Wu, Zhong, and
adulteration with NOR 1,790 plus 2,800 to 3,050 cm−1 , the Rcal 2 Yang (2018)
Heilongjiang Salmon and Rcv 2 values were 0.99 and 0.98,
respectively
Offal content in ground FT-IRS 4,000 to 550 cm−1 2nd derivative LDA, PCA-DA, The accuracy>99% identified the type of Hu et al. (2017)
beef meat PLS-DA, KNN, offal in the sample with >80% confidence,
SIMCA, and and identified the five types of offal (heart,
PLSR kidney, liver, omasum, and honeycomb
tripe) accurately (R2 > 0.81) in the LOD <
10% (w/w)
Turkey meat FT-IRS 12,500 to 3,750 SNV, MSC, and PCA, PLS, PLS-DA, PLS regression models with R2 in prediction Alamprese et al.
adulteration in fresh, cm−1 1st/2nd and latent higher than 0.884 and RMSEP lower than (2016)
frozen-thawed, and derivative variables 10.8; PLS-DA discriminates each type of
cooked minced beef sample in two classes (adulteration
threshold = 20%) by the sensitivity and
specificity in prediction higher than 0.84
and 0.76, respectively
(Continues)
DETECTION METHODS OF MEAT ADULTERATION…
TABLE 4 (Continued)
Detection Spectral
Detection items technology Wavenumber range pretreatment Data analysis Performance summary References
Pork adulteration in beef FT-IRS 4,000 to 700 cm−1 – LDA, SIMCA, and LDA model should be better than SIMCA Kuswandi et al.
jerkys SVM and SVM. It could classify and predict (2015)
100% accuracy of the sample test
Rat meat in meatball FT-IRS 4,000 to 400 cm−1 – PCA and PLS 750 to 1,000 cm−1 was selected during PLS Rahmania et al.
products and PCA modeling, R2 and RMSEC were (2015)
0.993 and 1.79%, respectively
Pork adulteration in the FT-IR instrument 12,500 to SNV, MSC, PCA and SVM SVM-PCA is an optimal technique to Schmutzler et al.
meat and fat part of 4,000 cm−1 PCA, and automate discriminate between adulterated (2015)
DETECTION METHODS OF MEAT ADULTERATION…

veal sausages 1st/2nd/3rd samples. Using this method, 10% of the

derivative adulteration could be onsite detected
Rainbow trout RS 2,000 to 500 cm−1 1st/2nd RFE, GA, SA, KM, MSC-GA–KM–Cubist machine learning Chen et al.
adulteration in derivative, PLSR, and model showed optimal results for the (2019)
Atlantic Salmon MSC, and machine learning Rainbow Trout adulteration, R2 and
SNV RMSEP were 0.87 and 10.93, respectively
Bovine meat by the RS 3,600 to 710 cm−1 NOR PLS-DA The PLS-DA model showed a great potential Nunes et al.
addition of salts and applicable in real situations, the sensitivity, (2019)
carrageenan false positive rate, reliability, or efficiency
rate were 91.5%, 11.7%, and 79.8%,
respectively
Beef tallow, pork lard, RS 1,800 to 700 cm−1 NOR PCA Standard Raman peak ratio, that is (I1,655 × Lee et al. (2018)
chicken fat, and duck I968 )/(I1,442 × I1,442 ) as an oil gauge,
oil followed by discrimination between duck
oil, chicken fat, pork lard, and beef tallow
Offal adulteration of RS 3,380 to 250 cm−1 NOR and 1st/2nd PCA, PLS-DA, PLS-DA predicted 90% to 100% of Zhao et al.
thawed beef burgers derivative SIMCA, and calibration samples and 81% to 90% of (2015)
PLSR validation samples. In SIMCA model,
sensitivity values based on PCA were
higher than PLS
Beef adulteration with Vis/NIR HSI 400 to 1,000 nm – PLSR, SVM, LS-SVM based on IWO model performed the Zhao et al.
spoiled beef LS-SVM, ELM, best, where R2 was of 0.97 and 0.95, as (2019)
IWO, CARS, and well as RMSE was of 4.74% and 5.67% for
GA calibration and prediction, respectively
(Continues)
27
 28

TABLE 4 (Continued)
Detection Spectral
Detection items technology Wavenumber range pretreatment Data analysis Performance summary References
Duck meat adulteration Vis/NIR HSI 400 to 1,000 nm SR and 2nd MLR, SPA, and PLSR model with selected wavelengths (2nd Zheng et al.
