Microbial Pathogenesis 107 (2017) 54e61

Contents lists available at ScienceDirect

Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Loop-mediated isothermal amplification (LAMP): A novel rapid

detection platform for pathogens
Yanmei Li a, Penghui Fan b, Shishui Zhou c, *, Li Zhang a, **
a
Department of Haematology, Guangzhou Women and Children's Medical Centre, Guangzhou Medical University, Guangzhou 510623, PR China
b
School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, PR China
c
School of Bioscience and Bioengineering, South China University of Technology, 382 Zhonghuan Road East, Guangzhou 510006, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Foodborne bacterial infections and diseases have been considered to be a major threat for public health
Received 26 January 2017 in the worldwide. Increased incidence of human diseases caused by foodborne pathogens have been
Received in revised form correlated with growing world population and mobility. Loop-mediated isothermal amplification (LAMP)
7 March 2017
has been regarded as an innovative gene amplification technology and emerged as an alternative to PCR-
Accepted 16 March 2017
Available online 18 March 2017
based methodologies in both clinical laboratory and food safety testing. Nowadays, LAMP has been
applied to detection and identification on pathogens from microbial diseases, as it showed significant
advantage in high sensitivity, specificity and rapidity. The high sensitivity of LAMP enables detection of
Keywords:
Loop-mediated isothermal amplification
the pathogens in sample materials even without time consuming sample preparation. An overview of
Foodborne pathogens LAMP mainly containing the development history, reaction principle and its application to four kind of
Application foodborne pathogens detection are presented in this paper. As concluded, with the advantages of
rapidity, simplicity, sensitivity, specificity and robustness, LAMP is capable of applications for clinical
diagnosis as well as surveillance of infection diseases. Moreover, the main purpose of this paper is to
provide theoretical basis for the clinical application of LAMP technology.
© 2017 Published by Elsevier Ltd.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
1.1. 1The development history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2. Principle of LAMP assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2.1. Design of LAMP primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2.2. The principle of LAMP amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3. The advantages of LAMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4. Reaction system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5. Results determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
6. Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
6.1. Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
6.2. Salmonella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
6.3. Vibrio parahaemolyticus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
6.4. Listeria monocytogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
6.5. Staphylococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
7. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

* Corresponding author. School of Bioscience and Bioengineering, South China University of Technology, 382 Zhonghuan Road East, Guangzhou Higher Education Mega
Centre, Guangzhou 510006, PR China.
** Corresponding author. Department of Haematology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, 9 Jinsui Road, Guangzhou 510623,
PR China.
E-mail addresses: liyanmei20172017@163.com (Y. Li), fanpenghui1226@163.com (P. Fan), zhoushishui2017@163.com (S. Zhou), zhangligz06@163.com (L. Zhang).

http://dx.doi.org/10.1016/j.micpath.2017.03.016
0882-4010/© 2017 Published by Elsevier Ltd.
 Y. Li et al. / Microbial Pathogenesis 107 (2017) 54e61 55

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Nowadays, culture-based diagnostic detection and identifica- other technologies which can be combined with LAMP so the
tion procedure for potentially pathogenic bacteria may involve existing technology that can be further optimized. For example,
enrichment and enumeration in liquid media, subsequent recovery Wang et al. developed LAMP-LFD to detect roundup ready soybean
and isolation of colonies on selective culture broth and further in 2013 [9]. This new method combined immunochromatography
confirmation assays. However, the lengthy recovery time requiring with LAMP, and this experiment is a successful example which
3e7 days to reach a positive identification at the species level, false LAMP technology is applied to the detection of genetically modified
negative results due to bacterial starvation and physical stress, as food.
well as insufficient sensitivity, had been considered to be major
concerns. During the past decades, a number of polymerase chain 2. Principle of LAMP assay
reaction (PCR) and real-time quantitative PCR (RQ-PCR) based as-
says have been employed and proposed for rapid detection of food- 2.1. Design of LAMP primers
borne pathogens [1]. However, disadvantages for PCR (time con-
sumption for post determination, high risk of cross contamination Online programs (Primer explorer V4/V5, http://primerexplorer.
