ma a,b,

tivity of the assay. However, derivation of PCR primers, either from nuclear DNA (nDNA) or mitochondrialy affect the sensitivity and specicity of the reaction as well as the quantitative

. . .

. . .ation m

2.2. Genes used for species identication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

Food Research International 50 (2013) 330338

Contents lists available at SciVerse ScienceDirect

Food Research

j ourna l homepage: www.e ls3. Inuence of nature of target gene on real-time PCR based identication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3354. Inuence of the nature of target gene in real-time PCR based DNA quantication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3365. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

1. Introduction

1.1. Background

Identication of porcine species in food products has been a major

being marketed. The responsible authorities have been challenged bythe complexity of the food product itself and the availability of highlysensitive and specic porcine identication method.

The Muslim population has been very doubtful of the Halal status ofthe food products in themarket due to pork adulteration. Products thatconcern nowadays in determining the Halal st

Correspondence to: A.F El Sheikha, Halal Products ResMalaysia (UPM), 43400 Serdang, Selangor Darul Ehsan, Mfax: +60 3 8943 9745. Corresponding author. Tel.: +60 3 8947 1836; fax:

E-mail addresses: elsheikha_aly@yahoo.com (A.F. Elfadhilah132230@gmail.com (N.F.K. Mokhtar).

0963-9969/$ see front matter 2012 Elsevier Ltd. Allhttp://dx.doi.org/10.1016/j.foodres.2012.10.047ethod . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3322. Nuclear and mitochondrial DNA . . .2.1. General characteristics . . . .Contents

1. Introduction . . . . . . . . .1.1. Background . . . . . .1.2. PCR-based porcine identicinuence on species identication and quantication using real-time PCR. 2012 Elsevier Ltd. All rights reserved.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330nDNAmtDNAmeasurement. In this review, both types of DNA are compared in terms of their characteristics and theirReal-time PCRHalal verication DNA (mtDNA) can relativelKeywords:Gene nature

technique for species identimethod. Incorporation of aNurhidayatul Asma Mohamad , Aly Farag El Sheikha ,Shuhaimi Mustafa a,c, Nur Fadhilah Khairil Mokhtar a,a Halal Products Research Institute, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor Darul Ehsan, Malaysiab Department of Food Science and Technology, Faculty of Agriculture, Minuya University, Shibin El Kom, Minuya Government, Egyptc Department of Microbiology, Faculty Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor Darul Ehsan, Malaysia

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

Article history:Received 20 September 2012Accepted 31 October 2012

Pork adulteration has been a major concern nowadays for Halal verication. Unintentional pork inclusion bycontamination 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

cation that can serve this purpose besides providing a powerful quanticationhighly sensitive and specic probe can greatly improve the specicity and sensi-Review

Comparison of gene nature used in real-tiquantication: A reviewatus of the food products

earch Institute, Universiti Putraalaysia. Tel.: +60 3 8941 7344;

+60 3 8943 9745.Sheikha),

rights reserved.e PCR for porcine identication and

International

ev ie r .com/ locate / foodrescontain any amount of porcine will be considered as Haram and cannotbe consumed at any level by theMuslim. Thus, it is a great concern to beable to detect the presence of porcine at the smallest amount, empha-sizing the need of highly sensitive and specic porcine identicationmethod.

Porcine inclusion in food products can occur at any step in the pro-duction chain, either during or after processing and manufacturing.

1.2. PCR-based porcine identication method

331N.A. Mohamad et al. / Food Research International 50 (2013) 330338These include analysis of different components of the cell such asprotein, lipid and DNA molecules. Lipid analysis would include liquidchromatography using reverse phase column and a UV detector(Ibrahim, 2008) while protein-based methods such as enzyme-linkedimmunosorbent assay (ELISA) (Chen, Hsieh, & Bridgman, 2008) andhigh-performance liquid chromatography (HPLC) (Chou et al., 2007)have also been used.

Porcine detection at molecular level has been diversely used now-adays especially DNA-based methods. DNA based-methods includingPCR amplication, PCR-RFLP and microarray gene chips have beenused for species detection (Zhang, Fowler, Scott, Lawson, & Slater,2007). These methods are preferred over protein and lipid analysisdue to relative stability of DNA molecule under extreme conditionsand efcient amplication of DNA sequence by PCR reaction (Zhanget al., 2007). There are two major types of PCR analysis: conventionalPCR and real-time PCR.

In conventional PCR, gel electrophoresis is often used as theend-point analysis where the PCR products are visualized on agarosegel according to their size, giving no quantitative results. Besides, thePCR products band on the gel does not give any information on theamount the sequence amplied. Carry over contamination can poten-tially occur while transferring DNA sample into the gel as opposed tothe reaction of real-time PCR assay that occurs in a closed tubethroughout the process which can also avoid the occurrence offalse-positive results (Lpez-Calleja et al., 2007). Species-specicprimers 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 variabilitycan also be distinguished by developing species-specic primers forthree deer species including red deer, fallow deer and roe deer spe-Direct inclusion results from using any part of pig, for example, porcinemeat as one of the ingredients or using ingredients that are producedfrom any part of pig as raw material. On the other hand, indirect inclu-sion occurswhen the product is contaminatedwith 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 processedfood products, making the detection process more formidable.

