Abstract
A rapid and sensitive multiplex PCR assay was developed for simultaneous identification of the adulteration ingredients of chicken, duck and pork in beef. Specific primers for the mitochondrial genes of Cyt b, CO III, ATPase subunit 8/6 and Cyt b of chicken, duck, pork, and beef, respectively, were adopted in the assay. DNA exaction from meat samples was carried out by using magnetic nanoparticles as rapid separation substrates. The multiplex PCR assay showed that the limit of detection was 0.05% for each species. Moreover, the multiplex PCR specifically identified five beef samples adulterated with pork and one beef samples adulterated with chicken among the 35 commercial samples examined, indicating the practicability of this multiplex PCR method for identifying adulterated ingredients of chicken, duck, and pork in commercial beef products.
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs13197-019-03591-2/MediaObjects/13197_2019_3591_Fig1_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs13197-019-03591-2/MediaObjects/13197_2019_3591_Fig2_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs13197-019-03591-2/MediaObjects/13197_2019_3591_Fig3_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs13197-019-03591-2/MediaObjects/13197_2019_3591_Fig4_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs13197-019-03591-2/MediaObjects/13197_2019_3591_Fig5_HTML.png)
Similar content being viewed by others
References
Blake RD, Delcourt SG (1998) Thermal stability of DNA. Nucleic Acids Res 26:3323–3332
Chinuki Y, Ishiwata K, Yamaji K, Takahashi H, Morita E (2015) Haemaphysalis longicornis, tick bites are a possible cause of red meat allergy in Japan. Allergy 71:421–425
Choi SJ, Hur GY, Shin SY, Park HS (2007) A case of adult onset cow’s milk allergy presenting beef and pork meat allergy. Korean J Asthma Allergy Clin Immunol 27(3):200–203
Das A, Spackman E, Thomas C, Swayne DE, Suarez DL (2008) Detection of H5N1 high-pathogenicity avian influenza virus in meat and tracheal samples from experimentally infected chickens. Avian Dis 52:40–48
Fajardo V, González I, Rojas M, García T, Martín R (2010) A review of current PCR-based methodologies for the authentication of meats from game animal species. Trends Food Sci Technol 21:408–421
Fan L, Li P, Ding H, ** P, Fu C (2013) Detection for bovine-derived ingredients in foods with real-time polymerase chain reaction method. Sci Technol Food Ind 34:65–67 (in Chinese)
Girish PS, Anjaneyulu AS, Viswas KN, Shivakumar BM, Anand M, Patel M, Sharma B (2005) Meat species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of mitochondrial 12S rRNA gene. Meat Sci 70:102–107
González-Mancebo E, Pastor C, González-de-Olano D, Gandolfo-Cano M, Melendez A, Cuesta J, Zapatero A, Bartolomé B (2011) Identification of allergens in chicken meat allergy. J Investig Allergol Clin Immunol 21:326–327
He H, Hong X, Feng Y, Wang Y, Ying J, Liu Q, Qian Y, Zhou X, Wang D (2015) Application of quadruple multiplex PCR detection for beef, duck, mutton and pork in mixed meat. J Food Nutr Res 3:392–398
Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH (1997) Multiplex PCR: critical parameter and step-by-step protocol. Biotechniques 23:504–511
Hitchcock CH, Crimes AA (1985) Methodology for meat species identification: a review. Meat Sci 15:215–224
Hou B, Meng X, Zhang L, Guo J, Li S, ** H (2015) Development of a sensitive and specific multiplex PCR method for the simultaneous detection of chicken, duck and goose DNA in meat products. Meat Sci 101:90–94
Huang L, Zheng L, Chen Y, Xue F, Cheng L, Adeloju SB, Chen W (2015) A novel GMO biosensor for rapid ultrasensitive and simultaneous detection of multiple DNA components in GMO products. Biosens Bioelectron 66:431–437
Izadpanah M, Mohebali N, Elyasi GZ, Farzaneh P, Vakhshiteh F, Shahzadeh Fazeli SA (2018) Simple and fast multiplex PCR method for detection of species origin in meat products. J Food Sci Technol 55:698–703
