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Food Research International 50 (2013) 330338

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Comparison of gene nature used in real-time PCR for porcine identication and quantication: A review
Nurhidayatul Asma Mohamad a, Aly Farag El Sheikha a, b,, Shuhaimi Mustafa a, c, Nur Fadhilah Khairil Mokhtar a,
a b c

Halal Products Research Institute, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor Darul Ehsan, Malaysia Department of Food Science and Technology, Faculty of Agriculture, Minuya University, Shibin El Kom, Minuya Government, Egypt Department of Microbiology, Faculty Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor Darul Ehsan, Malaysia

a r t i c l e

i n f o

a b s t r a c t
Pork adulteration has been a major concern nowadays for Halal verication. Unintentional pork inclusion by contamination in highly processed food materials involves a minute amount of porcine DNA to be detected, emphasizing the need of specic and sensitive method for porcine detection. Real-time PCR is a widely used technique for species identication that can serve this purpose besides providing a powerful quantication method. Incorporation of a highly sensitive and specic probe can greatly improve the specicity and sensitivity of the assay. However, derivation of PCR primers, either from nuclear DNA (nDNA) or mitochondrial DNA (mtDNA) can relatively affect the sensitivity and specicity of the reaction as well as the quantitative measurement. In this review, both types of DNA are compared in terms of their characteristics and their inuence on species identication and quantication using real-time PCR. 2012 Elsevier Ltd. All rights reserved.

Article history: Received 20 September 2012 Accepted 31 October 2012 Keywords: Gene nature Real-time PCR Halal verication nDNA mtDNA

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. PCR-based porcine identication method . . . . . . . . . . . . . . . 2. Nuclear and mitochondrial DNA . . . . . . . . . . . . . . . . . . . . . . 2.1. General characteristics . . . . . . . . . . . . . . . . . . . . . . . 2.2. Genes used for species identication . . . . . . . . . . . . . . . . . 3. Inuence of nature of target gene on real-time PCR based identication . . . . 4. Inuence of the nature of target gene in real-time PCR based DNA quantication 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 330 331 332 332 333 335 336 337 337

1. Introduction 1.1. Background Identication of porcine species in food products has been a major concern nowadays in determining the Halal status of the food products
Correspondence to: A.F El Sheikha, Halal Products Research Institute, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor Darul Ehsan, Malaysia. Tel.: +60 3 8941 7344; fax: +60 3 8943 9745. Corresponding author. Tel.: +60 3 8947 1836; fax: +60 3 8943 9745. E-mail addresses: (A.F. El Sheikha), (N.F.K. Mokhtar). 0963-9969/$ see front matter 2012 Elsevier Ltd. All rights reserved.

being marketed. The responsible authorities have been challenged by the complexity of the food product itself and the availability of highly sensitive and specic porcine identication method. The Muslim population has been very doubtful of the Halal status of the food products in the market due to pork adulteration. Products that contain any amount of porcine will be considered as Haram and cannot be consumed at any level by the Muslim. Thus, it is a great concern to be able to detect the presence of porcine at the smallest amount, emphasizing the need of highly sensitive and specic porcine identication method. Porcine inclusion in food products can occur at any step in the production chain, either during or after processing and manufacturing.

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Direct inclusion results from using any part of pig, for example, porcine meat as one of the ingredients or using ingredients that are produced from any part of pig as raw material. On the other hand, indirect inclusion occurs when the product is contaminated with porcine. In this case, porcine can be scarcely detected due to trace amount of DNA. Moreover, most of the high quality porcine DNA is destroyed in highly processed food products, making the detection process more formidable. Currently, there are a range of methods used for that purpose including DNA-based method, PCR. In PCR, a specic target sequence is amplied by the designated primers. Furthermore, the specicity and sensitivity of PCR can be improved by incorporating an internal hybridization probe (Mohd Hazim, Shuhaimi, Che Man, Abdul, & Nur Fadhilah, 2012). On top of that, the origin of the target nucleotide sequence, either from nuclear (nDNA) or mitochondrial DNA (mtDNA) has been thought to affect the specicity and sensitivity of PCR reaction (Lpez-Andreo, Lugo, Garrido-Pertierra, Prieto, & Puyet, 2005). In species identication, the nucleotide sequence is derived from the variable region of target gene that is specic to the species of interest (Lenstra, 2010). As specicity of a species-specic nucleotide sequence is the potential factor in determining the efcacy of the PCR to identify a particular species, so do the source of the gene. Intra- and interspecies sequence variability and copy number of the nuclear and mitochondrial DNA can inuence the performance of the PCR reaction in detection and quantication of particular target. Real-time PCR offers both a sensitive and specic automated species identication method, and a powerful DNA quantication tool to determine and compute the presence of porcine DNA in food products. It can also offer a clear picture to the manufacturer on this matter while taking appropriate measures to exclude porcine inclusion in their products either intentionally or not.