in minced lamb meat derivative PLSR derivative) achieved better results than (2019)
others with RP 2 of 0.995 and RMSEP of
2.51%
Duck meat adulteration Vis/NIR HSI 380 to 1,012 nm MSC, SNV, and PLSR, PCR, Model based on competent wavelengths Jiang et al.
in beef 1st/2nd/3rd LOOCV, and PCA selected from PC loadings resulted in good (2019)
derivative performance of Rp2 = 0.96, RMSEP =
6.58%, and RPD = 4.86 with LOD of
7.59%
Carrageenan Vis/NIR HSI 380 to 1,012 nm 1st/2nd PLSR and PCA PLSR model developed by wavelengths Zhang, Jiang, and
adulteration in derivative, selected from absorbance spectra (Rp2 = Wang (2019)
chicken meat MSC, and 0.85, RMSEP = 0.93, and RPD = 3.20)
SNV was successfully used to visualize the
carrageenan distribution in chicken meat
Differentiate ground NIR HSI 900 to 2,500 nm 1st/2nd PLSR and PCA PLSR models indicated that NIR-HIS Nolasco-Perez
samples from beef, derivative, provided better results than portable NIR et al. (2019)
pork, and chicken MSC, and spectrometer, the Rp2 was 0.83 and 0.94,
meat SNV RPD was 1.96 and 3.56, for samples of
chicken adulterated with pork and beef,
respectively, under selecting wavelengths
condition
Minced beef MSI 405 to 970 nm – PCA, PLS-DA, RF, Using a two-step SVM model, all pure and Ropodi et al.
adulteration with and SVM freshly ground samples were classified (2017)
horsemeat correctly and the overall correct
classification was equal to 95.31%
Chicken adulteration in Vis/NIR HSI 400 to 1,000 nm 1st/2nd PLSR Rp 2 was 0.97, 0.97, and 0.96, RMSEP was Kamruzzaman
minced beef derivative, SG 2.62%, 2.45%, and 3.18% (w/w) for et al. (2016)
smooth, MSC, reflectance, absorbance, and
and SNV Kubelkae–Munck units spectra,
respectively
(Continues)
DETECTION METHODS OF MEAT ADULTERATION…
TABLE 4 (Continued)
Detection Spectral
Detection items technology Wavenumber range pretreatment Data analysis Performance summary References
Identification of Vis/NIR HSI 328 to 1,115 nm MSC SPA, GLGCM, RBF-SVM model showed a slightly better Xiong et al.
free-range and broiler PLS-DA, and classification result than PLS-DA. The (2015)
chicken meats RBF-SVM correct classification rate for calibration
and the prediction of RBF-SVM model
was 100% and 93.33%, respectively
Pork adulteration in Vis/NIR HSI 420 to 1,000 nm 2nd derivative, PLSR, PCR, and PLSR showed better performance than PCR Kamruzzaman
minced beef MSC, and MLR for predicting pork adulteration in minced et al. (2015a)
SNV beef. PLSR-MLR showed Rc 2 of 0.992,
SEC of 1.831%, Rp 2 of 0.985, and SEP of
4.172%
DETECTION METHODS OF MEAT ADULTERATION…

Horse meat adulteration Vis/NIR HSI 400 to 1,000 nm 1st/2nd PCA and PLSR PLSR models based on raw spectra had Rc 2 , Kamruzzaman,
in minced beef derivative, SG Rcv 2 , and Rp 2 of 0.99, 0.99, and 0.98, Makino,
smooth, MSC, respectively, and SEC, SECV, and SEP of Oshita, and
and SNV 1.14%, 1.56%, and 2.23%, respectively Liu (2015)
Shrimp, chicken, beef, LIBS – MSC KNN The KNN model with MSC could identify Chu et al. (2018)
Scallop, and pig liver 100% of the adulteration with coefficient
of variance of 0.56%
Offal adulteration in LIBS – NOR and OSC PCA and PLS Adulteration ratio can be determined using Velioglu et al.
