Identification of chicken, duck, pigeon and pig

Identification of chicken,duck,pigeon and pig meat by species-specific markers of mitochondrial originSantosh Haunshi a,*,Rantu Basumatary b ,P.S.Girish c ,Sunil Doley a ,R.K.Bardoloi b ,Ashok Kumar daMolecular Genetics Laboratory,Poultry Science Division,ICAR Research Complex for N.E.H.Region,Umroi Road,Umiam 793103,Meghalaya,India bAnimal Production Division,ICAR Research Complex for N.E.H.Region,Umroi Road,Umiam 793103,Meghalaya,India cNational Research Centre on Meat,Chengicherla,PB No.19,Uppal Post,Hyderabad 500039,AP,India dAnimal Health Division,ICAR Research Complex for N.E.H.Region,Umroi Road,Umiam 793103,Meghalaya,Indiaa r t i c l e i n f o Article history:Received 20December 2008Received in revised form 2May 2009Accepted 15June 2009Keywords:Chicken Duck Pigeon Pig PCRMitochondria Speciation Markera b s t r a c tIn the present study,PCR based method for meat species identification of chicken,duck,pigeon and pig was achieved by developing species-specific ing mitochondrial sequences species-specific primers were designed and the sizes of them were 256bp,292bp,401bp and 835bp for chicken,duck,pigeon and pig,respectively.The species-specific PCR products were sequenced to confirm the specificity of the product amplified.These markers were subsequently tested for cross amplification by checking them with beef,mutton,chevon,pork,rabbit,chicken,duck,turkey and pigeon meat.DNA markers devel-oped in this study can help identify the species of fresh,cooked and autoclaved meat of chicken,duck and pigeon and fresh and cooked meat of pig.The process of identification is simple,economical and quick as compared to other methods such as RAPD,PCR-RFLP and sequencing method of species identification.Ó2009Elsevier Ltd.All rights reserved.1.IntroductionFraudulent or unintentional mislabeling of food products,which could not be detected using conventional techniques is still prevalent across the world.Certain section of population does not like to consume the meat of pigeon and pet animals due to ethical and compassionate reasons.Some religious groups such as Muslim and Jewish do not accept pork in their diet.Vegetarians are another section of the society;they are totally against consumption of meat of any species.So,these groups of consumers demand methods to detect species (chicken,duck,pigeon,pork,etc.)of meat in the food.The species of meat present in processed,cooked or com-pounded mixtures is not always possible to identify by routine examination,and hence different analytical methods have been developed for meat species identification and to authenticate that the meat is genuine and acceptable to the consumers.Chemomet-rics is one such method which is valuable,rapid and non-destruc-tive method used for authentication of meat and meat products (Arvanitoyannis &Van Houwelingen-Koukaliaroglou,2003)and it was used for authentication of beef samples using mid infraredspectroscopy with a chemometric technique of soft independent modelling of class analogy (SIMCA)analysis (Al-Jowder,Defernez,Kemsley,&Wilson,1999)and meat of pig breeds using visible and near infrared spectroscopy with SIMCA chemometrics (Cozzolino,Vadell,Ballesteros,Galietta,&Barlocco,2006).How-ever,chemometrics may not be a good choice when it comes to the cooked or thermal processed meat samples.Therefore,DNA based methods are a way out for meat species identification and authentication of processed and cooked meats .There are numer-ous nuclear DNA based procedures available to identify precisely the origin of meat species such as DNA hybridization (Chikuni,Ozustume,Hoishi Kawa,&Kato,1990),cloth based hybridization array system for detection of species origin of meat meals in feed grains (Murphy,Armour,&Blais,2007),random amplified poly-morphic DNA polymerase chain reaction (RAPD-PCR)fingerprints (Calvo,Zaragoza,&Osta,2001;Koh,Lim,Chua,Chew,&Phang,1998;Lee &Chang,1994),species-specific satellite DNA probes (Buntjer,Nijman,Zijlstra,&Lenstra,1998).However,Rastogi et al.