1.2. PCR-based porcine identication method These include analysis of different components of the cell such as protein, lipid and DNA molecules. Lipid analysis would include liquid chromatography using reverse phase column and a UV detector (Ibrahim, 2008) while protein-based methods such as enzyme-linked immunosorbent assay (ELISA) (Chen, Hsieh, & Bridgman, 2008) and high-performance liquid chromatography (HPLC) (Chou et al., 2007) have also been used. Porcine detection at molecular level has been diversely used nowadays especially DNA-based methods. DNA based-methods including PCR amplication, PCR-RFLP and microarray gene chips have been used for species detection (Zhang, Fowler, Scott, Lawson, & Slater, 2007). These methods are preferred over protein and lipid analysis due to relative stability of DNA molecule under extreme conditions and efcient amplication of DNA sequence by PCR reaction (Zhang et al., 2007). There are two major types of PCR analysis: conventional PCR and real-time PCR. In conventional PCR, gel electrophoresis is often used as the end-point analysis where the PCR products are visualized on agarose gel according to their size, giving no quantitative results. Besides, the PCR products band on the gel does not give any information on the amount the sequence amplied. Carry over contamination can potentially occur while transferring DNA sample into the gel as opposed to the reaction of real-time PCR assay that occurs in a closed tube throughout the process which can also avoid the occurrence of false-positive results (Lpez-Calleja et al., 2007). Species-specic primers have been designed for bovine (Maudet & Taberlet, 2001), horse, dog, cat, bovine, sheep, porcine, and goat (Lhak & Arslan, 2007). As reported by Fajardo et al. (2007) intra-species variability can also be distinguished by developing species-specic primers for three deer species including red deer, fallow deer and roe deer species that amplify different size of amplicons.

On the other hand, PCR-RFLP refers to digestion of PCR products by a restriction enzyme that cleaves at a species-specic (restriction) site. The specicity of the method is limited as it cannot potentially differentiate intra-species variability, for instance, between domestic pig and wild boar (Maede, 2006). Besides, the possibility of the restriction site to be polymorphic will eventually prevent the detection of all individuals from a species (Lenstra, 2010). Moreover, ambiguous results of PCR-RFLP method requires further analysis like DNA sequencing (Maede, 2006). Enzymatic digestion also requires large PCR products, restricting the use of this technique for species identication of thermally processed foods that consist of highly fragmented DNA (Fajardo et al., 2007). Besides, meat admixtures like sausages, pates and minced meat products are not suitable to be analyzed by PCR-RFLP as the digestion results may show a combination of miscellaneous restriction patterns for all possible species contained in the adultered sample (Fajardo et al., 2007). Real-time PCR involves real-time monitoring of the increment of uorescence intensity developed during the reaction (Heid, Stevens, Livak, & Williams, 1996) due to the binding of uorescent dye to double stranded DNA strands of the PCR products. Eventually, the accumulation of PCR product will generate uorescence over the reaction cycles. Fluorescence intensity will reach the threshold where specic target are successfully detected. Threshold refers to the point that represents the log phase of product accumulation and is usually set at 10 times the standard deviation of the base-line (Heid et al., 1996). At threshold, the intensity of the uorescence generated is equal to the background and as it increases above background, more amplicons are being produced. Early detection indicates more target DNA templates are present in the sample. On top of that, real-time PCR is an automated process which eliminates the end-point analysis. It offers a quantitative result based on the measurement of uorescence intensity of the PCR products provided by the dye or probe. However, only short PCR products or amplicons, around 150 bp or shorter are suitable to be amplied using real-time PCR (Lenstra, 2010). Detection limits of real-time PCR assay are variable but mostly sufcient in detecting signicant adulterations or potentially harmful trace amounts (Lenstra, 2010). The most common detector used in real-time PCR is SYBR Green dye that emits uorescence upon binding non-specically to the minor groove of double-stranded DNA in the reaction mixture. As PCR products accumulate, the uorescence intensity of the dye increases. The use of this intercalating dye for species detection provides a exible method that does not require complicated probe design and optimization steps (Fajardo et al., 2008b). Besides, the dye is cheaper than designated probe, reducing the overall cost of the analysis (Fajardo et al., 2008b). However, it shows reduced specicity as compared to probe and not suitable for multiplex real-time PCR assay (Martn et al., 2009). SYBR Green dye has been reported to potentially interfere with PCR reactions, therefore, low dye concentration has to be used leading to dye redistribution problem during DNA melting curve analysis (Mao, Leung, & Xin, 2007). This problem can be overcome by using alternative intercalating dye namely EvaGreen with low PCR inhibition that allows the use of high dye concentration to develop robust PCR system (Mao et al., 2007). As compared to SYBR Green, EvaGreen is more stable to withstand intense PCR reaction condition and more sensitive due to better DNA binding prole (Mao et al., 2007). Application of EvaGreen dye in meat speciation has been demonstrated by Santos et al. (2012) while Wang, Chen, and Xu (2006b) showed the suitability of the dye for DNA quantication. Additionally, the use of sequence-specic probe has also been widely applied to provide enhanced specicity to the PCR system. TaqMan is a frequently used-oligonucleotide probe (Dooley, Paine, Garrett, & Brown, 2004; Heid et al., 1996; Kesmen, Gulluce, Sahin, & Yetim, 2009; Zhang et al., 2007) that consists of a reporter dye at 5 end and a quencher at the 3 end. The release of reporter due to 5