beef PLS with R2 of 0.947 and LOD of 3.8% for (2018)
offal mixture adulterated in beef samples
Offal adulteration in LIBS – SNV PLSR PLSR was performed to build a calibration Casado-Gavalda
beef and validation model. The Rcv 2 and et al. (2017)
RMSECV were 0.85 and 43.5 ppm,
respectively, the Rp 2 RMSEP was 0.85 and
36.8 ppm, respectively
Identification of pork, LIBS – NOR, 2nd PCA and PLS The PLS model showed R2 and LOD of 0.994 Bilge et al.
beef, and chicken derivative, and 4.4% for pork adulterated beef, and (2016)
OSC, and 0.999 and 2.0% for chicken adulterated
SNV beef, respectively
AF, fish swarm algorithm; ANN, artificial neural network; CARS, competitive adaptive reweighted sampling; CART, classification and regression tree; ELM, extreme learning machine; FFNN, feed forward artificial neural network;
FW, full wavelength model; GA, genetic algorithm; HCA, hierarchical cluster analysis; IPLS, interval PLS; IWO, invasive weed optimization; KM, K-means clustering; KNN, k-nearest neighbor; KS, Kennard and Stone; LDA,
linear discriminant analysis; LOD, limit of detection; LOO, leave-one-out evaluation method; LOOCV, leave-one-out cross-validation; LS-SVM, least squares-SVM; MC, median center; MLR, partial least square regression; MSC,
multiplicative, scatter correction; NB, naïve Bayes; NOR, normalization; OSC, orthogonal signal correction; PCA, principal component analysis; PCR, principal component regression; PLS, partial least square; PLS-DA, PLS
discriminant analysis; PLSR, PLS regression; PNN, probabilistic neural network; PW-AF, spectral pretreatment before wavelength selection-AF; RBF-SVM, radial basis function-support vector machine; RFE, recursive feature
elimination; RMSEP, root mean square error of prediction; RPD, ratio performance to deviation; SA, simulated annealing; SEC, standard errors in calibration; SECV, standard errors in cross-validation; SEP, standard errors in
prediction; SG, Savitzky–Golay; SNV, standard normal variation; SPA, successive projections algorithm; SR, stepwise regression; SVM-C, support vector machine classification.
29
30
5.1 Infrared spectroscopy
IRS is an optical technique that detects molecular bond (such
as C-H, O-H, N-H, C-O, etc.) vibrations and rotations upon
absorption of infrared light. Because different chemical func-
tional groups absorb infrared light at different frequencies,
IRS can be used for meat adulteration identification because
the composition of various meats is different, and its absorp-
tion spectrum is specific (Fu & Ying, 2016). The most com-
monly used infrared spectrum technologies in meat adulter-
ation are near-infrared spectroscopy (NIRS), from 4,000 to
12,500 cm−1 , and mid-infrared spectroscopy, from 400 to
4,000 cm−1 (Abu-Ghoush et al., 2017; Wu et al., 2018). Using
Fourier transform infrared spectroscopy (FT-IRS), 10% of
animal offal in ground beef could be detected, and the accu-
racy was as high as 99% (Hu et al., 2017). Recently, Wu
et al. (2018) developed an FT-IR method combined with
improved PLS-DA to detect Norwegian salmon adulteration
with Chinese Heilongjiang salmon. Absorption peaks of CH2 ,
C = O, and C–O–C stretching vibrations from lipids or
protein were identified to distinguish Chinese Heilongjiang
salmon from Norwegian salmon. Moreover, IRS methods also
showed a certain application prospect in meat quality detec-
tion. Sinanoglou et al. (2018) established an FT-IRS method
that suggested that the absorbance bands of proteins, triglyc-
erides, fatty acids, and carbohydrates of raw meat and process-
ing meat products are different, and the refrigeration storage
time could be predicted by the partial degradation of triglyc-
erides and proteins.
To improve the accuracy and simplicity of IRS meth-
ods, spectral pretreatment and multivariate analysis are very
important. Infrared scanning can obtain a number of spec-
tra, including sample information and interference informa-
tion. Spectral pretreatment eliminates irrelevant information
and noise. Commonly used spectral pretreatment methods
include normalization, smoothing, derivation, standard nor-
mal variation (SNV), multiplicative scatter correction (MSC),
and so on (Alamprese et al., 2016; Chen et al., 2018). Multi-
variate analysis is used to calculate the useful sample infor-
mation and quantitative analysis of the target components.