(2007)have concluded that mitochondrial markers were more efficient than nuclear markers (RAPD finger printing and Actin gene barcoding)in species identification and authentication Further,Eriksson et al.(2008)demonstrated that domestic chick-ens inherited the mitochondrial genome from red jungle fowl,therefore,it is possible to identify the species of domestic chicken0309-1740/$-see front matter Ó2009Elsevier Ltd.All rights reserved.doi:10.1016/j.meatsci.2009.06.026*Corresponding author.Tel.:+913642570305;fax:+913642570362.E-mail address:santoshi575@ (S.Haunshi).Meat Science 83(2009)454–459Contents lists available at ScienceDirectMeat Sciencejo ur na l h o me pa ge :w w w.e ls ev ie r.c o m/lo c a t e/me a t s ciirrespective of its genetic origin(monophyletic or polyphyletic) using DNA markers of mitochondrial genome.Therefore,PCR amplification and sequencing of cytochrome b(Forrest&Carnegie, 1994)and12S rRNA genes(Girish et al.,2004;Prakash,Patole, Ghumatkar,Nandode,&Yogesh,2000)of mitochondria were help-ful for meat species identification.Recently,restriction fragment length polymorphism(RFLP)of PCR products of cytochrome b and12S rRNA genes of mitochondria has been developed(Girish et al.,2005,2007;Meyer,Hofelein,Luthy,&Candrian,1995; Verkaar,Nijman,Boutaga,&Lenstra,2002).Although earlier methods proved to be accurate and useful in identification of species origin of meat but they suffer from certain disadvantages.While reproducibility of RAPD-PCR is a matter of concern due to requirement of high stringent conditions(Koh et al.,1998),sequencing of mitochondrial genes and PCR-RFLP pro-cedures are expensive,requires more time and more analytical work and interpretation of the results is somewhat complex.Fur-ther,intricacies involved in interpretation of the results of PCR-RFLP in admixture meat and meat products make the procedure unfeasible(Ilhak&Arslan,2007).Therefore,meat species identification through PCR amplifica-tion of D-loop sequence of mitochondria is gaining importance more recently(Mane et al.,2007).Identification of origin of meat species by PCR using species-specific markers of D-loop origin of mitochondria is relatively quick,precise,sensitive and economical as compared to other PCR based assays.Keeping these points in view the present study was carried out to develop PCR based pro-tocols for identification of meat of chicken,duck,pigeon and pigs using species-specific DNA markers of mitochondrial origin.2.Materials and methods2.1.Collection of blood and meat samplesBlood samples from chicken,duck,turkey and pigeon species were collected from either jugular or wing veins aseptically.Blood samples from Hampshire and Khasi-local varieties of pig were col-lected from anterior vena cava.Meat samples from cattle,sheep, goat,pig,rabbit,chicken,duck,turkey and pigeon were collected aseptically and randomly from Institute farm,local retail meat shops or slaughterhouses.Meat samples were either cooked at 80°C with a holding period of30min or autoclaved at121°C tem-perature,15-psi pressure for20min duration.Autoclaving is a sim-ulation of canning of meat and meat products.2.2.Extraction and purification of DNA from blood and meat samplesTotal DNA was initially extracted from whole blood of chicken, duck and pigeon and white blood cells of pig by phenol–chloro-form–isoamyl alcohol method(Sambrook&Russell,2001)for stan-dardization of amplification of species-specific markers.Total DNA from fresh,cooked and autoclaved meat samples of chicken,duck, pigeon,turkey,cattle,sheep,goat,pig and rabbit was extracted by PCI method as described by Girish et al.