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nuclease degradation during DNA extension/polymerization causes the dye to uoresce. The probe, generally 27 nucleotide long (Lpez-Andreo et al., 2005), binds to a complementary sequence in between the forward and reverse primers and collision of Taq polymerase and the probe resulting in cleavage of the probe by activity of the 5 exonuclease. In designing the probe, the melting temperature of the probe must be 810 C higher than that of the primers. Besides, the annealing temperature may have to be reduced in 710 C if there is a mismatch in the middle third of the probe upon binding that will compromise the specicity of the primers (Lpez-Andreo et al., 2005). The high specicity of TaqMan probe has been exploited by Zhang et al. (2007) to identify bovine in meats, milks and cheeses. High sensitivity of the probe allows detection of bovine DNA at a very low level. Kesmen et al. (2009) have also used the same type of probe for detection of donkey, horse and pig species in raw and cooked products. The detection limit has been found to be 0.0001 ng of template DNA of raw meat. The closely related species, donkey and horse can also be differentiated using the PCR system developed. Real-time PCR system using TaqMan probe has been enhanced by Lpez-Andreo et al. (2005) by conjugating the probe with minor groove binder (MGB). The designated probe has increased afnity to target sequence which allows the use of shorter sequence and broadens the variety of sequences that are specic to a particular species or group (Lpez-Andreo et al., 2005). Compared to other conventional oligonucleotide probes, molecular beacon probe specicity is enhanced by the hairpin or stem-and-loop structure that permits detection of single-nucleotide variations of the target sequence. The stem structure also offers more efcient quenching that reduces initial uorescence background (Mohd Hazim et al., 2012). The sequence of the loop region is complementary to the target sequence while the stems are complementary to each other. Bound covalently on each stem, a uorophore and a quencher are in close proximity that prevents release of uorescent but is restored upon binding of the loop region to target sequence as the stems separate, pulling apart the two molecules. The 1530 (Mhlanga & Malmberg, 2001) or 1825 nucleotides long loop region is designed to melt at a slightly higher temperature than the annealing temperature of the PCR (Marras, Kramer, & Tyagi, 2003). On the other hand, the stem region may contain 57 nucleotides that separate at 710 C higher than the annealing temperature of the DNA polymerase (Mhlanga & Malmberg, 2001). The stability of the perfectly complementary probe-target hybrid over a wide range of temperature is important to overcome stem hybrid binding, ensuring complete quenching of mismatched probe-target hybrid and only highly stable, perfect match of probe-target hybrid produces uorescent. (Mhlanga & Malmberg, 2001). Furthermore, detection of multiple targets in the same solution can be done by using different types of uorophore on the probe molecule (Marras et al., 2003; Mhlanga & Malmberg, 2001). Species identication by real-time PCR employing molecular beacon probe includes the work done by Sandhya, Chen, and Mulchandani (2008) on Escherichia coli detection in fresh produce and water. Besides, the probe has also been coupled with real-time PCR in detection of single nucleotide polymorphism using EDTA-treated blood sample (Mhlanga & Malmberg, 2001). Porcine detection and quantication using molecular beacon probe has also been done by Mohd Hazim et al. (2012) showing very high specicity against a number of game species tested. The use of molecular probe together with the speciesspecic primers also allows detection of porcine DNA at a very low level of 0.0001 ng porcine DNA and 0.1% (w/w) pork in meat mixtures, indicating great sensitivity of the PCR system developed. On the other hand, scorpion probe refers to a tailed primer that serves simultaneously as a PCR primer and as a molecular beacon in the assay (Broude, 2005). The 5 end of the primer is extended with a hairpin structure of molecular beacon probe which is linked by a blocker, typically hexethylene glycol (HEG) monomer. The blocker prevents the extended region from being copied during PCR reaction

(Whitcombe, Theaker, Guy, Brown, & Little, 1999). Extension of the primer and synthesis of target sequence causes the stem and loop structure to unfold, allowing hybridization of the loop sequence with the amplied target and eventually, generating uorescence (Broude, 2005). The design of this probe permits unimolecular target detection that promotes faster kinetics and higher stability of the complex compared to bimolecular reaction (Broude, 2005). Furthermore, the production of probe-target hybrid by the extension of the probe is concentration independent (zero order) and highly specic detection down to single base change can be achieved (Whitcombe et al., 1999). The Scorpion probe containing species-specic primer has been applied for cow detection and quantication in binary meat mixtures. The highly selective duplex PCR system is able to amplify cow species against sheep at a high sensitivity of 0.1% (w/w) (Sawyer, Wood, Shanahan, Gout, & McDowell, 2003). Despite the high specicity and sensitivity offered, there is not much application of Scorpion probe in meat speciation studies, most probably due to complicated design of the probe (Whitcombe et al., 1999). 2. Nuclear and mitochondrial DNA 2.1. General characteristics In eukaryotic cells, DNA is a genetic material contained in each cell, within the nucleus (nuclear DNA) while mitochondrial DNA (mtDNA) is referred to the DNA molecule that resides within the cytoplasmic organelle of the cell, the mitochondria that present in variable amount depending on the types of the cell (Hartwell et al., 2008). The double membrane compartment provides some kind of protection to the DNA upon mitochondria isolation process from the cell, prior to mtDNA extraction (Bogenhagen, 2009). Cellular location of the DNA has been reported to inuence vulnerability to DNA degradation. It has been found that nDNA from whole tissue tends to degrade faster than mtDNA but oppositely in homogenized tissue after treatments in a study on relative DNA degradation (Foran, 2006). In the nucleus, the long and linear double-stranded DNA is wrapped around histone proteins in the supercoiled structure of chromosomes with phosphate group at the 5 end and hydroxyl group at the 3end (Hartwell et al., 2008). On the other hand, mtDNA of most species are circular and largely free of bound proteins, which contained in highly condensed structures called nucleoids (Bogenhagen, 2009). The circular shape of mtDNA contributes to greater stability over time as it is less susceptible to degradation compared to nDNA (Gefrides & Welch, 2011). This results in better survival of mtDNA in highly processed food that undergo extreme conditions of food processing. In addition, a few copies of the mtDNA genomes, approximately 210 copies are organized in nucleoids (Bogenhagen, 2009) resulting in the existence of very high copy number of mtDNA in a particular cell. In contrast, only one copy of nDNA exists in the nucleus of each cell. This provides more sensitivity in mtDNA detection compared to nDNA testing (Gefrides & Welch, 2011). The mammal nDNA consists of about 20,000 genes or proteincoding regions (Moran, 2011). According to C-value paradox, 98% or more of the nuclear genome of many organisms consists of non-coding and repetitive DNA fractions which have unknown function and are often regarded as junk DNA that signicantly differs in amount between species (Moran, 2011). Other regions of nDNA would include introns and spacer sequences between the genes. The presence of introns, repetitive DNA as well as spacer sequences between the genes in nDNA is one of the factors that distinguish nDNA from mtDNA (Magoulas, 2005). Studies have shown that nDNA elements including satellite DNA (Guoli, Mingguang, Zhijiang, Hongsheng, & Qiang, 1999), introns (Whittall, Medina-Marino, Zimmer, & Hodges, 2006), internal transcribed spacer (ITS) region (Wang et al., 2006) and repetitive elements such as short-interspersed elements,