Commonly used multivariate tools include PLS-DA, PLS,
multiple linear regression (MLR), principal component
regression, support vector machine (SVM), and so on
(Rohman, 2019; Wang, Peng et al., 2018) (Table 4). Alam-
prese et al. (2016) applied FT-NIR coupled with PCA and the
PLS-DA model to discriminate turkey adulteration in fresh,
frozen-thawed, and cooked minced beef. PLS-DA classifi-
cation models are able to distinguish between low (<20%)
and high (≥20%) levels of adulteration. Recently, Chen et al.
(2018) developed UV-Vis-NIR in tandem with a novel syn-
chronous wavelength selection and pretreatment methods,
artificial fish swarm algorithm (AF), to detect beef adul-
teration with spoiled beef. By using spectral pretreatment
DETECTION METHODS OF MEAT ADULTERATION…
before wavelength selection (PW-AF model), 29 wavelengths
were selected for PLS regression analysis, and the root-
mean-square errors (RMSEs) were 0.10 and 0.12 for cross-
validation and prediction, respectively. The study also found
that the sequence of spectral preprocessing and wavelength
selection is very important for the final detection results.
5.2 Raman spectroscopy
RS is a technique for analyzing the composition of a substance
and its structure based on the Raman scattering effect (Hu,
He, Zhang, Yang, & Liu, 2019). Raman bands are indepen-
dent of the intensity of the incident light and are related to the
polarizability of the sample constituent itself. The vibrational
frequency fingerprint and intensity in RS could be applied in
the analysis of the nature of molecules and their concentra-
tions (Lee et al., 2017), and, more importantly, this technique
is noninvasive, requires small sample volumes, is insensitive
to water, does not require sample preparation, and is suitable
for opaque samples (Hu et al., 2019; Lee et al., 2017; Lee et al.,
2018). By using RS and the PLS-DA model based on func-
tional groups of amino acids, lipids, and proteins, the non-
meat ingredients sodium chloride, sodium tripolyphosphate,
and carrageenan could be determined in pork products (Nunes
et al., 2019). Recently, Lee et al. (2018) successfully estab-
lished an RS method to distinguish beef tallow, pork lard,
chicken fat, and duck oil when the lard contents ranged from
0% to 100% (v/v), and the results showed good correlated lin-
ear relationships.
Similar to other vibrational spectroscopic methods, chemo-
metric operations, such as spectral data preprocessing and
multivariate analysis, are also very important for Raman
spectroscopic methods (Teixeira & Sousa, 2019). The com-
mon preprocessing techniques mainly include first derivation,
second derivation, MSC, SNV, and so on, and commonly
used multivariate analytical methods include PCA, hierarchi-
cal cluster analysis, PLS-DA, soft independent modeling of
class analogy (SIMCA), artificial neural networks, and so on
(Table 4). Chen et al. (2019) applied different data prepro-
cessing methods to quickly identify rainbow trout adulteration
in Atlantic salmon, and the results indicated that the MSC
method was better than first derivation (FD), second deriva-
tion (SD) and SNV. By using RS in tandem with PLS-DA and
SIMCA, offal-adulterated and authentic beef burgers could be
identified, and the accuracy rate was as high as 90% (Zhao
et al., 2015).
5.3 Hyperspectral imaging
IRS and RS are single-point detection methods and only col-
lect information about local areas of the samples (Feng & Sun,
2012), which suggests that the spectral information cannot
fully represent the information of the samples due to uneven
distribution of the adulterated ingredients (Zhao et al., 2019;
DETECTION METHODS OF MEAT ADULTERATION…
F I G U R E 5 Flowchart for meat adulteration detection and visualization of the HIS method. Figure derived from Kamruzzaman et al. (2015a),
Li et al. (2020), and Zhao et al. (2019). Abbreviations: ROI, regions of interest; Rp 2 , coefficient of the prediction model
Zheng et al., 2019). Moreover, these methods could also not
intuitively reflect the real aspect of the samples. Therefore,
the combined application of spectroscopic and imaging tech-
nologies, such as multispectral imaging (MSI) and HSI, devel-
oped in recent years further overcomes these shortcomings
(Ropodi et al., 2017; Zheng et al., 2019). MSI technology
is limited in meat adulteration detection because few wave-
lengths could be applied (Zheng et al., 2019). HSI technol-
ogy has been proven to be a promising method in meat adul-
teration detection (Al-Sarayreh, Reis, Yan, & Klette, 2018;
Kamruzzaman, Makino, & Oshita 2015a; Kamruzzaman et al.