(2007).DNA samples were dissolved in nuclease free water and kept atÀ20°C until further use.2.3.Checking quality and purity of DNAThe quality of genomic DNA was checked by horizontal submar-ine agarose gel electrophoresis using0.8%agarose strength.The quantity of total DNA extracted from various sources was esti-mated by taking optical density(O.D.)readings at260nm and the purity of DNA was checked by taking ratio of O.D.readings at 260nm and280nm using spectrophotometer.2.4.Design and custom synthesis of species-specific primersMitochondrial D-loop DNA sequences of chicken,duck and pig and cytochrome b gene of pigeon were retrieved from Genbank ()and species-specific DNA primers were designed using computer software(DNA STAR).List of sequences of species-specific primers designed using computer software is given in Table1.2.5.Standardization of PCR procedures and sequencing of PCR productsPCR chemistry conditions like template concentration,primer concentration and PCR amplification conditions like denaturation,Table1Species-specific primers designed for the present study.S.No.Species Primer name Primer sequence Length base pair1Chicken GgD-For50-CTCGCCCTACTTGCCTTCC-30192GgD-Rev50-TAGGACGCAACGCAGGTGT-30193Duck ApD-For50-CACAATCTAAGCCTGGACAC-30204ApD-Rev50-TCGTCTTTAGTGCTGGAGTT-30205Pigeon ClCytb-For50–TTACTACTCGCCGCACATTACA-30226ClCytb-Rev50–TGAAGGGTGAAGAATCGTGTTA-30227Pig SsD-For50-TACTTCAGGACCATCTCACC-30208SsD-Rev50-TATTCAGATTGTGGGCGTAT-3020Table2Optimized PCR thermal profile of various primers of species-specific DNA markers. of DNA marker Species Denaturation temperatureand duration Annealing temperatureand durationExtension temperature anddurationNumberof cycles1GgD marker Chicken Initial denaturation of95°Cfor5min and95°C for30s 54°C for30s72°C for30s andfinalextension of72°C for5min302ApD marker Duck Initial denaturation of94°Cfor5min and94°C for45s 54°C for45s72°C for45s andfinalextension of72°C for7min303ClCytb marker Pigeon Initial denaturation of94°Cfor5min and94°C for30s 51°C for30s72°C for45s andfinalextension of72°C for5min304SsD marker Pig Initial denaturation of94°Cfor3min and94°C for30s 54°C for30s72°C for30s andfinalextension of72°C for3min30S.Haunshi et al./Meat Science83(2009)454–459455annealing and extension temperatures and their durations and number PCR cycles for each new pair of primers designed to amplify species-specific DNA markers for chicken,ducks,pigeon and pig were standardized initially.PCR reactions were carried out in a volume of25l l reaction mixture consisting of12.5l l Qiagen PCR master mix,50ng of genomic DNA,1.0l M of each for-ward and reverse primer.Optimized thermal profile of PCR assays were given in Table2.PCR reactions were carried out in program-mable Thermal Cycler(Applied Biosystems,Foster city,USA).After amplification PCR products were analyzed on1.75%agarose gel in 1X TAE buffer and bands were visualized under UV light after ethi-dium bromide staining(Sambrook&Russell,2001).The size of the PCR products was determined with the help of Kodak1D software.The PCR products from each species were purified using sample exonuclease shrimp alkaline phosphatase digestion method.Direct sequencing of purified PCR products was carried out using Big Dye terminator V3.1(ABI)chemistry in ABI prism3100genetic ana-lyzer to ascertain the specificity of primers.Sequencing of PCR products was carried out at Labindia Sequencing Facility,Gurgaon (India).3.ResultsThe species-specific primers designed for chicken,duck,and pigeon were initially subjected to basic local alignment search tool (/Blast.cgi)for sequence alignment in other species of poultry and livestock.It was observed that these primers were unique to the species of interest in our study.Spe-cies-specific marker sequences of mitochondria were initially tested with total DNA extracted from blood samples of each spe-cies and they successfully amplified unique fragments for chicken, duck,pigeon and pig.The size of species-specific markers was 256bp for chicken,292bp for ducks,401bp for pigeon and 835bp for pig(Fig.1).These markers were subsequently tested in different varieties such as Miri,Mizo-local and White Leghorn chicken,Khaki Campbell and Indian Runner breeds of ducks,domestic pigeons (Columba livia)and Khasi-local and Hampshire breeds of pig.The results were consistent across different varieties or