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SINE (Walker et al., 2003) have been useful for species identication. The species-specic primers developed by (Guoli et al., 1999) based on satellite DNA for bovine can also amplify buffalo and yak but no other species, suggesting the satellite DNA to have interspecies variability but no intra-species variability. Diversity of ITS sequences between species but conserved within a species make the region as an ideal marker for species identication. However, closely related species may be distinguished by the variability of the ITS region due to very high polymorphism that results in variation of the sequence from geographically separated populations (Wang, Bao, et al., 2006). On the other hand, SINE-based PCR system has been developed to quantitatively identify bovine, porcine, chicken and ruminant species. Variability of the elements allows designing species-specic primers, whereas recombination of some of the sequence variants that form satellites of the original SINE families permits detection of closely related species (Walker et al., 2003). From another point of view, the size of mtDNA is much shorter than that of nDNA and varies greatly among organisms (Hartwell et al., 2008). Typical size of higher animal mtDNA has been reported to be about 16,000 base pairs (bp) (Magoulas, 2005). The coding regions of the mtDNA include 13 genes code for proteins involved in enzyme subunits productions for electron transport and oxidative phosphorylation, two genes for ribosomal RNAs (12S and 16S rRNA) and 22 genes for transfer RNAs (tRNAs). Besides, the rRNAs and tRNAs function in protein translation process by the ribosomes. One non-coding control region of about 1000 bp long can also be found in the mtDNA sequence that is referred to displacement loop (D-loop) in vertebrates and contains the replication origin of mtDNA (Magoulas, 2005). nDNA is transmitted to the offspring through Mendelian inheritance that refers to paternal lineage. The process involves genetic recombination which commonly occurs in nDNA where new allelic combination or different nucleotide sequence than that of either parent is produced, usually as a result of crossing-over (Hartwell et al., 2008). Genetic recombination and gene conversion cause the nuclear introns to evolve very slowly, to be over-shufed and susceptible to incomplete lineage sorting that reduces the capability of nDNA in resolving interspecic phylogenies (Whittall et al., 2006). However, there are several studies reporting successful usage of introns in determining intra-species phylogenies among diverse animal groups (Whittall et al., 2006). On the other hand, mtDNA is almost exclusively maternally inherited and genetic recombination does not usually occur in mtDNA (Magoulas, 2005). Maternal inheritance of the mtDNA analysis allows better comparison of an individual to other more distantly related individuals (Gefrides & Welch, 2011). In addition, any occurrence of genetic recombination would not develop a new genotype due to homoplasmy (Magoulas, 2005). Homoplasmy is a condition where somatic and germ cells of an individual organism contain a single type of mtDNA and the two recombining molecule may be the same in most cases. Moreover, mtDNA is well-known to have higher mutation rate of about 10 times than in nDNA of the same vertebrates even though it is functionally crucial (Brown, George, & Wilson, 1979). It is suggested to introduce more errors in replication due to less efcient repair mechanisms of the DNA (Brown et al., 1979; Hartwell et al., 2008) and more mutagenic intracellular environment. Enhanced chance of xation in mtDNA given by low functional constraint on mitochondrial gene products may also be the potential reason for faster mutation rate (Brown et al., 1979). The variations in mtDNA among closely related organisms that share greatly similar nDNA explains the preference of using mtDNA in tracking evolutionary history of a particular species. In addition, different regions in mtDNA evolve at different rates providing a range selection of regions to be chosen as a target, depending on the purpose of study (Kvist, 2000).