and low detection limit levels) and real-time PCR (requirement for jp/elamp4.0.0/index.html) have been commonly used to design
trained personnel, operating space, expensive equipment and re- LAMP primers. A set of primers for LAMP reaction include forward
agents) posed significant obstacles for their broad application. inner primer (FIP), backward inner primer (BIP), two outer primers
were described as forward outer primer (F3) and backward outer
1. Introduction primer (B3), corresponding to target gene sequence of 6 different
regions (F3c, F2c, F1c at 30 side and B1, B2, B3 at 50 side, respec-
1.1. 1The development history tively) [10]. Two loop primers were designed to accelerate the
amplification reaction, forward loop primer (FLP) and backward
Nucleic acid amplification is commonly used in the field of life loop primer (BLP) (Fig. 1).
science research. With the development of molecular biology, Each of the basic primers (2 inner primers FIP and BIP, and 2
many new molecular diagnostic technologies have been developed outer primers F3 and B3) plays a key role in the LAMP reaction.
subsequently. Notomi developed a new isothermal nucleic acid Some of the design principles were as follows. If content of GC is
amplification method in 2000, Loop-mediated isothermal amplifi- abundantly of the sequence, the GC content of the primers should
cation (LAMP) [2]. The initial studies are considered as a variety of be about 50e60%, and the GC content of primers should be about
validation studies to appraise the feasibility of LAMP technology. It 40e50% in case of AT rich. Second, the primers should not be
has been widely used in microbial identification and diagnosis area designed to form secondary structures easily, the 30 end of primer
since then for LAMP has the advantage of high specificity, efficiency should better not be AT rich or complementary to other primers.
and easy operation. And LAMP kits for detecting Salmonella, Third, the melting temperature (Tm) for primer region should be
Escherichia coli and Listeria monocytogenes have been commercial- about 60e65  C in case of GC rich as well as the Tm should be
ized in the initial phase development. Maruyama et al. is the first 55e60  C in case of AT rich. At the same time, Tm of F1c and B1c
one who used LAMP to detect bacteria [3]. To detect the Myco- should be higher than the Tm of F2 and B2 for the loop structure
bacterium tuberculosis and Mycobacterium avium, Iwamoto et al. was formed immediately when the single strand was released.
designed primers and succeeded in detecting the bacteria above There is another thing that we must take into account, the free
from sputum specimen by LAMP methods based on the gene energy (△G) of the primers we choose should be less than -4 kcal/
sequence of gyrb, respectively [4]. The DNA virus which were mol for the sake of the stability of the primers [11].
detected successfully by LAMP assay at present include HSV,
Adenovirus and HBV, etc. In 2005, Kaneko et al. succeeded in 2.2. The principle of LAMP amplification
detecting HSV-1, HSV-2 and VZV-1 by using LAMP [5]. And he drew
a conclusion that we could make accurate judgement as a result of The LAMP amplification can be divided into two phases. At the
the established assay through the detection of 18 clinical samples initial phase, F2 of FIP hybridises to F2c of template DNA strand,
which was confirmed by real-time Q-PCR. As for detection of RNA guiding the synthesis of complementary DNA strand. The F3 primer
virus, Enomoto et al. established RT-LAMP technology by anneals to the F3c region of template, outside of FIP. With the help
combining LAMP with reverse transcription, and Parida et al. of Bst DNA polymerase, starting the chain substitution reaction and
detected the envelope gene of West Nile (WN) virus with RT-LAMP releasing the FIP-linked complementary strand, double-stranded
[6]. By adding RNA and reverse transcriptase into the tube, RT- DNA is synthesized from the F3 primer and the template DNA
LAMP could achieve the goal of amplifying RNA directly on the strand [12e14]. Because the complementary F1c and F1 region
basis of LAMP technique. exists at the chain of 50 end of the released single strand, stem-loop
With more and more scientists focusing their attention on the structure is formed by complementary base. And then this single
application of LAMP technology, the range of its use is not limited to strand is regarded as a template in turn, under the action of the BIP
the bacteria detection and identification any more. It was also and B3, occurring the similar reaction, another new stem-loop
applied to the parasites and embryonic appraisal domain. Lau Y structure at the other end of the DNA is assembled. Thus looking
et al. introduced the LAMP into the detection of toxoplasmosis [7]. like a dumbbell structure is formed, this structure serves as the
Recently, LAMP reagents for detecting certain parasites have been template for cycle amplification during the subsequent reaction
commercialized. Zoheir et al. took LAMP method for sex identifi- (Fig. 2).