Currently, there are a range of methods used for that purposeincluding DNA-based method, PCR. In PCR, a specic target sequenceis amplied by the designated primers. Furthermore, the specicityand sensitivity of PCR can be improved by incorporating an internalhybridization probe (Mohd Hazim, Shuhaimi, Che Man, Abdul, &Nur Fadhilah, 2012).

On top of that, the origin of the target nucleotide sequence, eitherfrom nuclear (nDNA) or mitochondrial DNA (mtDNA) has beenthought to affect the specicity and sensitivity of PCR reaction(Lpez-Andreo, Lugo, Garrido-Pertierra, Prieto, & Puyet, 2005). Inspecies identication, the nucleotide sequence is derived from thevariable region of target gene that is specic to the species of interest(Lenstra, 2010). As specicity of a species-specic nucleotidesequence is the potential factor in determining the efcacy of thePCR to identify a particular species, so do the source of the gene.Intra- and interspecies sequence variability and copy number of thenuclear and mitochondrial DNA can inuence the performance ofthe PCR reaction in detection and quantication of particular target.

Real-time PCR offers both a sensitive and specic automated spe-cies identication method, and a powerful DNA quantication tool todetermine and compute the presence of porcine DNA in food prod-ucts. It can also offer a clear picture to the manufacturer on this mat-ter while taking appropriate measures to exclude porcine inclusion intheir products either intentionally or not.cies that amplify different size of amplicons.On the other hand, PCR-RFLP refers to digestion of PCR productsby a restriction enzyme that cleaves at a species-specic (restriction)site. The specicity of the method is limited as it cannot potentiallydifferentiate intra-species variability, for instance, between domesticpig and wild boar (Maede, 2006).

Besides, the possibility of the restriction site to be polymorphicwill eventually prevent the detection of all individuals from a species(Lenstra, 2010). Moreover, ambiguous results of PCR-RFLP methodrequires further analysis like DNA sequencing (Maede, 2006). Enzy-matic digestion also requires large PCR products, restricting the useof this technique for species identication of thermally processedfoods that consist of highly fragmented DNA (Fajardo et al., 2007).Besides, meat admixtures like sausages, pates and minced meat prod-ucts are not suitable to be analyzed by PCR-RFLP as the digestionresults may show a combination of miscellaneous restriction patternsfor all possible species contained in the adultered sample (Fajardo etal., 2007).

Real-time PCR involves real-time monitoring of the increment ofuorescence intensity developed during the reaction (Heid, Stevens,Livak, &Williams, 1996) due to the binding of uorescent dye to doublestranded DNA strands of the PCR products. Eventually, the accumula-tion of PCR product will generate uorescence over the reaction cycles.Fluorescence intensitywill reach the thresholdwhere specic target aresuccessfully detected. Threshold refers to the point that represents thelog phase of product accumulation and is usually set at 10 times thestandard deviation of the base-line (Heid et al., 1996). At threshold,the intensity of the uorescence generated is equal to the backgroundand as it increases above background, more amplicons are being pro-duced. Early detection indicatesmore target DNA templates are presentin the sample.

On top of that, real-time PCR is an automated process which elim-inates the end-point analysis. It offers a quantitative result based onthe measurement of uorescence intensity of the PCR products pro-vided by the dye or probe. However, only short PCR products oramplicons, around 150 bp or shorter are suitable to be ampliedusing real-time PCR (Lenstra, 2010). Detection limits of real-timePCR assay are variable but mostly sufcient in detecting signicantadulterations or potentially harmful trace amounts (Lenstra, 2010).

Themost common detector used in real-time PCR is SYBRGreen dyethat emits uorescence upon binding non-specically to the minorgroove of double-stranded DNA in the reaction mixture. As PCR prod-ucts accumulate, the uorescence intensity of the dye increases. Theuse of this intercalating dye for species detection provides a exiblemethod that does not require complicated probe design and optimiza-tion steps (Fajardo et al., 2008b). Besides, the dye is cheaper than desig-nated probe, reducing the overall cost of the analysis (Fajardo et al.,2008b). However, it shows reduced specicity as compared to probeand not suitable for multiplex real-time PCR assay (Martn et al., 2009).

SYBR Green dye has been reported to potentially interfere withPCR reactions, therefore, low dye concentration has to be used lead-ing to dye redistribution problem during DNA melting curve analysis(Mao, Leung, & Xin, 2007). This problem can be overcome by usingalternative intercalating dye namely EvaGreen with low PCR inhibi-tion that allows the use of high dye concentration to develop robustPCR system (Mao et al., 2007). As compared to SYBR Green, EvaGreenis more stable to withstand intense PCR reaction condition and moresensitive due to better DNA binding prole (Mao et al., 2007). Appli-cation of EvaGreen dye in meat speciation has been demonstrated bySantos et al. (2012) while Wang, Chen, and Xu (2006b) showed thesuitability of the dye for DNA quantication.

Additionally, the use of sequence-specic probe has also beenwidely 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

332 N.A. Mohamad et al. / Food Research International 50 (2013) 330338nuclease degradation during DNA extension/polymerization causes thedye touoresce. The probe, generally 27 nucleotide long (Lpez-Andreoet al., 2005), binds to a complementary sequence in between the for-ward and reverse primers and collision of Taq polymerase and theprobe resulting in cleavage of the probe by activity of the 5 exonucle-ase. In designing the probe, the melting temperature of the probemust be 810 C higher than that of the primers. Besides, the annealingtemperature may have to be reduced in 710 C if there is a mismatchin the middle third of the probe upon binding that will compromisethe specicity of the primers (Lpez-Andreo et al., 2005).