2015b; Kamruzzaman et al., 2016; Velásquez, Cruz-Tirado,
Siche, & Quevedo, 2017; Xiong et al., 2015; Zhao et al., 2019;
Zheng et al., 2019). A hyperspectral image contains a great
deal of information in a three-dimensional (3D) form called
a “hypercube”: two of the dimensions are coordinate infor-
mation of spatial pixels (x and y), and the third dimension
(𝜆) is wavelength information (Ropodi, Panagou, & Nychas,
2016; Wang, Peng et al., 2018; Xiong et al., 2015) (Figure 5).
As a combination and extension of traditional spectroscopy
31
and digital imaging, HSI provides detailed information about
external attributes (such as shape, size, and color of the sam-
ples) and internal attributes (such as chemical composition)
(Kamruzzaman et al. 2015b; Wang, Peng et al., 2018). Kam-
ruzzaman and coworkers (2015a) used a visible near-infrared
hyperspectral imaging (Vis/NIR-HSI) system to detect pork
adulteration in minced beef. Wavelengths of 430, 605, 665,
and 705 nm were selected to replace the full-range spectra to
build the MLR model. This method could predict 2% to 50%
pork adulteration in minced beef, and the correlation coeffi-
cient and standard errors were 0.985 and 4.172%, respectively.
Recently, Zheng et al. (2019) established a Vis/NIR-HSI sys-
tem for the detection of duck meat in minced lamb. By using
the second derivative by Savitzky-Golay to reduce dimension-
ality, 14 effective wavelengths were identified to establish the
partial least square regression (PLSR) model, and the corre-
lation coefficient and standard errors were 0.98 and 2.51%,
respectively.
To date, HSI technology has seldom been applied in indus-
try settings due to technical challenges. One of the most
32
challenging aspects is data processing (Reis et al., 2018).
The rich information in hyperspectral images also results in
difficulties in data processing, and a complex data models
are needed for dimension reduction (Ropodi et al., 2016).
In addition, the establishment of a quantitative prediction
model requires a large number of samples. Al-Sarayreh et al.
(2018) developed a self-extraction of spectral and spatial fea-
tures by using a deep convolution neural network model to
detect fresh and processed red-meat adulteration. This model
showed a 94.4% overall classification accuracy, and it is sim-
pler and more time-saving than the manual extraction of spec-
tral and spatial features by using the SVM model. Recently,
Zhao et al. (2019) compared the application of four multi-
variate statistical analysis methods, including PLSR, SVM,
least squares SVM (LS-SVM), and extreme learning machine,
in beef adulteration. The results indicated that the LS-SVM
model showed good coefficients of determination (0.94 and
0.94, respectively) and RMSEs (5.39% and 6.29%, respec-
tively) for calibration and prediction.
5.4 Laser-induced breakdown spectroscopy
LIBS is a laser-based optical emission spectroscopy tech-
nique used to detect elemental emission signals from organic
and inorganic molecules (Bilge et al., 2016; Nespeca, Vieira,
Junior, Neto, & Ferreira, 2020). Because different meat prod-
ucts have different elemental composition characteristics, the
spectral fingerprint of LIBS can predict adulterations. The
greatest advantages of LIBS were efficiency and little to no
sample pretreatment (Caceres et al., 2013; Casado-Gavalda
et al., 2017). By using LIBS coupled with PLS analysis, Bilge
et al. (2016) investigated the K, Na, Ca, Mg, and Zn composi-
tion characteristics to identify pork and chicken adulteration
in beef. The LODs for pork and chicken in beef were 4.4% and
2.0%, respectively, and the correlation coefficients (R2 ) were
as high as 0.994 and 0.999, respectively. Chu et al. (2018)
developed an LIBS method in tandem with MSC spectral pre-
treatment and a K-nearest neighbor identification model to
improve the accuracy and stability of meat adulteration iden-
tification. Approximately 240 spectra of scallop, shrimp, pig
liver, chicken, beef, and mixed samples (shrimp powder in
scallop powder at a 1:1 ratio) were acquired for metallic ele-
ments (Mg, Na, K, Ca, and Al) and nonmetallic elements (C,
H, O, N, and C-N), 120 processed spectra were selected to
build the model, the identification rate improved to 100%,
and the prediction coefficient of variance decreased to 0.56%.
Recently, Casado-Gavalda et al. (2017) developed an LIBS
method based on the copper content in beef and beef liver.