cation of bovine embryos by using EB and CuSO4 as indicator of The second phase was described as cyclic amplification.
reaction [8]. At the same time, the scientists are seeking for some Dumbbell structure serves as the template, this dumbbell-like DNA
56 Y. Li et al. / Microbial Pathogenesis 107 (2017) 54e61

Fig. 1. Schematic representation of primer design for LAMP assay showing the position of the four primers spanning the target.

structure is quickly converted into a stem-loop DNA by self-primed
DNA synthesis. F2 of FIP hybridises to the loop area of F2c of the
product. At the same time, F1c combined with F1, dissociativing out
the double-stranded nucleic acid guided by F1 and releasing the
previously synthesized single strand guided by F2, respectively.
This released single strand formed a stem-loop structure at the 30
side because of B1c complementary to B1. Then, initiating from the
30 end of B1, self-structure serves as a template for DNA synthesis,
and releases FIP-linked strand. Later a dumbbell structure is formed
at both ends because of having complementary F1eF1c and B1c-B1,
respectively. In the same way, B2 of BIP hybridises to the loop area
of B2c to generate a new DNA sequence, and primes strand sub-
stitution DNA synthesis, releasing the B1-primed DNA strand. Such
repeated amplification eventually leads to so much dumbbell
structure DNA are formed. The final products are stem-loop DNAs
with several repeats of target and cauliflower-like structures with
multiple loops (Fig. 3).
3. The advantages of LAMP
LAMP has been regarded as a kind of new detection technology,
and it is widely used resulting from its outstanding advantages.
Compared with traditional detection methods, the specificity of the
LAMP is extremely high [15]. The four primers are specifically
designed for 6 different regions of the target, respectively. Once any
of the primers does not match exactly will lead to the reaction
doesn't happen as well as the phenomenon of the nonspecific
amplification in theory. Second, the LAMP assay was found to be
10e100 fold more sensitive than PCR with a detection limit of 10
copy or even more less template. In other words, LAMP assay can be
used for the detection of disease in the early stage of the infection
or the clinical symptom which is not obvious. Moreover, it takes less
time than conventional PCR to detect virus. The high amplification
efficiency of LAMP is attributed to it is not necessary to choose
complex-variable temperature conditions and set different
response procedure [16]. With DNA being amplified 109-1010 times
in 15e60 min, it can save 1 h to complete the reaction compared
with PCR. That is to say the LAMP has great advantage over PCR in
terms of time consuming. Furthermore, what attract us most are
the advantages of low-cost and easy operation. For example, we can
directly take animal diseased tissue and nucleic acid amplification
when we detect of pathogenic microorganism. It is not necessary to
cultivate bacteria separately and avoid the tedious nucleic acid
extraction step which we did during conventional PCR assay [17]. At
the same time, its ability to amplify nucleic acid under isothermal
conditions in the range of 65  C allowing the use of simple and cost
effective reaction equipment [18]. Finally, the most important thing
is that we can judge the result of reaction by our eyes, the favorable

Fig. 2. Principles of LAMP amplification. Non-Cyclic Step [1e8]: generation of stem loop DNA with dumbbell-shaped structure at both ends and then it will enter into cyclic step.