The high specicity of TaqMan probe has been exploited by Zhanget al. (2007) to identify bovine in meats, milks and cheeses. High sen-sitivity of the probe allows detection of bovine DNA at a very lowlevel. Kesmen et al. (2009) have also used the same type of probefor detection of donkey, horse and pig species in raw and cookedproducts. The detection limit has been found to be 0.0001 ng of tem-plate DNA of raw meat. The closely related species, donkey and horsecan also be differentiated using the PCR system developed. Real-timePCR system using TaqMan probe has been enhanced by Lpez-Andreoet al. (2005) by conjugating the probe with minor groove binder(MGB). The designated probe has increased afnity to targetsequence which allows the use of shorter sequence and broadensthe variety of sequences that are specic to a particular species orgroup (Lpez-Andreo et al., 2005).

Compared to other conventional oligonucleotide probes, molecularbeacon probe specicity is enhanced by the hairpin or stem-and-loopstructure that permits detection of single-nucleotide variations of thetarget sequence. The stem structure also offersmore efcient quenchingthat reduces initial uorescence background (MohdHazim et al., 2012).The sequence of the loop region is complementary to the targetsequence while the stems are complementary to each other. Boundcovalently on each stem, a uorophore and a quencher are in closeproximity that prevents release of uorescent but is restored uponbinding 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 aslightly higher temperature than the annealing temperature of thePCR (Marras, Kramer, & Tyagi, 2003). On the other hand, the stemregion may contain 57 nucleotides that separate at 710 C higherthan the annealing temperature of the DNA polymerase (Mhlanga &Malmberg, 2001). The stability of the perfectly complementaryprobe-target hybrid over a wide range of temperature is important toovercome stem hybrid binding, ensuring complete quenching ofmismatched probe-target hybrid and only highly stable, perfect matchof probe-target hybrid produces uorescent. (Mhlanga & Malmberg,2001). Furthermore, detection of multiple targets in the same solutioncan be done by using different types of uorophore on the probe mole-cule (Marras et al., 2003; Mhlanga & Malmberg, 2001).

Species identication by real-time PCR employingmolecular beaconprobe includes the work done by Sandhya, Chen, and Mulchandani(2008) on Escherichia coli detection in fresh produce andwater. Besides,the probe has also been coupled with real-time PCR in detection ofsingle nucleotide polymorphism using EDTA-treated blood sample(Mhlanga & Malmberg, 2001). Porcine detection and quanticationusing molecular beacon probe has also been done by Mohd Hazim etal. (2012) showing very high specicity against a number of gamespecies tested. The use of molecular probe together with the species-specic primers also allows detection of porcine DNA at a very lowlevel 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 thatserves simultaneously as a PCR primer and as a molecular beacon inthe assay (Broude, 2005). The 5 end of the primer is extended witha hairpin structure of molecular beacon probe which is linked by ablocker, 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 theprimer and synthesis of target sequence causes the stem and loopstructure to unfold, allowing hybridization of the loop sequencewith the amplied target and eventually, generating uorescence(Broude, 2005). The design of this probe permits unimolecular targetdetection that promotes faster kinetics and higher stability of thecomplex compared to bimolecular reaction (Broude, 2005). Further-more, the production of probe-target hybrid by the extension of theprobe is concentration independent (zero order) and highly specicdetection down to single base change can be achieved (Whitcombeet al., 1999). The Scorpion probe containing species-specic primerhas been applied for cow detection and quantication in binarymeat mixtures. The highly selective duplex PCR system is able toamplify cow species against sheep at a high sensitivity of 0.1% (w/w)(Sawyer, Wood, Shanahan, Gout, & McDowell, 2003). Despite the highspecicity and sensitivity offered, there is not much application of Scor-pion probe in meat speciation studies, most probably due to complicat-ed 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 eachcell, within the nucleus (nuclear DNA) while mitochondrial DNA(mtDNA) is referred to the DNA molecule that resides within thecytoplasmic organelle of the cell, the mitochondria that present invariable amount depending on the types of the cell (Hartwell et al.,2008). The double membrane compartment provides some kind ofprotection to the DNA upon mitochondria isolation process from thecell, prior to mtDNA extraction (Bogenhagen, 2009). Cellular locationof the DNA has been reported to inuence vulnerability to DNA deg-radation. It has been found that nDNA from whole tissue tends todegrade faster than mtDNA but oppositely in homogenized tissueafter treatments in a study on relative DNA degradation (Foran,2006).

In the nucleus, the long and linear double-stranded DNA is wrappedaround histone proteins in the supercoiled structure of chromosomeswith phosphate group at the 5 end and hydroxyl group at the 3end(Hartwell et al., 2008). On the other hand, mtDNA of most species arecircular and largely free of bound proteins, which contained in highlycondensed structures called nucleoids (Bogenhagen, 2009). The circularshape of mtDNA contributes to greater stability over time as it is lesssusceptible to degradation compared to nDNA (Gefrides & Welch,2011). This results in better survival of mtDNA in highly processedfood that undergo extreme conditions of food processing. In addition,a few copies of the mtDNA genomes, approximately 210 copies areorganized in nucleoids (Bogenhagen, 2009) resulting in the existenceof 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 providesmore sensitivity in mtDNA detection compared to nDNA testing(Gefrides & Welch, 2011).

