Meat Species Identification: Amplification Refractory Mutation System-Polymerase Chain Reaction–Based Assay
- 19 Downloads
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.
KeywordsACTB 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.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
- 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. https://doi.org/10.1007/s12161-011-9311-4 CrossRefGoogle Scholar
- 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. https://doi.org/10.1136/jclinpath-2019-205699
- 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. https://doi.org/10.14202/vetworld.2016.226-230
- 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. https://doi.org/10.2527/2001.7982108x
- 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. https://doi.org/10.1371/journal.pone.0139688
- 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. https://doi.org/10.1007/s12033-016-9984-4 CrossRefGoogle Scholar
- Garg N (2008) PCR Primer Design: DREB Genes. J Comput Sci Syst Biol 01. https://doi.org/10.4172/jcsb.1000002
- 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. https://doi.org/10.1016/S0309-1740(03)00158-X
- 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. https://doi.org/10.1016/j.foodchem.2016.12.062 CrossRefGoogle Scholar
- 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. https://doi.org/10.1371/journal.pone.0191558 CrossRefGoogle Scholar
- 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. https://doi.org/10.1371/journal.pone.0041256
- 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 https://doi.org/10.1016/j.jviromet.2004.01.001
- 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. https://doi.org/10.1515/pjfns-2015-0051 CrossRefGoogle Scholar