Meat Species Identification: Amplification Refractory Mutation System-Polymerase Chain Reaction–Based Assay

  • Muhammad WaqasEmail author
  • Zahid Hussain
  • Awais Ihsan


True verification of meat species is critical for religious, economical, legal, and/or public health concerns. Current methods available for verification of meat species are very time-consuming and/or costly. So considering the necessity, a novel polymerase chain reaction assay (PCR)–based ARMS (amplification refractory mutation system) type identification assay for detection of 6 most common mammalian species—Equus asinus (donkey), Equus caballus (horse), Bos Taurus (cow), Bubalus bubalis (buffalo), Capra hircus (goat), and Canis lupus familiaris (dog)—was described in present study by designing species-specific forward primers against variable regions and a single universal reverse primer against a very conserved region of nuclear beta actin (ACTB) gene by investigation of areas of homology and variation. As compared with conventional designing of primers, ARMS type primer designing is a better alternative as it allows more room for choice and offers differentiation of closely related species by exploiting just a single nucleotide base difference. PCR bands of 128, 229, 273, 362, 710, and 796 bp were generated on electrophoretic gel for buffalo, donkey, cow, horse, dog, and goat meat species respectively. Besides singleplexing, duplex (multiplex) PCR for donkey and horse, donkey and goat, and donkey and buffalo were also performed which successfully generated corresponding bands. The method is a simple and straightforward setup; results can be interpreted easily in a short time and do not need validation by sequencing. Results of the present study clearly demonstrate that the method can be used as an identification tool for differentiation between cow, buffalo, goat, donkey, horse, and dog species.


ACTB gene (beta actin) ARMS (amplification refractory mutation system) PCR (polymerase chain reaction) Meat adulteration Meat authenticity Species identification 



The authors would like to thank Higher Education Commission (HEC) Pakistan, GCU Lahore Pakistan and COMSATS Institute of Information Technology Sahiwal for financial and experimental support. We would also like to express special thanks to Dr. Sumaira Kousar (GCWU, Faisalabad) and Dr. Waqar Anjum (UVAS, Lahore, Pakistan) for their cooperation and support in providing samples for the research.

Compliance with Ethical Standards

Conflict of Interest

Muhammad Waqas declares that there is no conflict of interest. Zahid Hussain declares that there is no conflict of interest. Awais Ihsan declares that there is no conflict of interest.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Informed Consent

Not applicable.