The mammal nDNA consists of about 20,000 genes or protein-coding regions (Moran, 2011). According to C-value paradox,98% or more of the nuclear genome of many organisms consists ofnon-coding and repetitive DNA fractions which have unknown func-tion and are often regarded as junk DNA that signicantly differs inamount between species (Moran, 2011). Other regions of nDNAwould include introns and spacer sequences between the genes.

The presence of introns, repetitive DNA as well as spacersequences between the genes in nDNA is one of the factors that distin-guish nDNA from mtDNA (Magoulas, 2005). Studies have shown thatnDNA 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,

333N.A. Mohamad et al. / Food Research International 50 (2013) 330338SINE (Walker et al., 2003) have been useful for species identication.The species-specic primers developed by (Guoli et al., 1999) basedon satellite DNA for bovine can also amplify buffalo and yak but noother species, suggesting the satellite DNA to have interspecies variabil-ity but no intra-species variability. Diversity of ITS sequences betweenspecies but conserved within a species make the region as an idealmarker for species identication. However, closely related speciesmay be distinguished by the variability of the ITS region due to veryhigh polymorphism that results in variation of the sequence from geo-graphically separated populations (Wang, Bao, et al., 2006). On theother hand, SINE-based PCR systemhas beendeveloped to quantitative-ly identify bovine, porcine, chicken and ruminant species. Variability ofthe elements allows designing species-specic primers, whereasrecombination of some of the sequence variants that form satellites ofthe original SINE families permits detection of closely related species(Walker et al., 2003).

From another point of view, the size of mtDNA is much shorterthan that of nDNA and varies greatly among organisms (Hartwell etal., 2008). Typical size of higher animal mtDNA has been reported tobe about 16,000 base pairs (bp) (Magoulas, 2005). The coding regionsof the mtDNA include 13 genes code for proteins involved in enzymesubunits productions for electron transport and oxidative phosphory-lation, two genes for ribosomal RNAs (12S and 16S rRNA) and 22genes for transfer RNAs (tRNAs). Besides, the rRNAs and tRNAs func-tion in protein translation process by the ribosomes. One non-codingcontrol region of about 1000 bp long can also be found in the mtDNAsequence that is referred to displacement loop (D-loop) in verte-brates and contains the replication origin of mtDNA (Magoulas,2005).

nDNA is transmitted to the offspring through Mendelian inheri-tance that refers to paternal lineage. The process involves geneticrecombination which commonly occurs in nDNA where new alleliccombination or different nucleotide sequence than that of either par-ent is produced, usually as a result of crossing-over (Hartwell et al.,2008). Genetic recombination and gene conversion cause the nuclearintrons to evolve very slowly, to be over-shufed and susceptible toincomplete lineage sorting that reduces the capability of nDNA inresolving interspecic phylogenies (Whittall et al., 2006). However,there are several studies reporting successful usage of introns indetermining intra-species phylogenies among diverse animal groups(Whittall et al., 2006).

On the other hand, mtDNA is almost exclusively maternallyinherited and genetic recombination does not usually occur in mtDNA(Magoulas, 2005). Maternal inheritance of the mtDNA analysis allowsbetter comparison of an individual to other more distantly relatedindividuals (Gefrides & Welch, 2011). In addition, any occurrenceof genetic recombination would not develop a new genotype due tohomoplasmy (Magoulas, 2005). Homoplasmy is a condition wheresomatic and germ cells of an individual organism contain a single typeof mtDNA and the two recombining molecule may be the same inmost cases.

Moreover, mtDNA is well-known to have higher mutationrate of about 10 times than in nDNA of the same vertebrates eventhough it is functionally crucial (Brown, George, & Wilson, 1979). Itis suggested to introduce more errors in replication due to lessefcient repair mechanisms of the DNA (Brown et al., 1979;Hartwell et al., 2008) and more mutagenic intracellular environ-ment. Enhanced chance of xation in mtDNA given by low functionalconstraint on mitochondrial gene products may also be the potentialreason for faster mutation rate (Brown et al., 1979). The variations inmtDNA among closely related organisms that share greatly similarnDNA explains the preference of using mtDNA in tracking evolution-ary history of a particular species. In addition, different regions inmtDNA evolve at different rates providing a range selection ofregions 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 thesequence variability of the genes. Inter- and intra-species identica-tion using PCR employs at least one specic primer set to specicallyrecognize nucleotide sequence unique to one species. Another primerset that is able to detect all species in the sample, called the universalprimer is also used and usually made as a positive control to amplifya conserved region for all species (Lenstra, 2010). The positive ampli-cation control of the PCR system serves as a tool to exclude false-negative results possibly due to inhibition of DNA polymerase andto check the quality of the DNA extracted, which can be run prior tospecies-specic PCR systems (Laube et al., 2003).

The most common target gene for species-specic PCR amplica-tion is mitochondrial-encoded cytochrome b gene. It is a gene thatis often used for phylogenetic studies and as reference gene inspecies-specic PCR. It contains both variable and conserved regionsthat are sufcient to resolve divergence at population level and clari-cation of deeper evolutionary relationship respectively (Kvist,2000). Zhang et al. (2007) has developed a quantitative real-timePCR system using TaqMan probe based on cytochrome b gene todetect and quantify bovine DNA in meats, milks and cheeses. Theinterspecies variability of cytochrome b sequence has been exploitedto design bovine-specic primers that are highly selective againstsheep, pig, goat, turkey, chicken and water buffalo but the ovine-specic primers can still detect goat's DNA at low efciency. Thegene has also been used to specically detect ve animal speciesincluding 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 ovineDNA (approximately 50 pg DNA) in DNA admixtures can be detectedby Dooley et al. (2004). Even lower detection limit of 1 pg can beachieved in the detection of hare meat using cytochrome b-derivedprimers and probe that are specic against a wide range of differentgame species but not closely related species (Santos et al., 2012).

