Journal of Food and Nutrition Research
ISSN (Print): 2333-1119 ISSN (Online): 2333-1240 Website: http://www.sciepub.com/journal/jfnr Editor-in-chief: Prabhat Kumar Mandal
Open Access
Journal Browser
Go
Journal of Food and Nutrition Research. 2015, 3(6), 392-398
DOI: 10.12691/jfnr-3-6-6
Open AccessArticle

Application of Quadruple Multiplex PCR Detection for Beef, Duck, Mutton and Pork in Mixed Meat

Haining He1, Xia Hong1, Yusheng Feng1, Yongsong Wang1, Jin Ying1, Qi Liu1, Yingwen Qian1, Xinkui Zhou1 and Dongshuai Wang1

1Institute of Business and Technology, Lianchuang square of high-tech zones, No. 449, Lanzhou, Gansu, P. R. China

Pub. Date: July 06, 2015

Cite this paper:
Haining He, Xia Hong, Yusheng Feng, Yongsong Wang, Jin Ying, Qi Liu, Yingwen Qian, Xinkui Zhou and Dongshuai Wang. Application of Quadruple Multiplex PCR Detection for Beef, Duck, Mutton and Pork in Mixed Meat. Journal of Food and Nutrition Research. 2015; 3(6):392-398. doi: 10.12691/jfnr-3-6-6

Abstract

In this study, we have developed quadruple multiplex PCR assay for meat (beef, duck, mutton and pork) in processed meat products. By mixing four primers in appropriate ratios could be identified by the PCR. A forward primer was designed on a conserved DNA sequence in the mitochondrial ND2 and 16S rDNA gene in sheep and duck genes, and reverse primers on species-specific DNA sequences for each species. PCR primers were designed to give different length fragments from the four meats. The products showed species-specific DNA fragments of 116, 212, 177 and 322 bp from beef, pork, mutton and duck. Optimal PCR conditions were established. The assay sensitivity under these conditions was 0.1ng, and its specificity was 100 %. The results of the study suggest that PCR represents a simple, efficient test method as a practical alternative for the rapid detection and identification of meat.

Keywords:
multiplex PCR; beef; duck; mutton; pork; identification

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References:

[1]  P.D. Brodmann, D. Moor, Sensitive and semi-quantitative TaqMan real-time polymerase chain reaction systems for the detection of beef (Bos taurus) and the detection of the family Mammalia in food and feed. Meat Science, 65(1), 599-607, 2003.
 
[2]  WANG Jian-chang, WANG Jin-feng, CHEN Rui-chun, TIAN Zhen-xiang, Molecular Biological Detection of Adulteration of Mutton and Beef with Duck. MEAT RESEARCH, 26(6), 20-23, 2012.
 
[3]  M.E. Ali, U. Hashim, S. Mustafa, Y.B.C. Man, T.S. Dhahi, M. Kashif, M.K. Uddin, S.B.A. Hamid, Analysis of pork adulteration in commercial meatballs targeting porcine-specific mitochondrial cytochrome b gene by TaqMan probe real-time polymerase chain reaction. Meat Science, 91 454-459, 2012. (Chinese).
 
[4]  XU Yuan-Cong , DONG Kai , HUANG Kun-Lun , XU Wen-Tao, PCR on Adulteration Detection of Pork, Beef and Mutton. Journal of Agricultural Biotechnology, 21(12), 1504-1508, 2013. (Chinese).
 
[5]  J.J. Dooley, K.E. Paine, S.D. Garrett, H.M. Brown, Detection of meat species using TaqMan real-time PCR assays. Meat Science, 68, 431-438, 2004.
 
[6]  M.A. Rodriguez, T. Garcia, I. Gonzalez, P.E. Hernandez, R. Martin, TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures. Meat Science, 70, 113-120, 2005.
 
[7]  M. Montowska, E. Pospiech, Species-specific expression of various proteins in meat tissue: proteomic analysis of raw and cooked meat and meat products made from beef, pork and selected poultry species. Food Chemistry, 136, 1461-1469, 2013.
 
[8]  J.H. Calvo, P. Zaragoza, R. Osta, 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. Journal of Animal Science, 79, 2108-2112, 2001.
 
[9]  P.S. Girish, A.S. Anjaneyulu, K.N. Viswas, M. Anand, N. Rajkumar, B.M. Shivakumar, S. Bhaskar, Sequence analysis of mitochondrial 12S rRNA gene can identify meat species. Meat Science, 66, 551-556, 2004.
 
[10]  P. Ulca, H. Balta, I. Cagin, H.Z. Senyuva, Meat species identification and Halal authentication using PCR analysis of raw and cooked traditional Turkish foods. Meat Science, 94, 280-284, 2013.
 