breeds of each species.Meat species identification of chicken,duck,pigeon,and pig was further validated by checking them for cross amplification in other species such as cattle,sheep,goat,pig,rabbit,chicken,duck, pigeon and turkeys.It can be seen from Fig.2that the chicken pri-mer set(GgD-For/GgD-Rev)amplified successfully the256bp DNA fragment from chicken meat,where as no amplification products were obtained with DNA from cattle,sheep,goat,pig,rabbit,duck, pigeon and turkey meat samples.Similarly,DNA samples from duck meat successfully amplified with duck primer set(ApD-For/ ApD-Rev),generating a specific PCR product of292bp unique to this species(Fig.3).Likewise,pigeon primers(ClCytb-For/ClCytb-Rev)amplified a unique401bp product from pigeon meat while not amplifying any DNA fragment from other meat species (Fig.4).Finally,as expected the(SsD-For/SsD-Rev)primer pair amplified a fragment of835bp from pork samples without show-ing any amplification with the DNA from rest of the meat species analyzed(Fig.5).It was observed that all the four markers were highly specific for the species to which they weredesigned.Fig.1.PCR amplification of chicken,duck,pigeon(cytochrome b)and pig specificD-loop region of mitochondrial DNA nes M are molecular size marker(100bp ladder),lane1is chicken,lane2is duck,lane3is pigeon and lane4is a pigspecificmarker.Fig. 2.PCR amplification of species-specific marker from chicken and crosschecking with other meat nes M are MS marker,lane1is cattle,lane2is sheep,lane3is goat,lane4is pig,lane5is rabbit,lane6is chicken,lane7is duck,lane8is turkey and lane9is pigeon and lane B isblank.Fig.3.PCR amplification of species-specific marker from duck and cross checkingwith other meat nes M are MS marker,lane1is cattle,lane2is sheep,lane3is goat,lane4is pig,lane5is rabbit,lane6is duck,lane7is chicken,lane8isturkey and lane9is pigeon and lane B isblank.Fig.4.PCR amplification of species-specific marker from pigeon and cross checkingwith other meat nes M are MS marker,lane1is cattle,lane2is sheep,lane3is goat,lane4is pig,lane5is rabbit,lane6is pigeon,lane7is chicken,lane8is duck,lane9is turkey and lane B is blank.456S.Haunshi et al./Meat Science83(2009)454–459The meat samples of chicken,duck,pigeon and pig were ob-tained randomly and heat treated at 80°C with a holding period of 30min or autoclaved.All markers could be successfully ampli-fied from the fresh,heated (80°C)and autoclaved meat samples (Figs.6–8)except for the pig specific marker,which could not be amplified from the autoclaved meat samples (Fig.9).Thespecies-specific PCR products from chicken,duck,pigeon and pig,were sequenced to confirm the specificity of the products ampli-fied and these partial sequences were deposited in Genbank (Table 3).It was observed in the present study that for some markers such as chicken,pigeon and ducks as little as 25number of PCR cycles with denaturation,annealing and extension duration of 20s each was enough to amplify the species-specific markers (figures not shown).4.DiscussionIn this study we have utilized the D-loop portion of mitochon-drial genome to design species-specific markers for chicken duck and pig and cytochrome b gene for pigeon.Since D-loop region is a hyper variable region of mitochondrial DNA and hence it is possible to select the sequences,which are specific toparticularFig. 6.PCR amplification of species-specific marker from fresh,cooked and autoclaved chicken meat nes M are MS marker,lane 1is fresh meat sample,lane 2is heated sample (80°C)with a holding period of 30min,lane 3is autoclaved sample (121°C)and lane 4isblank.Fig.7.PCR amplification of species-specific marker from fresh and cooked duck meat nes M are MS marker,lane 1is fresh meat,lane 2is heated sample (80°C)with a holding period of 30min,lane 3is autoclaved sample (121°C)and lane 4isblank.Fig.8.PCR amplification of species-specific marker from fresh,cooked and autoclaved meat of nes M are MS marker,lane 1is fresh meat sample,lane 2is heated sample (80°C)with a holding period of 30min,lane 3is autoclaved sample (121°C)and lane 4isblank.Fig.9.PCR amplification of species-specific marker from fresh and cooked pork nes M are MS marker,lane 1is fresh pork sample,lane 2is heated sample (80°C)with holding period of 30min,lane 3is autoclaved sample (120°C)and lane 4isblank.Fig.5.PCR amplification of species-specific marker from pig and cross checking with other meat nes M are MS marker,lane 1is cattle,lane 2is sheep,lane 3is goat,lane 4is rabbit,lane 5is pig,lane 6is chicken,lane 7is duck,lane 8is turkey,lane 9is pigeon and lane B is blank.S.Haunshi et al./Meat Science 83(2009)454–459457species.Besides,mitochondrial DNA is maternally inherited so nor-mally only one allele exists in an individual and thus no sequence ambiguities are to be expected from the presence of more than one allele(Unseld,Beyermann,Brandt,&Hiesel,1995).The chicken specific primers of D-loop control region of mito-chondrial genome used in our study are conserved in all four spe-cies of jungle fowls namely red,grey,Ceylon and green jungle fowls including domestic chicken,further,mitochondrial DNA of domestic chicken is originated from red jungle fowl therefore,it is possible to identify the species of domestic chicken irrespective of its genetic origin[monophyletic(red jungle fowl)or polyphy-letic(Eriksson et al.,2008)].For pigeon species,since we could notfind reliable primers to amplify the D-loop region of mitochon-dria we went with the sequence of cytochrome b gene that is spe-cific to the pigeons.We have tested these markers in several samples and the re-sults were consistent and reproducible across the different samples tested.Cooking of meat samples at80°C with a holding period of 30min did not have any adverse effects on amplification of spe-cies-specific markers from chicken,duck,pigeon and pig.Further, even autoclaving of meat samples at121°C temperature and 15psi pressure for15–20min duration did not affect the PCR amplifications of chicken,duck and pigeon specific markers(Figs. 6–8).Thisfinding is in line with the established fact that the heat stability and large copy number of mitochondrial DNA in meat tis-sue contribute to the protection and survivability of the fragments of DNA that are sufficient enough to be amplified by PCR(Girish et al.,2004).On an average800–1000mitochondria are present in an animal cell and each mitochondrion will have6–8copies of mitochondrial genome.Hence,chances of survival of copies of mitochondrial genome during extreme conditions of heating,pro-cessing and storage are very high.Further,it was proved that mito-chondrial markers were more efficient than nuclear markers(RAPD finger printing and Actin gene barcoding)in species identification and authentication purposes(Rastogi et al.,2007).Therefore,these characteristics make the D-loop region or any other genes of mito-chondrial origin ideal for identification of species origin of fresh, cooked and autoclaved meat and meat products.Other workers also suggested that mitochondrial markers are more efficient than nuclear markers for the purpose of identification and authentica-tion meat species(Hopwood,Fairbrother,Lockley,&Bardsley, 1999).Murphy et al.(2007)have developed the cloth based hybridiza-tion array system for detection of species origin of meat meals in feed grains by amplifying mitochondrial sequences and subse-quent probing with species-specific oligonucleotide capture probes embedded on polyester cloth,followed by immunoenzymatic assay of the bound PCR products.This method was sensitive and permitted precise detection of meat meals in animal feeds. However,the process offixing the probes on polyester cloth, hybridization with amplified probe and subsequent immunoenzy-matic assay of the bound PCR products is laborious,technically demanding and expensive as compared to the species-specific PCR markers.Spectroscopy methods coupled with chemometrics analysis of protein of meat offer economical,quick,and reliable methods for