Another mitochondrial-encoded gene that has been used to iden-tify specic species is 12S rRNA. The gene has been used to generatespecies-specic primers for a few deer species, indicating the pres-ence of intra-species variability of the gene sequence Fajardo et al.(2008b). Besides, PCR system employing a range of species-specicprimers for a range of game birds species demonstrates both interand intra-species variability contained in the sequence (Rojas et al.,2010). Porcine-specic primers have also been designed based onthe gene giving a highly specic PCR system against a wide range ofspecies including both animals and plants that present in feedstuff(Martn et al., 2009). The values of detection limit of the 12SrRNA-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 speciationdue to high substitution rate and most rapidly evolving region inmitochondrial genome Fajardo et al. (2008a). As suggested by singlenucleotide polymorphism analysis, the mutations within a populationof animals and between individuals are very frequent in this region(Fajardo et al., 2008a). This would indicate the potential use of mito-chondrial D-loop region to differentiate inter- and intra-species ani-mals. Quantitative determination of beef in mixed samples usingspecies-specic primers and probe targeting D-loop region has beenreported by (Sawyer et al., 2003). The primers are able to specicallydetect bovine DNA in beef and lamb admixtures at 0.1% and inunknown DNA sample.

Besides, the genes of the subunits of NADH dehydrogenase havealso been useful in identifying a particular species using real-timePCR. The NADH dehydrogenase subunit 5 (ND5) gene has also beenused to design porcine-specic primers for highly specic PCR system(Farihah Liyana et al., 2009; Kesmen et al., 2009). NADH dehydroge-

nase subunit 2 (ND2)-based specic primer for donkey shows

334 N.A. Mohamad et al. / Food Research International 50 (2013) 330338sufcient intra-species variability to distinguish donkey from horsemeat (Kesmen et al., 2009) as species differentiation between horseand donkey is quite challenging. On the other hand, horse-specicprimers derived from ATPase6/ATPase8 gene sequence are also ableto target horse from donkey, however, at high Ct value of more than30, porcine DNA can be detected leading to ambiguous result(Kesmen et al., 2009). The detection limit of the PCR system devel-oped based on ND2, ND5 and ATPase6/ATPase8 genes is very low;0.1 pg DNA in water solution which gives a highly sensitive speciesdetection system.

16S rRNA genes are usually used as universal primers as theirsequences are well conserved (Dalmasso et al., 2004) and they codefor proteins that play role in vital function of the cell, resulting inless mutation rate of the genes. The conserved region in 16S rRNAsequence is likely to be maintained in closely related species, thusthe gene sequences of distantly related species (cow and chicken)are aligned to generate the universal primer that will amplify bothmammalian and avian species (Sawyer et al., 2003). The 16S rRNA-based universal primer for mammals and poultry is also used byKesmen et al. (2009) as an amplication control. Whilst, ruminant-specic primers for the 16S rRNA gene has been developed to detectand quantify ruminant species including sheep, cattle and goat in avery complex pool of DNA mixtures in meat and bone meal (MBM)(Chiappini et al., 2005). It is chosen as the target sequence since thelevel of intra-species variability is much lower than that of interspe-cies variability (Chiappini et al., 2005).

Despite that, species-specic primers and probes may also bedesigned based on nuclear genes. Every mammalian order has a sig-nicant number of these short interspersed elements (SINE) as eachof the SINE families within different genomes is derived independent-ly (Walker et al., 2003). This provides SINE as a potential targetsequence to identify species in PCR system. The specic primersdeveloped for bovine, chicken, porcine and ruminant using the SINEcan effectively detect the target species against other template DNAof 14 species (Walker et al., 2003). However, some cross-speciesamplication has been found in bovine and porcine assays at highCt value of 26 and 29 respectively which would still allow efcientquantitation range of these assays using complex DNA sources. Theability 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 DNAin DNA mixtures of the three species indicates signicantly sensitivedetection systems. Even lower detection limit of 10 fg of bovineDNA can be achieved by the SINE-based primers designed by(Mendoza-Romero et al., 2004) that are optimized for bovine butcan also detect several other ruminants including sheep, goat, deerand giraffe. More sensitive PCR assays may be due to different probeused for amplication.

Species identication by real-time PCRusing chromosomal-encodedgenes has utilized cyclic guanosine monophosphate (cyclic GMP)phospohodiesterase, ryanodin receptor and interleukin-2 (IL-2) precur-sor genes. The DNA sequence in the non-coding regions of cyclic GMPphosphodiesterase gene have been used in species identication forcattle, sheep and goat, ryanodin receptor gene for pig and IL-2 precursorgene for chicken, turkey and duck (Laube et al., 2007). With the excep-tion of the cattle-specic PCR system, all of the primers and probesdesigned show no cross-reactions between 27 species of animals andplants tested but intra-species variability cannot be distinguished.Non-specic amplication of bison and three deer species by cattle-specic primers and probe have been found. Although deer speciesdetection has been overcome by designing an additional cattle-specic reverse primer, bison DNA amplication still cannot be elimi-nated. On top of that, the limit of detection of the assays obtained isten genome copies of the respective animal species. Quantitative PCRsystem has also been developed using the same primers and probesequence for cattle, goat, sheep, pig, chicken and turkey (Laube,