[11]  F. Bellagamba, F. Valfre, S. Panseri, V.M. Moretti, Polymerase chain reaction-based analysis to detect terrestrial animal protein in fish meal. J Food Prot, 66 , 682-685, 2003.
 
[12]  A. Arslan, O.I. Ilhak, M. Calicioglu, Effect of method of cooking on identification of heat processed beef using polymerase chain reaction (PCR) technique. Meat Science, 72, 326-330, 2006.
 
[13]  S. Manzano, B. Bailey, J.B. Girodias, J. Cousineau, E. Delvin, A. Gervaix, Comparison of procalcitonin measurement by a semi-quantitative method and an ultra-sensitive quantitative method in a pediatric emergency department. Clinical Biochemistry, 42, 1557-1560, 2009.
 
[14]  J.A. Loo, Statistics of Chi-square: X2. Journal of Statistics, 63, 2488-2499, 1991.
 
[15]  I. Pfeiffer, J. Burger, B. Brenig, Diagnostic polymorphisms in the mitochondrial cytochrome b gene allow discrimination between cattle, sheep, goat, roe buck and deer by PCR-RFLP. BMC Genetics, 5(30), 1-5, 2004.
 
[16]  C. Murugaiah, Z.M. Noor, M. Mastakim, L.M. Bilung, J. Selamat, S. Radu, Meat species identification and Halal authentication analysis using mitochondrial DNA. Meat Science, 83, 57-61, 2009.
 
[17]  P.S. Girish, A.S. Anjaneyulu, K.N. Viswas, B.M. Shivakumar, M. Anand, M. Patel, B. Sharma, Meat species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of mitochondrial 12S rRNA gene. Meat Science, 70, 107-112, 2005.
 
[18]  A. Macedo-Silva, S.F. Barbosa, M.G. Alkmin, A.J. Vaz, M. Shimokomaki, A. Tenuta-Filho, Hamburger meat identification by dot-ELISA. Meat Science, 56, 189-192, 2000.
 
[19]  M. Safdar, M.F. Abasiyanik, Simultaneous identification of pork and poultry origins in pet foods by a quick multiplex real-time PCR assay using EvaGreen florescence dye. Appl Biochem Biotechnol, 171, 1855-1864, 2013.
 
[20]  L. Cuttell, H. Owen, A.E. Lew-Tabor, R.J. Traub, Bovine cysticercosis--development of a real-time PCR to enhance classification of suspect cysts identified at meat inspection. Veterinary Parasitology, 194, 65-69, 2013.
 
[21]  S. Soares, J.S. Amaral, M.B. Oliveira, I. Mafra, A SYBR Green real-time PCR assay to detect and quantify pork meat in processed poultry meat products. Meat Science, 94, 115-120, 2013.
 
[22]  J.J. Dooley, K.E. Paine, S.D. Garrett, H.M. Brown, Detection of meat species using TaqMan real-time PCR assays. Meat Science, 68, 431-438, 2004.
 
[23]  F. Bellagamba, S. Comincini, L. Ferretti, F. Valfre, V.M. Moretti, Application of quantitative real-time PCR in the detection of prion-protein gene species-specific DNA sequences in animal meals and feedstuffs. J Food Prot, 69, 891-896, 2006.
 
[24]  M. Rojas, I. Gonzalez, M.A. Pavon, N. Pegels, A. Lago, P.E. Hernandez, T. Garcia, R. Martin, Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds. Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 27, 749-763, 2010.
 
[25]  Z. Kesmen, A.E. Yetiman, F. Sahin, H. Yetim, Detection of chicken and turkey meat in meat mixtures by using real-time PCR assays. Journal of Food Science, 77, 167-173, 2012.
 
[26]  A. Rodriguez, M. Rodriguez, M.I. Luque, A. Martin, J.J. Cordoba, Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods. Food Microbiology, 31, 89-99, 2012.
 
[27]  A. Abdulmawjood, N. Grabowski, S. Fohler, S. Kittler, H. Nagengast, G. Klein, Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Sensitive Identification of Ostrich Meat. PLoS One, 9(6), e100717. 2014.
 
[28]  T. Kitpipit, K. Sittichan, P. Thanakiatkrai, Direct-multiplex PCR assay for meat species identification in food products. Food Chemistry, 163, 77-82, 2014.
 
[29]  A. Dalmasso, E. Fontanella, P. Piatti, T. Civera, S. Rosati, M.T. Bottero, A multiplex PCR assay for the identification of animal species in feedstuffs. Mol Cell Probes, 18, 81-87, 2004.
 