First, with the help of Bst DNA polymerase, the strand displacement activity of synthesizing a complementary DNA strand begin, starting with FIP. The F3 then displaces the FIP-
linked complementary strand, which forms a rings structure at the 50 end. This regard as a template for BIP-initiated DNA synthesis and subsequent B3 strand displacement DNA
synthesis. Finally, a structure with stem-loops will be formed at both end [14]. Copyright: 2005, Eiken Chemical Co. Ltd., Japan.
 Y. Li et al. / Microbial Pathogenesis 107 (2017) 54e61
Fig. 3. Principles of LAMP amplification. Cyclic Amplification Step [9e11]: exponential amplification of original dumbbell-shaped stem-loop DNA employing internal primers. The
product is the differently sized structures consisting of alternately inverted repeats of the target sequence on the same strand [14]. Copyright:2005, Eiken Chemical Co. Ltd., Japan.
peculiarity of this method is that we can demonstrate the absence
of the target gene by the production of white precipitate of mag-
nesium pyrophosphate, making it ideal for easy monitoring
through naked eye. LAMP assay has the great advantage of moni-
toring amplification by adding SYBR Green I into reaction tube and
real-time monitoring by using an inexpensive turbid meter ac-
cording to the situation.
Except for the advantages we talked above, we can incubate the
mixture of primers, gene sample, DNA polymerase with strand
displacement activity and substrates at a constant temperature.
That is why we can achieve the goal of amplification and detection
of the target in a single step. On the other hand, the existing of the
target gene sequence can easily be detected just by judging pres-
ence of amplified products because of its high specificity.
4. Reaction system
This assay used the optimized primers to explore the levels and
proportions of different components for the 4 kinds of foodborne
bacteria which we will talk about in this paper according to Zhao
et al. [19]. And detailed reaction system as follows:
LAMP assay was carried out in a total of 25 ml reaction mixture
containing 1.6 mM (each) of the primers FIP and BIP, 0.2 mM (each) of
the primers F3 and B3, 0.8 mM (each) of primers LF and LB, 1.6 mM of
deoxynucleoside triphosphates, 6 mM MgSO4, 1 M betaine,
10  Thermopol buffer, and the specified amounts of target
genomic DNA. The reaction was heated at 95  C for 3 min, then
chilled on ice, 1 ml (8 U) of Bst DNA polymerase (large fragment) was
added, after incubation at 65  C for 45 min, the reaction was
terminated by heating at 80 Cfor 2 min. Heating and isothermal
amplification were performed on a heating block and water bath,
and positive LAMP reactions were measured by several qualitative
criteria.
57
5. Results determination
Measures we take mostly at present to judge the reaction results
include visual inspection, ultraviolet fluorescence and agarose gel
electrophoresis, etc. Visual inspection mainly depends on the
turbidity degree of reaction tube to the naked eye, specific ampli-
fication of LAMP products will present muddy phenomenon. White
magnesium pyrophosphate precipitates, generated during the
strand displacement auto-cycling reaction, could be seen at the
bottom of microfuge tubes (Fig. 4, a). However, there is no visible
precipitate to the naked eye in the control [20]. Adding fluorescent
dye SYBR Green Ⅰ into the reaction tube leads to the color change,
positive reaction tube will present fluorescent color under the
condition of the ultraviolet radiation, negative control will not
happen (Fig. 4, b). And the third measure we take at present is that
the products of LAMP are detected by agarose gel electrophoresis
[21], as the result of positive tube after amplification will form the
stem loop structure of DNA mixture, the LAMP positive reaction
tube will appear ladder-like banding, that's why we could choose
UV gel imaging analysis system for interpretation results (Fig. 4, c).
In practice, the visual inspection for amplification is performed
through observation of color change by adding 1 ml of SYBR Green I
to the tube. In case of positive amplification, the original orange
color of the dye will change into green that can be judged under
normal light as well as under UV light (302 nm) with the help of UV
lamp. In the control, the original color of the dye will be remained.