Zagon, & Broll, 2007). The same value of limit of detection has beenachieved in unprocessed products and ultra-high heat-treated cannedfoods. In addition, very low quantication limit of 0.1% in low-processed products and in normal canned foods while 1% value isobtained 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 forintra-species differentiation of several animals including pig, cattle,horse, chicken and sheep as the gene sequences are highly polymorphicand the strong relation between MC1R gene mutations and variationswith coat colour phenotype (Fajardo et al., 2008a). For instance, for por-cine breeds, the wild boar possesses a unique MC1R receptor variantthat involves the expression of the wild-type coat colour and cannotbe found in domestic pig (Fajardo et al., 2008a). However, canine-specic primers and probe developed by Evans et al. (2007) are not suf-ciently specic for closely related species such as fox and coyote.Whilst interspecic variability of the primers and probe sequencerestricts 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 indicatedby a very low detection limit of 5 pg of DNA (Evans et al., 2007).

Universal primers used for amplication control in a particularPCR system can be generated from several types of nuclear genes.For example, myostatin gene has been applied for mammals andpoultry PCR system that serves as control (Laube, Zagon, & Broll,2007; Laube, Zagon, Spiegelberg, et al., 2007; Laube et al., 2003). Suc-cessful amplication of all mammalian and avian species tested usingthe myostatin-based primers and probe has been reported (Laube etal., 2003). While more plant species tested results in negative detec-tion, salmon and human DNAs failed to be detected by the sameprimers and probe. Variation in Ct values for mammalian and avianDNA amplied has been suggested to be due to sequence differencesin primers- and probe-binding region. The efciency has beenimproved by a slight modication of the forward primer as well asthe probe (Laube, Zagon, Spiegelberg, et al., 2007). For quanticationpurpose, using myostatin-based amplication control, comparablestandard curves has been obtained from both control and species-specic PCR systems (Laube, Zagon, & Broll, 2007).

The 18S rRNA gene has been used to design universal primers for acontrol system that is commonly applied in conjunction with 12SrRNA (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 etal., 2005, 2012) species-specic PCR systems. The frequent use ofthe gene to derive the universal primers is due to the presence ofregions that are conserved among eukaryotes in the gene sequence.Furthermore, the amplication of all eukaryotic species tested in aparticular PCR system occurs at comparable efciency. In addition,18S rRNA-based control system enables detection of all eukaryoticDNA including traces of DNA that cannot be detected by species-specic PCR system present in the sample providing a positive controlof a wide range of species (Lpez-Andreo et al., 2005; Rojas et al.,2010).

Highly conserved regions in growth hormone gene sequence areideal for designing universal primers for all mammalian specieswhile bovine- and porcine-specic primers are also derived fromthe same gene but from highly variable regions among mammalssuch as specic intron sequence for the bovine (Brodmann & Moor,2003). The mammalian-specic PCR system developed allows detec-tion of all species tested with a slight difference between the Ct valuesobtained for each species. On the other hand, the beef-specicprimers and probe show cross reactions with several deer speciesand sheep, indicating an insufcient degree of specicity to cattle.The bovine-specic PCR system has a very low detection limit of0.02 ng of DNA, however, unsatisfactory value of detection limit of

1% is obtained for MBM sample (Brodmann & Moor, 2003).

Based on the various studies in meat speciation, a number of bothchromosomal- and mitochondrial-encoded genes can be used todetect a particular animal species, exploiting inter and intra-speciesvariability present in the gene sequence. Amplication control thatis based on PCR reaction of universal primers and probe usuallytargets nuclear genes as the sequences are more conserved among agroup of species. Besides, the specicity and sensitivity of species-specic PCR systems targeting genes from either nDNA or mtDNAare comparable for some genes.

Table 1 shows briey the advantages and disadvantages of differ-ent PCR targeting genes.

3. Inuence of nature of target gene on real-time PCR basedidentication

Despite the specicity and sensitivity offered by the probe andprimers in real-time PCR in determining a particular species, it canbe potentially compromised by other factors such as the gene source.Gene selection from either mtDNA or nDNA can relatively affect thesensitivity and specicity of the PCR analysis (Lpez-Andreo et al.,2005; Martn et al., 2009).

The specicity of the gene sequence exploited for PCR amplica-tion is provided by higher genetic variations that occur in mtDNAamong species compared to nDNA that enables designing ofspecies-specic PCR primers (Kortbaoui, Locas, Imbeau, Payment, &Villemur, 2009). Rapidly evolved mtDNA provides a range of variablesequences in the gene that is specic to the species of interest. Forexample, mitochondrial cytochrome b gene sequence is highly vari-able between different species (Mohd Hazim et al., 2012; Santos et

al., 2012; Zhang et al., 2007) while intra-species variability of 12SrRNA gene sequence is usually exploited to solve divergence betweenbreeds of a particular species (Fajardo et al., 2008b; Rojas et al., 2010).ND5 gene sequence also shows sufcient interspecies variability todifferentiate porcine while intra-species variability of ND2 andATPase6/ATPase8 genes are able to differentiate between horse anddonkey (Kesmen et al., 2009).