meat species authentication(Arvanitoyannis&Van Houwelingen-Koukaliaroglou,2003)and they have been used to authenticate the beef samples using mid infrared spectroscopy with a chemometric technique of soft independent modelling of class analogy(SIMCA) analysis(Al-Jowder et al.,1999)and meat of pig breeds using visible and near infrared spectroscopy with SIMCA chemometrics(Cozzoli-no et al.,2006).However,they cannot be used for authentication of cooked or thermal processed meat samples due to denaturation of muscle proteins in high temperature processing.The effect of heat treatment of meat on successful amplification of species-specific markers was tested through PCR assay of DNA extracted from fresh,cooked and autoclaved meat samples.Aga-rose gel electrophoresis of total DNA extracted after heat treatment of meat samples revealed typical smearing of disintegrated DNA due to thermal processing(figures not shown).On the other hand DNA of blood samples and fresh meat samples seem to be intact and was of high molecular weight.Nonetheless,our PCR results indicate successful amplification of the desired DNA segments using DNA extracted from fresh and thermal processed meat sam-ples,confirming the ability of the PCR protocol to amplify the spe-cies-specific markers of mitochondrial origin.It was seen in the present study that pig specific marker could not be amplified using the DNA extracted from autoclaved meat samples(Fig.9).It was probably due to the larger size of the mar-ker(835bp),which might have been disintegrated during auto-claving process.Thisfinding reiterated the fact that for meat speciation by species-specific PCR,lesser the size of the marker better is the chance of its amplification from autoclaved or pro-cessed meat products(Arslan,Ilhak,&Calicioglu,2006).Therefore,the DNA markers developed in this study could be useful in identifying the species origin of fresh,cooked,as well as autoclaved meat samples of the species mentioned above and the process of identification is simple,economical and quick as com-pared to any other method such as RAPD-PCR,hybridization by species-specific probes,sequencing analysis of nuclear or mito-chondrial genes,or PCR-RFLP method of species identification. The proposed PCR assay represents a rapid,straightforward,and economical method for possible detection of misrepresentations in meat and meat products in high throughput manner.AcknowledgementsAuthors would like to sincerely thank the Director of the Insti-tute for providingfinancial assistance to carry out this b-oratory facilities provided from Veterinary Parasitology and Plant Biotechnology divisions of our Institute to carry out this study is gratefully acknowledged.ReferencesAl-Jowder,O.,Defernez,M.,Kemsley,E.K.,&Wilson,R.H.(1999).Mid-infrared spectroscopy and chemometrics for the authentication of meat products.Journal of Agricultural Food Chemistry,47(8),3210–3218.Table3Details of mitochondrial D-loop sequence used in the present study.S.No.Species Common name Breed/variety Accession No.1Gallus gallus Chicken Mizo-local FM2075612Gallus gallus Chicken White Leghorn FM2075603Gallus gallus Chicken Miri FM2075624Sus scrofa Pig Hampshire FM2075595Sus scrofa Pig Khasi-local FM2075586Anas platyrhynchos Duck Indian Runner FM2094947Anas platyrhynchos Duck Khaki Campbell FM2094958Columba livia(cytochrome b gene)Domestic pigeon Meghalaya-local FM207563 458S.Haunshi et al./Meat Science83(2009)454–459Arslan,A.,Ilhak,O.I.,&Calicioglu,M.(2006).Effect of method of cooking on identification of heat processed beef using polymerase chain reaction(PCR) technique.Meat Science,72,326–330.Arvanitoyannis,I.S.,&Van Houwelingen-Koukaliaroglou,M.(2003).Implementation of chemometrics for quality control and authentication of meat and meat products.Critical Reviews in Food Science and Nutrition,43(2), 173–218.Buntjer,J.B.,Nijman,I.J.,Zijlstra,C.,&Lenstra,J.A.(1998).A satellite DNA element specific for roe deer(Capreolus capreolus).Chromosoma,107(1),1–5.Calvo,J.H.,Zaragoza,P.,&Osta,R.