2.2. Genes used for species identication Gene selection for species-specic PCR analysis depends on the sequence variability of the genes. Inter- and intra-species identication using PCR employs at least one specic primer set to specically recognize nucleotide sequence unique to one species. Another primer set that is able to detect all species in the sample, called the universal primer is also used and usually made as a positive control to amplify a conserved region for all species (Lenstra, 2010). The positive amplication control of the PCR system serves as a tool to exclude falsenegative results possibly due to inhibition of DNA polymerase and to check the quality of the DNA extracted, which can be run prior to species-specic PCR systems (Laube et al., 2003). The most common target gene for species-specic PCR amplication is mitochondrial-encoded cytochrome b gene. It is a gene that is often used for phylogenetic studies and as reference gene in species-specic PCR. It contains both variable and conserved regions that are sufcient to resolve divergence at population level and clarication of deeper evolutionary relationship respectively (Kvist, 2000). Zhang et al. (2007) has developed a quantitative real-time PCR system using TaqMan probe based on cytochrome b gene to detect and quantify bovine DNA in meats, milks and cheeses. The interspecies variability of cytochrome b sequence has been exploited to design bovine-specic primers that are highly selective against sheep, pig, goat, turkey, chicken and water buffalo but the ovinespecic primers can still detect goat's DNA at low efciency. The gene has also been used to specically detect ve animal species including pig, cattle, sheep, chicken and turkey in meat mixtures (Dooley et al., 2004). The detection limit reported by Zhang et al. (2007) is 35 pg bovine DNA while 0.1% of bovine, turkey and ovine DNA (approximately 50 pg DNA) in DNA admixtures can be detected by Dooley et al. (2004). Even lower detection limit of 1 pg can be achieved in the detection of hare meat using cytochrome b-derived primers and probe that are specic against a wide range of different game species but not closely related species (Santos et al., 2012). Another mitochondrial-encoded gene that has been used to identify specic species is 12S rRNA. The gene has been used to generate species-specic primers for a few deer species, indicating the presence of intra-species variability of the gene sequence Fajardo et al. (2008b). Besides, PCR system employing a range of species-specic primers for a range of game birds species demonstrates both inter and intra-species variability contained in the sequence (Rojas et al., 2010). Porcine-specic primers have also been designed based on the gene giving a highly specic PCR system against a wide range of species including both animals and plants that present in feedstuff (Martn et al., 2009). The values of detection limit of the 12S rRNA-based PCR system reported in the three studies are variable, ranging from less than 5 fg to 10 pg of DNA. D-loop region of mtDNA is frequently chosen for meat speciation due to high substitution rate and most rapidly evolving region in mitochondrial genome Fajardo et al. (2008a). As suggested by single nucleotide polymorphism analysis, the mutations within a population of animals and between individuals are very frequent in this region (Fajardo et al., 2008a). This would indicate the potential use of mitochondrial D-loop region to differentiate inter- and intra-species animals. Quantitative determination of beef in mixed samples using species-specic primers and probe targeting D-loop region has been reported by (Sawyer et al., 2003). The primers are able to specically detect bovine DNA in beef and lamb admixtures at 0.1% and in unknown DNA sample. Besides, the genes of the subunits of NADH dehydrogenase have also been useful in identifying a particular species using real-time PCR. The NADH dehydrogenase subunit 5 (ND5) gene has also been used to design porcine-specic primers for highly specic PCR system (Farihah Liyana et al., 2009; Kesmen et al., 2009). NADH dehydrogenase subunit 2 (ND2)-based specic primer for donkey shows


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sufcient intra-species variability to distinguish donkey from horse meat (Kesmen et al., 2009) as species differentiation between horse and donkey is quite challenging. On the other hand, horse-specic primers derived from ATPase6/ATPase8 gene sequence are also able to target horse from donkey, however, at high Ct value of more than 30, porcine DNA can be detected leading to ambiguous result (Kesmen et al., 2009). The detection limit of the PCR system developed based on ND2, ND5 and ATPase6/ATPase8 genes is very low; 0.1 pg DNA in water solution which gives a highly sensitive species detection system. 16S rRNA genes are usually used as universal primers as their sequences are well conserved (Dalmasso et al., 2004) and they code for proteins that play role in vital function of the cell, resulting in less mutation rate of the genes. The conserved region in 16S rRNA sequence is likely to be maintained in closely related species, thus the gene sequences of distantly related species (cow and chicken) are aligned to generate the universal primer that will amplify both mammalian and avian species (Sawyer et al., 2003). The 16S rRNAbased universal primer for mammals and poultry is also used by Kesmen et al. (2009) as an amplication control. Whilst, ruminantspecic primers for the 16S rRNA gene has been developed to detect and quantify ruminant species including sheep, cattle and goat in a very complex pool of DNA mixtures in meat and bone meal (MBM) (Chiappini et al., 2005). It is chosen as the target sequence since the level of intra-species variability is much lower than that of interspecies variability (Chiappini et al., 2005). Despite that, species-specic primers and probes may also be designed based on nuclear genes. Every mammalian order has a signicant number of these short interspersed elements (SINE) as each of the SINE families within different genomes is derived independently (Walker et al., 2003). This provides SINE as a potential target sequence to identify species in PCR system. The specic primers developed for bovine, chicken, porcine and ruminant using the SINE can effectively detect the target species against other template DNA of 14 species (Walker et al., 2003). However, some cross-species amplication has been found in bovine and porcine assays at high Ct value of 26 and 29 respectively which would still allow efcient quantitation range of these assays using complex DNA sources. The ability of the assays to detect very low level of target species DNA; 0.05 pg porcine DNA, 0.5 pg bovine DNA and 5 pg chicken DNA in DNA mixtures of the three species indicates signicantly sensitive detection systems. Even lower detection limit of 10 fg of bovine DNA can be achieved by the SINE-based primers designed by (Mendoza-Romero et al., 2004) that are optimized for bovine but can also detect several other ruminants including sheep, goat, deer and giraffe. More sensitive PCR assays may be due to different probe used for amplication. Species identication by real-time PCR using chromosomal-encoded genes has utilized cyclic guanosine monophosphate (cyclic GMP) phospohodiesterase, ryanodin receptor and interleukin-2 (IL-2) precursor genes. The DNA sequence in the non-coding regions of cyclic GMP phosphodiesterase gene have been used in species identication for cattle, sheep and goat, ryanodin receptor gene for pig and IL-2 precursor gene for chicken, turkey and duck (Laube et al., 2007). With the exception of the cattle-specic PCR system, all of the primers and probes designed show no cross-reactions between 27 species of animals and plants tested but intra-species variability cannot be distinguished. Non-specic amplication of bison and three deer species by cattlespecic primers and probe have been found. Although deer species detection has been overcome by designing an additional cattlespecic reverse primer, bison DNA amplication still cannot be eliminated. On top of that, the limit of detection of the assays obtained is ten genome copies of the respective animal species. Quantitative PCR system has also been developed using the same primers and probe sequence for cattle, goat, sheep, pig, chicken and turkey (Laube, Zagon, & Broll, 2007). The same value of limit of detection has been