Sequence variability between different species has also beenshown 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 asnDNA elements like SINE (Walker et al., 2003) and intron(Brodmann & Moor, 2003). Despite the specicity against a widerange of species achieved by the derived primers from these nucleargenes and elements, some non-specic amplications have alsobeen detected (Brodmann & Moor, 2003; Laube, Zagon, Spiegelberg,et al., 2007; Laube et al., 2003; Walker et al., 2003) and selectivitybetween 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 tohigher sensitivity of mitochondrial-encoded genes as genetic markers(Kortbaoui et al., 2009). It has been reported that mtDNA analysis ofselected gene required only 10 mtDNA molecules for successfuldetection but much more nDNA copies were required, indicating bet-ter sensitivity of mtDNA than nDNA in real-time PCR (Andrasson,Gyllensten, & Allen, 2002). Furthermore, the presence of multiplecopies of mtDNA molecules in a cell signicantly improves the sensi-tivity of the PCR system when the cells experience extreme process-ing condition (Rojas et al., 2010). The limit of detection achieved by

Table 1Advantages and disadvantages of various target genes.

Genenature

Target gene Advantages Disadvantages References

mtDNA Cytochrome b Usually used in phylogenetic studies. Low interspecies variability. Kvist (2000)

, av

d a

als.

335N.A. Mohamad et al. / Food Research International 50 (2013) 330338High interspecies variability.

12S rRNA Sufcient inter and inter-species variability.

D-loop region High substitution rate.Most rapidly evolving region in mtDNA.

ND5 High interspecies variability.

ND2 Sufcient inter- and intraspecies variability.ATPase6/ATPase8 Sufcient inter species variability.16S rRNA Contains well conserved regions.

Very low interspecies variability.Often used to develop universal primers for mammalianruminant species.

nDNA SINE High interspecies variability.

Cyclic GMP High interspecies variability.PhosphodiesteraseRyanodin receptorIL-2 precursorMCIR Highly polymorphic sequences.

High interspecies variability.Myostatin Often serves as amplication control for mammalian an

species.Low inter- and intraspecies variability.

18S rRNA Contains well conserved regions among eukaryotes.Very low interspecies variability.

Growth hormone Contain both conserved and variable regions for mammSufcient interspecies variabilityUnable to resolve closely related species. 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)

Low interspecies variability.

ian and

Sawyer et al. (2003)Dalmasso et al. (2004)Chiappini et al. (2005)Kesmen et al. (2009)

Low interspecies variability. Walker et al. (2003)Mendoza-Romero et al. (2004)

Low interspecies variability. Laube, Zagon, and Broll(2007a)

Different intraspecies variabilitydepending on species.

Evans et al. (2007)Fajardo et al. (2008a)

vian Laube et al. (2003)Laube, Zagon, and Broll (2007)Laube, Zagon, Spiegelberg, etal. (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)

On top of that, primers that are designed from nuclear genes

336 N.A. Mohamad et al. / Food Research International 50 (2013) 330338would have broad specicity and less sensitivity in binding to thetarget sequence (Lenstra, 2010). Consequently, the species-specicPCR system is compromised. This suggests the use of nuclear genelike 18S rRNA, myostatin and growth hormone genes to deriveuniversal primers for control that will amplify a group of species.Designated primers from the conserved regions of these genes havesuccessfully amplied a wide range of species of both animalsand plants. However, mitochondrial 16S rRNA has also been usedto derive universal primers from the conserved regions. The use ofmitochondrial-encoded gene to derive universal primers may elimi-nate the sensitivity problem caused by the use of nuclear genes.

The effect of gene source on real-time PCR is not really welldiscussed but it is clear that the important factors of detection system,sensitivity and specicity can be potentially reduced with the use ofnDNA to derive the primers probes. The mtDNA-based detection sys-tem offers absolute specicity of a particular species that restrictscross-reactions with other non-target species. Signicantly improvedsensitivity has been observed in the detection system targeting arange of mitochondrial-encoded genes. Although using nuclear ele-ment, SINE to derive species-specic primers may also give compara-ble sensitivity, specicity of the assay may be compromised.

4. Inuence of the nature of target gene in real-time PCR basedDNA quantication

Information on the amount of DNA contained in a particular sam-ple is very useful for various purposes in molecular biology elds.Methods to quantify DNA include quantitative competitive PCR, den-sitometry and real-time PCR procedures (Lpez-Calleja et al., 2007).DNA quantication using real-time PCR is a common techniqueused by researchers due to a highly accurate, specic, sensitive andrelatively fast process.