6. Application
6.1. Escherichia coli
As one of the most commonly found microorganisms, Escher-
ichia coli are Gram-negative, rod-shaped bacterium. Shiga-toxin
producing E. coli (STEC) is an important human pathogen, and the
O157: H7 has been the best known and most studied serotype,
58 Y. Li et al. / Microbial Pathogenesis 107 (2017) 54e61
Fig. 4. Detection of the LAMP products was confirmed by visual inspection, white precipitate(a) and SYBR Green І (b). Agarose gel analysis revealing the typical electrophoresis
pattern of LAMP amplified product. (c).
which is responsible for the majority of cases of diarrhea prevalent
all over the world [22,23]. Delayed recovery of E. coli O157: H7 may
lead to serious kidney damage and even death, and the current
“golden standard” identification of E. coli O157: H7 and its Shiga
toxins requires more than 3 days [24e27]. Zhao et al. took advan-
tage of the E. coli specific rfbE gene, as well as main toxins produced
by E. coli O157: H7, Shiga-toxin 1 (Stx1) and Shiga-toxin 2 (Stx2)
genes, to develop 3 LAMP rapid assays [28]. Thirty-four reference
strains were included, with 19 E. coli strains with different sero-
types, 8 Gram-negative non E. coli and 7 Gram-positive strains. The
optimal reaction was found to be 65  C for 45 min and the detection
limits were 100 fg DNA/tube for rfbE, 100 fg DNA/tube for stx1 and
100 fg DNA/tube and for stx2, with 100% specificity for 34 reference
strains. Four hundred and seventeen E. coli strains, including 154
O157 and 263 non-O157 isolates, were subjected to detection by
the aforementioned LAMP assays. The sensitivity of LAMP assays for
the rfbE, stx1 and stx2 was 100% (417/417), 95.3% (282/296) and
96.3% (312/324), and the negative predictive value (NPV) was 100%
96.7% and 97.1% respectively; with a 100% specificity and positive
predictive value (PPV).
6.2. Salmonella
Regarded as a major food-borne pathogen worldwide and the
leading microbe in food contamination, Salmonella is responsible
for various food poisoning cases of humans and the main cause of
human gastrointestinal and other related diseases [29e32]. As its
identification is concerned, 4e6 days is required for the conven-
tional detection method to obtain a confirmed result [33]. In a
preliminary study, a LAMP-based detection on Salmonella had been
developed [34], which required 60 min under 65  C to reach the
maximum of amplification. This LAMP assay had been improved by
using loop primers to reduce the reaction time to 45 min, with the
detection limits as 1 pg DNA/tube and 100 CFU/reaction and a 100%
specificity within 39 reference strains. In addition, this improved
LAMP assay had been applied to detect 214 Salmonella strains, and
the sensitivity was found to be 97.7% (209/214), with no false
positive observed.
6.3. Vibrio parahaemolyticus
As widely distributed in coastal and estuarine environments
globally, Vibrio parahaemolyticus has been regarded as the causative
agent in 50e70% of all cases of diarrhea and one of the major forms
of food-borne gastroenteritis, mostly associated with the con-
sumption of raw or undercooked seafood [35]. Up to date, routine
identification of V. parahaemolyticus may require long as 7 days, and
false negative as well as physical stress had posed significant ob-
stacles for its application [36]. In a previous study, Zhao et al
developed 3 LAMP assays for the detection of V. parahaemolyticus,
as well as 2 significant virulent factors associated with gastroen-
teritis related pathogenicity [37]. Three targets included thermol-
able haemolysin gene (tlh) which is a species-specific marker for
V. parahaemolyticus, thermostable direct haemolysin (TDH) which
is an inducing factor of Kanagawa phenomenon and considered to
be the major virulence factor, and TDH-related haemolysin (TRH)
which is also involved in the production of gastroenteritis. The
optimal reaction was found to be 65  C for 45 min and detection
limits of tlh, tdh and trh were detected to be 100 fg, 100 fg and 1 pg
DNA/tube, with 100% specificity when subjected to 45 reference