achieved in unprocessed products and ultra-high heat-treated canned foods. In addition, very low quantication limit of 0.1% in lowprocessed products and in normal canned foods while 1% value is obtained in canned foods for use under tropical conditions. Species identication for dog has applied melanocortin receptor 1 (MCIR) gene (Evans, Wictum, Cecilia, Penedo, & Kanthaswamy, 2007) in a real-time PCR system. The gene is often used in studies for intra-species differentiation of several animals including pig, cattle, horse, chicken and sheep as the gene sequences are highly polymorphic and the strong relation between MC1R gene mutations and variations with coat colour phenotype (Fajardo et al., 2008a). For instance, for porcine breeds, the wild boar possesses a unique MC1R receptor variant that involves the expression of the wild-type coat colour and cannot be found in domestic pig (Fajardo et al., 2008a). However, caninespecic primers and probe developed by Evans et al. (2007) are not sufciently specic for closely related species such as fox and coyote. Whilst interspecic variability of the primers and probe sequence restricts amplication of several other species including goat, cattle, sheep, elk, deer, human, domestic cat, or bobcat. On the other hand, high sensitivity of the MC1R-based PCR system developed is indicated by a very low detection limit of 5 pg of DNA (Evans et al., 2007). Universal primers used for amplication control in a particular PCR system can be generated from several types of nuclear genes. For example, myostatin gene has been applied for mammals and poultry PCR system that serves as control (Laube, Zagon, & Broll, 2007; Laube, Zagon, Spiegelberg, et al., 2007; Laube et al., 2003). Successful amplication of all mammalian and avian species tested using the myostatin-based primers and probe has been reported (Laube et al., 2003). While more plant species tested results in negative detection, salmon and human DNAs failed to be detected by the same primers and probe. Variation in Ct values for mammalian and avian DNA amplied has been suggested to be due to sequence differences in primers- and probe-binding region. The efciency has been improved by a slight modication of the forward primer as well as the probe (Laube, Zagon, Spiegelberg, et al., 2007). For quantication purpose, using myostatin-based amplication control, comparable standard curves has been obtained from both control and speciesspecic PCR systems (Laube, Zagon, & Broll, 2007). The 18S rRNA gene has been used to design universal primers for a control system that is commonly applied in conjunction with 12S rRNA (Fajardo et al., 2008b; Lpez-Andreo, Aldeguer, Guillen, Gabaldon, & Puyet, 2012; Lpez-Andreo et al., 2005; Martn et al., 2009; Rojas et al., 2010) and cytochrome b-based (Lpez-Andreo et al., 2005, 2012) species-specic PCR systems. The frequent use of the gene to derive the universal primers is due to the presence of regions that are conserved among eukaryotes in the gene sequence. Furthermore, the amplication of all eukaryotic species tested in a particular PCR system occurs at comparable efciency. In addition, 18S rRNA-based control system enables detection of all eukaryotic DNA including traces of DNA that cannot be detected by speciesspecic PCR system present in the sample providing a positive control of a wide range of species (Lpez-Andreo et al., 2005; Rojas et al., 2010). Highly conserved regions in growth hormone gene sequence are ideal for designing universal primers for all mammalian species while bovine- and porcine-specic primers are also derived from the same gene but from highly variable regions among mammals such as specic intron sequence for the bovine (Brodmann & Moor, 2003). The mammalian-specic PCR system developed allows detection of all species tested with a slight difference between the Ct values obtained for each species. On the other hand, the beef-specic primers and probe show cross reactions with several deer species and sheep, indicating an insufcient degree of specicity to cattle. The bovine-specic PCR system has a very low detection limit of 0.02 ng of DNA, however, unsatisfactory value of detection limit of 1% is obtained for MBM sample (Brodmann & Moor, 2003).

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Based on the various studies in meat speciation, a number of both chromosomal- and mitochondrial-encoded genes can be used to detect a particular animal species, exploiting inter and intra-species variability present in the gene sequence. Amplication control that is based on PCR reaction of universal primers and probe usually targets nuclear genes as the sequences are more conserved among a group of species. Besides, the specicity and sensitivity of speciesspecic PCR systems targeting genes from either nDNA or mtDNA are comparable for some genes. Table 1 shows briey the advantages and disadvantages of different PCR targeting genes. 3. Inuence of nature of target gene on real-time PCR based identication Despite the specicity and sensitivity offered by the probe and primers in real-time PCR in determining a particular species, it can be potentially compromised by other factors such as the gene source. Gene selection from either mtDNA or nDNA can relatively affect the sensitivity and specicity of the PCR analysis (Lpez-Andreo et al., 2005; Martn et al., 2009). The specicity of the gene sequence exploited for PCR amplication is provided by higher genetic variations that occur in mtDNA among species compared to nDNA that enables designing of species-specic PCR primers (Kortbaoui, Locas, Imbeau, Payment, & Villemur, 2009). Rapidly evolved mtDNA provides a range of variable sequences in the gene that is specic to the species of interest. For example, mitochondrial cytochrome b gene sequence is highly variable between different species (Mohd Hazim et al., 2012; Santos et