Quantitative detection and quantication by real-time PCR hasbeen 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 mitochon-drial encoded genes or both. Several species identication studieswould develop several PCR systems detecting a range of speciesmtDNA-based PCR system is generally very low, down to less than 5 fgof DNA by targeting 12S rRNA gene as demonstrated by Rojas et al.(2010) and 40 fg of DNA by cytochrome b-based PCR assay developedby Lpez-Andreo, Garrido-Pertierra, & Puyet (2006). In addition,species-specic PCR primers derived from other mitochondrial-encoded 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 SINEare advantageous in species identication (Walker et al., 2003) as itprovides comparable sensitivity to that of mtDNA-based PCR assay.Target species detection at very low level of 10 fg of bovine DNAusing SINE sequence (Mendoza-Romero et al., 2004) shows muchgreater sensitivity compared to other nDNA-based PCR systemsemploying either single-copy MC1R (Evans et al., 2007) or growthhormone (Brodmann & Moor, 2003) genes. Whilst highly sensitivedetection systems using cyclic GMP phosphodiesterase, ryanodinand IL-2 precursor can detect down to 10 genome copies (Laube,Zagon, Spiegelberg, et al., 2007). Although high sensitivity can beobtained by targeting nuclear genes and elements, cross-reactionswith other species may occur, reducing the specicity of the PCRsystem.(Laube, Zagon, & Broll, 2007; Rojas et al., 2010; Walker et al., 2003).DNA quantication is achieved by absolute quantication (Sawyeret al., 2003; Walker et al., 2003; Zhang et al., 2007) or by normaliza-tion 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 am-plied using the developed PCR system. Standard curve is constructedby plotting the Ct values obtained from the real-time graphs againstlog10 of DNA concentrations to perform regression analysis. Interpola-tion of the standard curve based on the Ct values obtained for thesample tested will give the amount of DNA of targeted species. Onthe other hand, normalization of the Ct values of unknown samplesinterpolated from the standard curve is done based on the endoge-nous control PCR system. In addition, linearity test, sensitivity, accu-racy and precision parameters of the species-specic PCR systemsare evaluated as well as assay validation.

Maede (2006) has stated the potential of nDNA in quanticationtechnique if the copy number of the target genes is stable to ensureaccuracy and avoid variation in measurement as well as to obtainreproducible results. Moreover, the use of mtDNA is incompatiblefor quantitative measurement in real-time PCR even though it isvery useful due to its high sensitivity for detection purpose. Single-copy target gene from nDNA is preferable to be used to derive bothspecies-specic and universal primers and probes to obtain compara-ble quantication results instead of the multi-copy target sequences(Lpez-Andreo et al., 2005; Martn et al., 2009). Using SINE thatpresent at high copy number as target sequence would also result invariable results as the quantitative measurement depends on theactual initial copy number (Walker et al., 2003). Restriction on theuse of mtDNA for quantication is strengthened as the accuracy ofthe yield of DNA obtained by the assay can be affected by the varia-tions in the amount of mitochondria contained in different tissues(Fajardo et al., 2008b). Types of species may also possess variableamount of mitochondria or yield different amount of DNA (Sawyeret al., 2003), affecting the reliability of quantication results. Inorder to overcome this issue, some researchers have applied nDNA-based endogenous control PCR system for amplication of total DNAtogether with mtDNA-based species-specic PCR systems (Fajardoet al., 2008b; Martn et al., 2009; Rojas et al., 2010).

The efciency of quantitative real-time PCR has been thought to beinuenced by the nature of the target sequence. For that purpose, aquantitative real-time PCR system has been developed by Andrassonet al. (2002) that amplies both mtDNA and nDNA targets. The type ofanalysis, either nDNA or mtDNA analysis is selected based on the esti-mated initial amount of DNA. Limited amount of DNA is not suitablefor nDNA analysis and the use ofmtDNA is preferable. For quanticationstandards, they have found that the amplication efciency for bothtargets is comparable depicted by similar Ct values of the linear stan-dard curves of both singleplex and multiplex reactions. In contrast,single-copy target gene,MC1R gene used in quantitative PCR measure-ment shows greater efciency compared to high copy number element,SINE (Evans et al., 2007). Furthermore, an efciency of 100% is achievedby a combination of nDNA-based amplication control and mtDNA-based species-specic PCR systems (Fajardo et al., 2008b). As theamount of eukaryotic DNA detected represents almost the total amountof DNA, the control system allows calculation of the percentages of dif-ferent species DNA in mixed samples (Lpez-Andreo et al., 2005). Inaddition to standard curve, the efciency of PCR system is also evaluat-ed by testing samples containing mixtures of DNA at different propor-tions as well as different types of tissues that will overcome variabilityof DNA yield due to different amount of mitochondria (Martn et al.,2009).

Uniformity of the copy number of target DNA sequence across thesamples tested is very important in DNA quantication. The prefer-ence towards single-copy rather than multi-copy target gene forquantication has been due to achieve analogous quantitative mea-

surements. Moreover, PCR efciency may also be affected by targeting

337N.A. Mohamad et al. / Food Research International 50 (2013) 330338a high copy number gene and the use of mtDNA for quanticationmay result in variable DNA yield due to variable mitochondria con-tent of different types of tissue.

5. Conclusion

Real-time PCR has been very useful in identifying species as wellas in quantifying DNA content in food products. However, the speci-city and sensitivity of detection and quantication can be affectedby the source of the selected target gene, either mtDNA or nDNA.Although mtDNA is often used in phylogeny and species identica-tion studies where it offers high sensitivity to the real-time PCRdetection system, some drawbacks have been observed in DNA quan-tication as the nature of mitochondrial-encoded genes that presentin multiple copies and variations in mitochondria content in differenttype of tissues restrict comparable quantitative results and reduce theaccuracy of the measurements respectively and nDNA genes on theother hand, has been proven for successful species detection. Conse-quently, in this case somehow, it is worth to study the use of bothsources in real-time PCR assay for porcine detection and quantica-tion for better comparison.

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Comparison of gene nature used in real-time PCR for porcine identification and quantification: A review1. Introduction1.1. Background1.2. PCR-based porcine identification method

2. Nuclear and mitochondrial DNA2.1. General characteristics2.2. Genes used for species identification

3. Influence of nature of target gene on real-time PCR based identification4. Influence of the nature of target gene in real-time PCR based DNA quantification5. ConclusionReferences