Karabasanavar NS, Singh SP, Kumar D, Shebannavar SN (2013) Development and application of highly specific PCR for detection of chicken (Gallus gallus) meat adulteration. Eur Food Res Technol 236:129–134
Kitpipit T, Sittichan K, Thanakiatkrai P (2014) Direct-multiplex PCR assay for meat species identification in food products. Food Chem 163:77–82
Li L, Xu J, Zhang Y, Wang Z (2004) Identification of components from animal speaies in meat and bone meals by PCR. Chin Anim Health Insp 21:29–31 (in Chinese)
Martinez I, Yman IM (1998) Species identification in meat products by RAPD analysis. Food Res Int 31:459–466
Matsunaga T, Chikuni K, Tanabe R, Muroyab S, Shibataa K, Yamadaa J, Shinmuraa Y (1999) A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Sci 51:143–148
Mutalib SA, Muin NM, Abdullah A, Muin M, Abdullah A, Hassan O, Mustapha WAW, Sani NA, Maskat MY (2015) Sensitivity of polymerase chain reaction (PCR)-southern hybridization and conventional PCR analysis for Halal authentication of gelatin capsules. LWT Food Sci Technol 63:714–719
Myers KP, Olsen CW, Gray GC (2007) Cases of swine influenza in humans: a review of the literature. Clin Infect Dis 44:1084–1088
Nakyinsige K, Man YB, Sazili AQ (2012) Halal authenticity issues in meat and meat products. Meat Sci 91:207–214
Rojas M, González I, Pavón MÁ, Pegels N, Hernández PE, García T, Martín R (2011) Development of a real-time PCR assay to control the illegal trade of meat from protected capercaillie species (Tetrao urogallus). Forens Sci Int 210:133–138
Safdar M, Abasıyanık MF (2013) 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
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
Safdar M, Junejo Y, Arman K, Abasıyanık MF (2014) A highly sensitive and specific tetraplex PCR assay for soybean, poultry, horse and pork species identification in sausages: development and validation. Meat Sci 98:296–300
Sun YH, Kim SH, Jang EJ, Kwon NH, Park YK, Koo HC, Jung WK, Kim JM, Park YH (2007) Novel multiplex PCR for the detection of the Staphylococcus aureus, superantigen and its application to raw meat isolates in Korea. Int J Food Microbiol 117:99–105
Teletchea F, Maudet C, Hänni C (2005) Food and forensic molecular identification: update and challenges. Trends Biotechnol 23:359–366
Verkaar EL, Nijman IJ, Boutaga K, Lenstra JA (2002) Differentiation of cattle species in beef by PCR-RFLP of mitochondrial and satellite DNA. Meat Sci 60:365–369
Xu R, Wei S, Zhou G, Ren J, Liu Z, Tang S, Pck C, Wu X (2018) Multiplex TaqMan locked nucleic acid real-time PCR for the differential identification of various meat and meat products. Meat Sci 137:41–46
Zhang J, Zong H, Zhang L (2008) PCR-mtDNA for detecting components of duck origin in foodstuff and feedstuff. Chin J Biotechnol 24:1832–1836
Zhang Y, Zhu Z, Yang W, Ren J, Tan X, Wang Y, Mao N, Xu S, Zhu S, Cui A, Zhang Y, Yan D, Li Q, Dong X, Zhang J, Zhao Y, Wan J, Feng Z, Sun J, Wang S, Li D, Xu W (2010) An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand foot and mouth disease in Fuyang city of China. Virol J 7:1–9
Zhao Y, Hao C, Yong Q, Qu C, Chen W, Peng C, Kuang H, Zhou H, Wang L, Xu C (2012) Systematic comparisons of genetically modified organism DNA separation and purification by various functional magnetic nanoparticles. Int J Food Sci Technol 47:910–917
Acknowledgements
This work is financially supported by the Grant of 2017YFF0208600, China Agriculture Research System-48 (CARS-48), Anhui Provincial Modern Argo-industry Tech. Research System (NYCYTX-2016-84), NSFC 21475030 and the National 10000 Talents-Youth Top-notch Talent Program.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Qin, P., Qu, W., Xu, J. et al. A sensitive multiplex PCR protocol for simultaneous detection of chicken, duck, and pork in beef samples. J Food Sci Technol 56, 1266–1274 (2019). https://doi.org/10.1007/s13197-019-03591-2
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13197-019-03591-2