  1. Ali ME, Hashim U, Dhahi TS, Mustafa S, Man YBC, Latif MA (2012) Analysis of pork adulteration in commercial burgers targeting porcine-specific mitochondrial cytochrome B gene by TaqMan probe real-time polymerase chain reaction. Food Anal Methods 5:784–794. CrossRefGoogle Scholar
  2. Ali ME, Razzak MA, Hamid SBA, Rahman MM, Amin MA, Rashid NRA, Asing (2015) Multiplex PCR assay for the detection of five meat species forbidden in Islamic foods. Food Chem 177:214–224. CrossRefGoogle Scholar
  3. Bai H, Xia J, Zhao X et al (2019) Detection of EGFR mutations using target capture sequencing in plasma of patients with non-small-cell lung cancer. J Clin Pathol 72(5):379–385.
  4. Bhat MM, Salahuddin M, Mantoo IA, et al (2016) Species-specific identification of adulteration in cooked mutton Rista ( a Kashmiri Wazwan cuisine product ) with beef and buffalo meat through multiplex polymerase chain reaction 9:226–230.
  5. Cai Y, Wang Q, He Y, Pan L (2017) Interlaboratory validation of a real-time PCR detection method for bovine- and ovine-derived material. Meat Sci 134:119–123. CrossRefGoogle Scholar
  6. Calvo JH, Zaragoza P, Osta R, De Gene L (2001) Technical note: a quick and more sensitive method to identify pork in processed and unprocessed food by PCR amplification of a new specific DNA fragment. J Anim Sci 79:2108–2112.
  7. Castigliego L, Armani A, Li X, Grifoni G, Gianfaldoni D, Guidi A (2010) Selecting reference genes in the white blood cells of buffalos treated with recombinant growth hormone. Anal Biochem 403:120–122. CrossRefGoogle Scholar
  8. Cheng J, Chou H, Lee M, Sheu S (2016) Development of qualitative and quantitative PCR analysis for meat adulteration from RNA samples. Food Chem 192:336–342. CrossRefGoogle Scholar
  9. Cieslak J, Mackowski M, Czyzak-Runowska G, et al (2015) Screening for the most suitable reference genes for gene expression studies in equine milk somatic cells. PLoS One 10:1–11.
  10. Chikuni K, Ozutsumi K, Koishikawa T, Kato S (1990) Species identification of cooked meats by DNA hybridization assay. Meat Sci 27:119–128. CrossRefGoogle Scholar
  11. Deb R, Sengar GS, Singh U, Kumar S, Alyethodi RR, Alex R, Raja TV, Das AK, Prakash B (2016) Application of a loop-mediated isothermal amplification assay for rapid detection of cow components adulterated in buffalo milk/meat. Mol Biotechnol 58:850–860. CrossRefGoogle Scholar
  12. Garg N (2008) PCR Primer Design: DREB Genes. J Comput Sci Syst Biol 01.
  13. Ghatak S, Muthukumaran RB, Nachimuthu SK (2013) A simple method of genomic DNA extraction from human samples for PCR-RFLP analysis. J Biomol Tech 24:224–231. Google Scholar
  14. Girish PS, Anjaneyulu ASR, Viswas KN et al (2004) Sequence analysis of mitochondrial 12S rRNA gene can identify meat species. Meat Sci 66:551–556.
  15. Gunning PW, Ghoshdastider U, Whitaker S, Popp D, Robinson RC (2015) The evolution of compositionally and functionally distinct actin filaments. J Cell Sci 128:2009–2019. CrossRefGoogle Scholar
  16. Haider N, Nabulsi I, Al-Safadi B (2012) Identification of meat species by PCR-RFLP of the mitochondrial COI gene. Meat Sci 90:490–493. CrossRefGoogle Scholar
  17. Hanukogle I, Tanese N, Fuchs E (1983) Complementary DNA sequence of a human cytoplasmic actin. Interspecies Divergence of 3′ non-coding regions. J Mol Biol 163:673–678. CrossRefGoogle Scholar
  18. Hossain MAM, Ali ME, Hamid SBA, Hossain SMA, Asing, Nizar NNA, Uddin MN, Ali L, Asaduzzaman M, Akanda MJH (2017) Tetraplex PCR assay involving double gene-sites discriminates beef and buffalo in Malaysian meat curry and burger products. Food Chem 224:97–104. CrossRefGoogle Scholar
  19. Iwobi A, Sebah D, Kraemer I, Losher C, Fischer G, Busch U, Huber I (2015) A multiplex real-time PCR method for the quantification of beef and pork fractions in minced meat. Food Chem 169:305–313. CrossRefGoogle Scholar
  20. Kaur R, Sodhi M, Sharma A, Sharma VL, Verma P, Swami SK, Kumari P, Mukesh M (2018) Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (Bubalus bubalis). PLoS One 13:e0191558. CrossRefGoogle Scholar
  21. Kumar D, Singh SP, Karabasanavar NS, Singh R, Umapathi V (2012) Authentication of beef, carabeef, chevon, mutton and pork by a PCR-RFLP assay of mitochondrial cytb gene. J Food Sci Technol 51:3458–3463. CrossRefGoogle Scholar
  22. Murugaiah C, Noor ZM, Mastakim M, Bilung LM, Selamat J, Radu S (2009) Meat species identification and Halal authentication analysis using mitochondrial DNA. Meat Sci 83:57–61. CrossRefGoogle Scholar
  23. Park JE, Kim MJ, Ha SK, et al (2012) Altered cell cycle gene expression and apoptosis in post-implantation dog parthenotes. PLoS One 7:1–11.
  24. Pulford D, Meyer H, Brightwell G et al (2004) Amplification refractory mutation system PCR assays for the detection of variola and Orthopoxvirus. J Virol Methods 117:81–90
  25. Saez R, Sanz Y, Toldrá F (2004) PCR-based fingerprinting techniques for rapid detection of animal species in meat products. Meat Sci 66:659–665. CrossRefGoogle Scholar
  26. Safdar M, Junejo Y (2016) The development of a hexaplex-conventional PCR for identification of six animal and plant species in foodstuffs. Food Chem 192:745–749. CrossRefGoogle Scholar
  27. Song K, Hwang HJ, Kim JH (2017) Ultra-fast DNA-based multiplex convection PCR method for meat species identification with possible on-site applications. Food Chem 229:341–346. CrossRefGoogle Scholar
  28. Spychaj A, Szalata M, Słomski R, Pospiech E (2016) Identification of bovine, pig and duck meat species in mixtures and in meat products on the basis of the mtDNA cytochrome oxidase subunit I (COI) gene sequence. Polish J Food Nutr Sci 66:31–36. CrossRefGoogle Scholar
  29. Tobe SS, Linacre AMT (2008) A multiplex assay to identify 18 European mammal species from mixtures using the mitochondrial cytochrome b gene. Electrophoresis 29:340–347. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Industrial BiotechnologyGovernment College UniversityLahorePakistan
  2. 2.Department of BiosciencesCOMSATS Institute of Information TechnologySahiwalPakistan

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