[30]  I. Martin, T. Garcia, V. Fajardo, I. Lopez-Calleja, P.E. Hernandez, I. Gonzalez, R. Martin, Species-specific PCR for the identification of ruminant species in feedstuffs. Meat Science, 75, 120-127, 2007.
 
[31]  A. Dalmasso, E. Fontanella, P. Piatti, T. Civera, S. Rosati, M.T. Bottero, A multiplex PCR assay for the identification of animal species in feedstuffs. Mol Cell Probes, 18, 81-87, 2004.
 
[32]  A. Abdulmawjood, C. Krischek, M. Wicke, G. Klein, Determination of pig sex in meat and meat products using multiplex real time-PCR. Meat Science, 91, 272-276, 2012.
 
[33]  Novel universal primers establish identity of an enormous number of animal species for forensic application. Molecular Ecology Notes, 3, 28-31, 2003.
 
[34]  J.J. Dooley, K.E. Paine, S.D. Garrett, H.M. Brown, Detection of meat species using TaqMan real-time PCR assays. Meat Science, 68, 431-438, 2004.
 
[35]  T. Matsunaga, K. Chikuni, R. Tanabe, S. Muroya, K. Shibata, J. Yamada, Y. Shinmura, A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Science, 51, 143-148, 1999.
 
[36]  K.F. Ebbehoj, P.D. Thomsen, Species differentiation of heated meat products by DNA hybridization. Meat Science, 30, 221-234, 1991.
 
[37]  K.F. Ebbehoj, P.D. Thomsen, Differentiation of closely related species by DNA hybridization. Meat Science, 30, 359-366, 1991.
 
[38]  GAO Dan- dan, CHEN Yan, WANG Ying- hua, CAO Yu- sheng, Extraction and purification of genomic DNA in the animal origin food. FOOD SCIENCE AND TECHNOLOGY, 8, 42-44, 2007. (Chinese).
 
[39]  O. Aslan, R.M. Hamill, T. Sweeney, W. Reardon, A.M. Mullen, Integrity of nuclear genomic deoxyribonucleic acid in cooked meat: Implications for food traceability. Journal of Animal Science, 87, 57-61, 2009.
 
[40]  T. Matsunaga, K. Chikuni, R. Tanabe, S. Muroya, K. Shibata, J. Yamada, Y. Shinmura, A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Science, 51, 143-148, 1999.
 
[41]  A. Arslan, O.I. Ilhak, M. Calicioglu, Effect of method of cooking on identification of heat processed beef using polymerase chain reaction (PCR) technique. Meat Science, 72, 326-330, 2006.
 
[42]  Z. Kesmen, A. Gulluce, F. Sahin, H. Yetim, Identification of meat species by TaqMan-based real-time PCR assay. Meat Science, 82, 444-449, 2009.
 
[43]  B.L. Herman, Determination of the animal origin of raw food by species-specific PCR. Journal of Dairy Research, 68, 429-436, 2001.
 
[44]  Z. Kesmen, F. Sahin, H. Yetim, PCR assay for the identification of animal species in cooked sausages. Meat Science, 77, 649-653, 2007.
 
[45]  N.S. Karabasanavar, S.P. Singh, D. Kumar, S.N. Shebannavar, Detection of pork adulteration by highly-specific PCR assay of mitochondrial D-loop. Food Chemistry, 145, 530-534, 2014.
 
[46]  J.S. Amaral, C.G. Santos, V.S. Melo, M.B.P.P. Oliveira, I. Mafra, Authentication of a traditional game meat sausage (Alheira) by species-specific PCR assays to detect hare, rabbit, red deer, pork and cow meats. Food Research International, 140-145, 2014.
 
[47]  X. Zhang, Z. Liu, J. Yang, Z. Chen, G. Guan, Q. Ren, A. Liu, J. Luo, H. Yin, Y. Li, Multiplex PCR for diagnosis of Theileria uilenbergi, Theileria luwenshuni, and Theileria ovis in small ruminants. Parasitology Research, 113, 527-531, 2014.
 
[48]  G. Rastogi, M.S. Dharne, S. Walujkar, A. Kumar, M.S. Patole, Y.S. Shouche, Species identification and authentication of tissues of animal origin using mitochondrial and nuclear markers. Meat Science, 76, 666-674, 2007.
 
[49]  M. Weissenberger, W. Reichert, R. Mattern, A Multiplex PCR assay to differentiate between dog and red fox. Forensic Sci Int Genet, 5, 411-414, 2011.
 
[50]  D. Sint, L. Raso, M. Traugott, Advances in multiplex PCR: balancing primer efficiencies and improving detection success. Methods in Ecology and Evolution, 3, 898-905, 2012.