strains. Application of LAMP assays were performed on 368 food-
borne V. parahaemolyticus strains, including 296 tdhþ and 247
trhþ isolates. The sensitivities were 100% (368/368), 95.6% (283/
296) and 96.4% (238/247) for tlh, tdh and trh, and the negative
predictive values (NPV) were 100%, 84.7% and 93.1%, respectively;
with a 100% specificity and positive predictive value (PPV). Never-
theless, this application had been limited by the pure culture of
isolates. In a further report, Wang et al had introduced a simple
sample processing and DNA preparation method, and applied the
LAMP assay on detection of 416 food borne V. parahaemolyticus
isolated from various seafood samples, including 160 marine fish
(38.5%), 92 shrimp (22.1%), 66 oyster (15.9%), 38 mussels (9.1%), 26
jellyfish (6.3%), 19 cuttlefish (4.6%) and 12 seaweeds (2.9%) samples
[38]. According to the results, the total detection rate was 96.2%
(400/416), with 96.3% (154/160) for marine fish, 98.9% (91/92) for
shrimp, 93.9% (62/66) for oyster, 89.5% (34/38) for mussle, 96.2%
(25/26) for jellyfish, 100% (22/22) for cuttlefish and 100% (12/12) for
seaweed, indicating the capability of the application of the LAMP
assay on real food samples.
6.4. Listeria monocytogenes
As a gram-positive, rod-shaped and facultative pathogen, Lis-
teria monocytogenes has been widely distributed in foods, the
environment, human and animal hosts, and is responsible for
serious invasive diseases with low incidence but high mortality rate
[39e41]. Considered to be an important health risk in the food
industry, the detection of L. monocytogenes, especially rapid and
easy operating detection assay may be of utmost significance and
urgent necessity. Most lately, a simple LAMP assay taking use of the
species specific target hlyA to differentiate L. monocytogenes and
non- L. monocytogenes strains had been developed [42]. With loop
primers included, this assay required as low as 45 min under 65  C.
High specificity had been demonstrated when subjected to 39
 Y. Li et al. / Microbial Pathogenesis 107 (2017) 54e61
reference strains, and the detection limits was found to be 1 pg
DNA/tube and 100 CFU/reaction. In addition, this detection had
been applied to detect 182 L. monocytogenes strains, of which 176
strains were detected to be positive by LAMP, totaling the sensi-
tivity and positive predictive value (PPV) as 96.7% and 100%.
6.5. Staphylococcus
As a group of gram-positive, facultative aerobic and usually
unencapsulated organisms, Staphylococcus are responsible for
various tissues infection and a multitude of diseases [43e50]. Up to
date, over 30 different types of staphylococci have been identified,
within which Staphylococcus aureus has been regarded as leading
issues both in medicine and food safety [51e60], and is responsible
for most infections and diseases, including skin infections, pneu-
monia, endocarditis, osteomyelitis, gastroenteritis, scalded skin
syndrome and toxic shock syndrome, etc.
Applications of LAMP (with other methodologies) to identify
possible S. aureus isolates using the spa and mecA genes as targets
have been demonstrated [61e64] and also showed the potential of
using the assays to detect MRSA directly from blood samples with
remarkable success. This detection procedure required the real-
time turbidimeter for LAMP reaction and result determination,
and the bacterial incubation solely required 120 min with DNA
preparation and result determination excluded, and the detection
limits reached 1000 copies for spa and 100 copies for mecA. As the
detection targets were concerned, the genetic target spa had been
chosen as the specific target of S. aureus. Due to its high variability,
the applicability of spa as an appropriate target for detection on
S. aureus may require further and insightful investigation, as it may
even lead to the lower sensitivity than PCR. As concluded, the
previous study may be limited, to some extended, by the sensitivity,
simplicity in operation, as well as applicability.