Using PLSR analysis, the LOD was 1 ppm with an RSD of 5%
to8%. Similarly, Velioglu and coworkers (2018) developed an
LIBS method based on Na, K, Mg, Ca, and Fe and combined
with the PLS analysis to distinguish beef and beef offal. The
test samples were only stored for 2 hr in a refrigerator, and
DETECTION METHODS OF MEAT ADULTERATION…
there was no need for sample preparation. The LOD was as
low as 3.8%, and the relative standard deviation (RSD) was
23.5%. However, the LIBS method is still in the early stages
of laboratory development, and there are many shortcomings,
such as low sensitivity, poor repeatability, and so on, that need
to be addressed. In addition, because the sample size is small,
the results may not be representative.
6 CONC LU S IO NS AND F UTUR E
TRENDS
As one of the main food safety issues, meat and meat produc-
tion adulteration has attracted increasing attention in recent
years. The literature review has shown that each of the tech-
niques has its advantages and disadvantages, so a method
should be selected or developed according to the actual appli-
cation. For example, for food safety monitoring and enforce-
ment agencies, high sensitivity and accuracy techniques are
required, and DNA- and protein-based methods are more
appropriate than spectroscopic methods. However, for market
screening or manufactory onsite monitoring, time savings and
ease of operation are required, so ELISA kit-based protein and
small portable device-based spectroscopic methods are more
suitable. In the future, as for the authors’ concerns, the devel-
opment of meat adulteration technologies should focus on the
following aspects.
First, for DNA-, protein-, and metabolic profiling-based
technologies, the mining of identification indicators/markers
is particularly important; markers directly determine the accu-
racy of the method, especially for highly processed meat prod-
ucts, such as high-temperature processing and emulsification
processing, or for distinction between similar species, such
as sheep and goat, chicken and turkey, and duck and goose.
For example, the N-glycosylation modification of proteins is
very stable and has high species specificity (Shi et al., 2019),
so future research could use glycosylation proteins as iden-
tification indicators for developing new technologies based
on proteins. Second, new interdisciplinary technologies, such
as biochips and biosensors (Mansouri et al., 2019; Wang,
Zhu, Chen, Xu, & Zhou, 2015), are promising applications
in the field of meat adulteration identification to improve sen-
sitivity and save time. For example, a recent study reported
that a DNA-based electrochemical genosensor was developed
to detect donkey adulteration in cooked sausages, and the
limit of quantitation reached a target probe concentration of
148 pM with an RSD of 0.16%, which indicated better sen-
sitivity and reproducibility than those of qRT-PCR (Man-
souri et al., 2019). Third, omics technologies, especially pro-
teomics, are important methods in laboratory detection. By
detecting multiple targets at once, the accuracy of detection
can be improved, and multispecies detection can be achieved.
DETECTION METHODS OF MEAT ADULTERATION…
More importantly, unknown meat species could be screened
by searching MS databases. Finally, regardless of any method,
effective data processing is essential. In the future, an effective
and convenient chemometric model should be developed to
popularize the application of these techniques. For example,
a deep convolutional neural network (DCNN) has shown out-
standing performance in image recognition for automatic self-
learning from large amounts of data, which is suitable for large
sets of spectral data (Weng et al., 2020), so it is a good choice
for large sample sizes in nondestructive technologies. How-
ever, the DCNN algorithm is relatively complicated and not
suitable for popularization, so it needs to be further improved
in the future.
ACKNOW LEDGMENTS
This work was supported by the Provincial Key Research
and Development Program of Sichuan (2019YFS0525) and
Sichuan Science and Technology Program (2019YFN0174
and 20ZDYF0467).
AU THOR CONT R I B U T I O N S
YC Li, SY Liu, and FB Meng collected the references and
wrote the manuscript. JM Zhang and Y Zhang prepared the
figures. DY Liu and W Wang revised the paper scientifically.
CONFLICT OF I N T E R E ST
The authors declare that there are no conflict of interest.
ORC ID
Fan-Bing Meng https://orcid.org/0000-0003-2166-0166
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How to cite this article: Li Y-C, Liu S-Y, Meng F-B,
et al. Comparative review and the recent progress
in detection technologies of meat product adulter-
ation. Compr Rev Food Sci Food Saf. 2020;1–41.
https://doi.org/10.1111/1541-4337.12579