(2001).Random amplified polymorphic DNA fingerprints for identification of species in poultry pate.Poultry Science,80, 522–524.Chikuni,K.,Ozustume,K.,Hoishi Kawa,T.,&Kato,S.(1990).Species identification of cooked meats by DNA hybridization assay.Meat Science, 27,199–208.Cozzolino,D.,Vadell,A.,Ballesteros,F.,Galietta,G.,&Barlocco,N.(2006).Combining visible and near-infrared spectroscopy with chemometrics to trace muscles from an autochthonous breed of pig produced in Uruguay:A feasibility study.Analytical Bioanalytical Chemistry,385,931–936.Eriksson,J.,Larson,G.,Gunnarsson,U.,Bed’hom,B.,Tixier-Boichard,M.,Strömstedt, L.,et al.(2008).Identification of the yellow skin gene reveals a hybrid origin of the domestic chicken.PLoS Genetics,4(2),e1000010.doi:10.1371/ journal.pgen.1000010.Forrest,A.R.R.,&Carnegie,P.R.(1994).Identification of gourmet using FINS (forensically informative nucleotide sequencing).Biotechniques,17(1),24–26. Girish,P.S.,Anjaneyulu,A.S.R.,Viswas,K.N.,Anand,M.,Rajkumar,N.,Shivakumar,B.M.,et al.(2004).Sequence analysis of mitochondrial12S rRNA gene canidentify meat species.Meat Science,66,551–556.Girish,P.S.,Anjaneyulu,A.S.R.,Viswas,K.N.,Shivakumar,B.M.,Anand,M.,Patel, M.,et al.(2005).Meat species identification by polymerase chain reaction–restriction fragment length polymorphism of mitochondrial12S rRNA gene.Meat Science,70,107–112.Girish,P.S.,Anjeneyulu,A.S.R.,Viswas,K.N.,Santosh,F.H.,Bhilegaonkar,K.N., Agarwal,R.K.,et al.(2007).Polymerase chain reaction–restriction fragment length polymorphism of mitochondrial12S r RNA gene:A simple method for identification of poultry meat species.Veterinary Research Communications, 31(4),447–455.Hopwood,A.J.,Fairbrother,K.S.,Lockley,A.K.,&Bardsley,R.G.(1999).An actin gene-related polymerase chain reaction test for identification of chicken in meat mixtures.Meat Science,53,227–231.Ilhak,O.I.,&Arslan,A.(2007).Identification of meat species by polymerase chain reaction(PCR)technique.Turkish Journal of Veterinary and Animal Science,31, 159–163.Koh,M.C.,Lim,C.H.,Chua,S.B.,Chew,S.T.,&Phang,S.T.W.(1998).Random amplified polymorphic DNA(RAPD)fingerprints for identification of red meat animal species.Meat Science,48,275–285.Lee,J.C.,&Chang,J.G.(1994).Random amplified polymorphic DNA polymerase chain reaction(RAPD-PCR)fingerprints in forensic species identification.Forensic Science International,67,103–107.Mane,B.G.,Mendiratta,S.K.,Tiwari,A.K.,Bhilegaokar,K.N.,Ravindra,P.V.,&Raut,A.A.(2007).Precise and rapid identification of ovine–caprine origin of meat byspecific PCR assay.Journal of Veterinary Public Health,5(2),107–110.Meyer,R.,Hofelein,C.,Luthy,J.,&Candrian,U.(1995).Polymerase chain reaction restriction fragment length polymorphism analysis:A simple method for species identification in food.Journal of Association of Official Analytical Chemistry,78,1542–1551.Murphy,J.,Armour,J.,&Blais, B.W.(2007).Research note:Cloth-based hybridization array system for expanded identification of the animal species origin of derived materials in feeds.Journal of Food Protection,70(12), 2900–2905.Prakash,S.,Patole,M.S.,Ghumatkar,S.V.,Nandode,S.K.,&Yogesh,S.S.(2000).Mitochondrial12S rRNA sequence analysis in wild life forensics.Current Science, 8,1239–1241.Rastogi,G.R.,Dharne,M.S.,Walujkar,S.,Kumar,A.,Patole,M.S.,&Shouche,Y.S.(2007).Species identification and authentication of tissues of animal origin using mitochondrial and nuclear markers.Meat Science,76(4),666–674. Sambrook,J.,&Russell,D.W.(2001).Molecular cloning–A laboratory manual(3rd ed.).New York:Cold Spring Harbour Laboratory Press.Unseld,M.,Beyermann,B.,Brandt,P.,&Hiesel,R.(1995).Identification of the species origin of highly processed meat products by mitochondrial DNA sequences.PCR Methods and Applications,4,241–243.Verkaar,E.L.C.,Nijman,I.J.,Boutaga,K.,&Lenstra,J.A.(2002).Differentiation of cattle species in beef by PCR-RFLP of mitochondrial and satellite DNA.Meat Science,60(4),365–369.S.Haunshi et al./Meat Science83(2009)454–459459。