al., 2012; Zhang et al., 2007) while intra-species variability of 12S rRNA gene sequence is usually exploited to solve divergence between breeds of a particular species (Fajardo et al., 2008b; Rojas et al., 2010). ND5 gene sequence also shows sufcient interspecies variability to differentiate porcine while intra-species variability of ND2 and ATPase6/ATPase8 genes are able to differentiate between horse and donkey (Kesmen et al., 2009). Sequence variability between different species has also been shown in nuclear genes such as MC1R (Evans et al., 2007), IL-2, ryanodin precursor and cyclic GMP phosphodiesterase (Laube, Zagon, Spiegelberg, et al., 2007; Laube et al., 2003) genes as well as nDNA elements like SINE (Walker et al., 2003) and intron (Brodmann & Moor, 2003). Despite the specicity against a wide range of species achieved by the derived primers from these nuclear genes and elements, some non-specic amplications have also been detected (Brodmann & Moor, 2003; Laube, Zagon, Spiegelberg, et al., 2007; Laube et al., 2003; Walker et al., 2003) and selectivity between breeds of the same species cannot be accomplished (Laube, Zagon, Spiegelberg, et al., 2007; Walker et al., 2003). Besides, the high copy number gene of mtDNA also contributes to higher sensitivity of mitochondrial-encoded genes as genetic markers (Kortbaoui et al., 2009). It has been reported that mtDNA analysis of selected gene required only 10 mtDNA molecules for successful detection but much more nDNA copies were required, indicating better sensitivity of mtDNA than nDNA in real-time PCR (Andrasson, Gyllensten, & Allen, 2002). Furthermore, the presence of multiple copies of mtDNA molecules in a cell signicantly improves the sensitivity of the PCR system when the cells experience extreme processing condition (Rojas et al., 2010). The limit of detection achieved by

Table 1 Advantages and disadvantages of various target genes. Gene nature Target gene Advantages Usually used in phylogenetic studies. High interspecies variability. Disadvantages Low interspecies variability. Unable to resolve closely related species. References Kvist (2000) Dooley et al. (2004) Zhang et al. (2007) Santos et al. (2012) Fajardo et al. (2008b) Martn et al. (2009) Rojas et al. (2010) Sawyer et al. (2003) Fajardo et al. (2008a) Farihah Liyana et al. (2009) Kesmen et al. (2009) Kesmen et al. (2009) Sawyer et al. (2003) Dalmasso et al. (2004) Chiappini et al. (2005) Kesmen et al. (2009) Walker et al. (2003) Mendoza-Romero et al. (2004) Laube, Zagon, and Broll (2007a)

mtDNA Cytochrome b

12S rRNA

Sufcient inter and inter-species variability.

D-loop region ND5 ND2 ATPase6/ATPase8 16S rRNA

High substitution rate. Most rapidly evolving region in mtDNA. High interspecies variability. Sufcient inter- and intraspecies variability. Sufcient inter species variability. Low interspecies variability. Contains well conserved regions. Very low interspecies variability. Often used to develop universal primers for mammalian, avian and ruminant species. High interspecies variability. Low interspecies variability. Low interspecies variability.



Cyclic GMP High interspecies variability. Phosphodiesterase Ryanodin receptor IL-2 precursor MCIR Highly polymorphic sequences. High interspecies variability. Myostatin Often serves as amplication control for mammalian and avian species. Low inter- and intraspecies variability. 18S rRNA Contains well conserved regions among eukaryotes. Very low interspecies variability.

Different intraspecies variability depending on species.

Growth hormone

Contain both conserved and variable regions for mammals. Sufcient interspecies variability

Evans et al. (2007) Fajardo et al. (2008a) Laube et al. (2003) Laube, Zagon, and Broll (2007) Laube, Zagon, Spiegelberg, et al. (2007) Fajardo et al. (2008b) Lpez-Andreo et al. (2012) Lpez-Andreo et al. (2005) Martn et al. (2009) Rojas et al. (2010) Lpez-Andreo et al. (2012) Brodmann and Moor (2003)


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mtDNA-based PCR system is generally very low, down to less than 5 fg of DNA by targeting 12S rRNA gene as demonstrated by Rojas et al. (2010) and 40 fg of DNA by cytochrome b-based PCR assay developed by Lpez-Andreo, Garrido-Pertierra, & Puyet (2006). In addition, species-specic PCR primers derived from other mitochondrialencoded genes can detect as low as 0.1 pg of DNA as reported by (Kesmen et al., 2009). Nuclear elements that present at high copy number such as SINE are advantageous in species identication (Walker et al., 2003) as it provides comparable sensitivity to that of mtDNA-based PCR assay. Target species detection at very low level of 10 fg of bovine DNA using SINE sequence (Mendoza-Romero et al., 2004) shows much greater sensitivity compared to other nDNA-based PCR systems employing either single-copy MC1R (Evans et al., 2007) or growth hormone (Brodmann & Moor, 2003) genes. Whilst highly sensitive detection systems using cyclic GMP phosphodiesterase, ryanodin and IL-2 precursor can detect down to 10 genome copies (Laube, Zagon, Spiegelberg, et al., 2007). Although high sensitivity can be obtained by targeting nuclear genes and elements, cross-reactions with other species may occur, reducing the specicity of the PCR system. On top of that, primers that are designed from nuclear genes would have broad specicity and less sensitivity in binding to the target sequence (Lenstra, 2010). Consequently, the species-specic PCR system is compromised. This suggests the use of nuclear gene like 18S rRNA, myostatin and growth hormone genes to derive universal primers for control that will amplify a group of species. Designated primers from the conserved regions of these genes have successfully amplied a wide range of species of both animals and plants. However, mitochondrial 16S rRNA has also been used to derive universal primers from the conserved regions. The use of mitochondrial-encoded gene to derive universal primers may eliminate the sensitivity problem caused by the use of nuclear genes. The effect of gene source on real-time PCR is not really well discussed but it is clear that the important factors of detection system, sensitivity and specicity can be potentially reduced with the use of nDNA to derive the primers probes. The mtDNA-based detection system offers absolute specicity of a particular species that restricts cross-reactions with other non-target species. Signicantly improved sensitivity has been observed in the detection system targeting a range of mitochondrial-encoded genes. Although using nuclear element, SINE to derive species-specic primers may also give comparable sensitivity, specicity of the assay may be compromised.