The LAMP assays, as well as the whole procedure, established by
Xu et al. had made significant progress, and remained a milestone
during the development of LAMP methodologies, as these meth-
odologies exhibited significant advantages on rapidity, simplicity
(for both reaction and template DNA preparation), cost-
effectiveness, sensitivity (detection limits), broadness and flexi-
bility of application [65e66]. As comparison, these detection
methodologies only require water bath (or heat block) for the
amplification reaction, and take only 60 min for the whole pro-
cedure from simple and rapid DNA preparation to result determi-
nation, showing the advantages on both rapidity and simplicity,
which may be key factor in terms of application. In addition, for the
3 LAMP assays developed by Xu et al., the detection limits were
reported to be 10 CFU/reaction, 10 CFU/reaction and 100 CFU/re-
action for 3 targets (16S rRNA, femA and mecA, respectively),
demonstrating at least 10 times higher sensitivity which is also a
very important factor for the application field. For such assays, the
genetic target femA was generally accepted as a species specific
target for S. aureus and had been commonly used and applied for
the detection of S. aureus. Together with other 2 genetic targets of
16S rRNA and mecA, these 3 LAMP assays showed advantages on the
broadness and flexibility in application, which are expectedly able
to be applied to either separate detection of staphylococci, S. aureus
and methicillin-resistance or combined use to differentiate MRSA,
MSSA, MRCNS, MSCNS and non-staphylococci. Also, another
improvement of these LAMP assays developed was rapidity, which
was partly contributed by the simple and rapid preparation of
template DNA. As generally accepted, the inhibited substances
would influence gene amplification, such as PCR and real-time PCR.
While LAMP takes use of the robust Bst enzyme and overcomes this
flaw. As seen from the previous study, the higher sensitivity and
specificity of LAMP assays showed none of significant difference as
59
compared with PCR assays, indicating the involvement of elaborate
DNA preparation which requires longer time length and more
expense. As generally accepted, the results determination of regular
LAMP assay involved either electrophoresis or real-time turbi-
dimeter, which significantly limits the further and broad applica-
tion of LAMP detection in clinical lab and medical field. As a
consequence, the employment of eye observation on the color
change for results determination, will aid in further application on
this simple operating assay. In another study, accuracy of the re-
action system designed by Su et al. has made much progress, 98.4%
vs 91.7% by real time PCR [67], the orfX was selected as specific
target for MRSA. Additionally, according to the result of detection of
food borne S. aureus strains by Zhao et al., the detection limits of
LAMP assay was found to be 100 fg DNA/tube, and PCR was 10 pg
DNA/tube, and high specificity was acquired when LAMP assay was
subjected to 105 reference strains, with no false positive amplifi-
cation observed [68].
7. Concluding remarks
LAMP is an innovative gene amplification tool by simplicity that
eliminates the need for highly sophisticated equipment for ther-
mocycling where all strand displacement reactions happened un-
der isothermal conditions. The reaction condition and accuracy of
LAMP have been optimized and improved with further research,
thus broadening the application of LAMP technology in the actual
clinical diagnosis of diseases and promoting the kit production.
Generally speaking, LAMP has been gradually developed into a
mature and reliable assay of molecular biology diagnosis, and it has
great potential for the study of local epidemics in endemic coun-
tries and regions because of its outstanding advantages. Further-
more, the practical realization of LAMP technology as a simple and
swift genetic test would be in high demand in many countries,
especially in most developing countries where many people are
facing the threat of a wide variety of infectious diseases. LAMP plays
a significance role in monitoring and controlling the spread of
infection disease in this area. However, there are some deficiencies
and defects that we can't ignore, such as the process is susceptible
to contamination and the results may present false positive phe-
nomenon. The existing problems have become the most important
reasons for its further popularization and application, the road to
the optimization of LAMP technology there is still a long way to go.
In the point of my view, further research and improvement is of
great needed in order to decrease the false positive rate of detection
to improve the detection accuracy and repeatability. LAMP is
considered to be effective as a gene amplification method for g-
POCT devices, which can be used for simple genetic testing
whenever and wherever necessary. In the future work, we can
combine LAMP with other technology to develop simple genetic
testing devices that have not been realized yet, overcoming the
weakness we talked above.
Acknowledgements
This work was supported by National Natural Science Founda-
tion of China (3120028).
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