4. Inuence of the nature of target gene in real-time PCR based DNA quantication Information on the amount of DNA contained in a particular sample is very useful for various purposes in molecular biology elds. Methods to quantify DNA include quantitative competitive PCR, densitometry and real-time PCR procedures (Lpez-Calleja et al., 2007). DNA quantication using real-time PCR is a common technique used by researchers due to a highly accurate, specic, sensitive and relatively fast process. Quantitative detection and quantication by real-time PCR has been used frequently in various works on many species such as deer (Fajardo et al., 2008b), goat (Lpez-Calleja et al., 2007), cattle (Brodmann & Moor, 2003; Evans et al., 2007; Lpez-Andreo et al., 2012; Sawyer et al., 2003; Walker et al., 2003; Zhang et al., 2007), pig (Lpez-Andreo et al., 2012; Martn et al., 2009; Rodrguez, Garca, Gonzlez, Hernndez, & Martn, 2005; Walker et al., 2003) and dog (Evans et al., 2007) using either chromosomal- or mitochondrial encoded genes or both. Several species identication studies would develop several PCR systems detecting a range of species (Laube, Zagon, & Broll, 2007; Rojas et al., 2010; Walker et al., 2003).

DNA quantication is achieved by absolute quantication (Sawyer et al., 2003; Walker et al., 2003; Zhang et al., 2007) or by normalization of the Ct values (Fajardo et al., 2008b; Lpez-Calleja et al., 2007; Martn et al., 2009; Rodrguez et al., 2005; Rojas et al., 2010). Basically, a range of dilution series of DNA concentration will be amplied using the developed PCR system. Standard curve is constructed by plotting the Ct values obtained from the real-time graphs against log10 of DNA concentrations to perform regression analysis. Interpolation of the standard curve based on the Ct values obtained for the sample tested will give the amount of DNA of targeted species. On the other hand, normalization of the Ct values of unknown samples interpolated from the standard curve is done based on the endogenous control PCR system. In addition, linearity test, sensitivity, accuracy and precision parameters of the species-specic PCR systems are evaluated as well as assay validation. Maede (2006) has stated the potential of nDNA in quantication technique if the copy number of the target genes is stable to ensure accuracy and avoid variation in measurement as well as to obtain reproducible results. Moreover, the use of mtDNA is incompatible for quantitative measurement in real-time PCR even though it is very useful due to its high sensitivity for detection purpose. Singlecopy target gene from nDNA is preferable to be used to derive both species-specic and universal primers and probes to obtain comparable quantication results instead of the multi-copy target sequences (Lpez-Andreo et al., 2005; Martn et al., 2009). Using SINE that present at high copy number as target sequence would also result in variable results as the quantitative measurement depends on the actual initial copy number (Walker et al., 2003). Restriction on the use of mtDNA for quantication is strengthened as the accuracy of the yield of DNA obtained by the assay can be affected by the variations in the amount of mitochondria contained in different tissues (Fajardo et al., 2008b). Types of species may also possess variable amount of mitochondria or yield different amount of DNA (Sawyer et al., 2003), affecting the reliability of quantication results. In order to overcome this issue, some researchers have applied nDNAbased endogenous control PCR system for amplication of total DNA together with mtDNA-based species-specic PCR systems (Fajardo et al., 2008b; Martn et al., 2009; Rojas et al., 2010). The efciency of quantitative real-time PCR has been thought to be inuenced by the nature of the target sequence. For that purpose, a quantitative real-time PCR system has been developed by Andrasson et al. (2002) that amplies both mtDNA and nDNA targets. The type of analysis, either nDNA or mtDNA analysis is selected based on the estimated initial amount of DNA. Limited amount of DNA is not suitable for nDNA analysis and the use of mtDNA is preferable. For quantication standards, they have found that the amplication efciency for both targets is comparable depicted by similar Ct values of the linear standard curves of both singleplex and multiplex reactions. In contrast, single-copy target gene, MC1R gene used in quantitative PCR measurement shows greater efciency compared to high copy number element, SINE (Evans et al., 2007). Furthermore, an efciency of 100% is achieved by a combination of nDNA-based amplication control and mtDNAbased species-specic PCR systems (Fajardo et al., 2008b). As the amount of eukaryotic DNA detected represents almost the total amount of DNA, the control system allows calculation of the percentages of different species DNA in mixed samples (Lpez-Andreo et al., 2005). In addition to standard curve, the efciency of PCR system is also evaluated by testing samples containing mixtures of DNA at different proportions as well as different types of tissues that will overcome variability of DNA yield due to different amount of mitochondria (Martn et al., 2009). Uniformity of the copy number of target DNA sequence across the samples tested is very important in DNA quantication. The preference towards single-copy rather than multi-copy target gene for quantication has been due to achieve analogous quantitative measurements. Moreover, PCR efciency may also be affected by targeting

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a high copy number gene and the use of mtDNA for quantication may result in variable DNA yield due to variable mitochondria content of different types of tissue. 5. Conclusion Real-time PCR has been very useful in identifying species as well as in quantifying DNA content in food products. However, the specicity and sensitivity of detection and quantication can be affected by the source of the selected target gene, either mtDNA or nDNA. Although mtDNA is often used in phylogeny and species identication studies where it offers high sensitivity to the real-time PCR detection system, some drawbacks have been observed in DNA quantication as the nature of mitochondrial-encoded genes that present in multiple copies and variations in mitochondria content in different type of tissues restrict comparable quantitative results and reduce the accuracy of the measurements respectively and nDNA genes on the other hand, has been proven for successful species detection. Consequently, in this case somehow, it is worth to study the use of both sources in real-time PCR assay for porcine detection and quantication for better comparison. References
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