Abstract
DNA-based methods are more reliable than the conventional protein-based techniques and have been applied to species identification in food of animal origin and meat fraud detection. We developed and evaluated dry reagent-based real-time PCR assays for identification of four commonly eaten meat species. A set of species-specific primers and probes were designed targeting transforming growth factor beta-3 (TGFB3), prolactin receptor (PRLR), NADH dehydrogenase (ND5), and beta actin (ACTB) genes to differentiate chicken, mutton, beef and pork, respectively. All the real-time PCR reagents along with the primers and probes were lyophilized into a pellet form in the presence of suitable stabilizers. The assay specificity was tested with the DNA of animal species other than the target species and also with plant species. The assay was found to be sensitive down to 0.002 ng of DNA from each target species. Thermostability studies of lyophilized mix showed that it was stable for 90 days at room temperature and 120 days at 4 °C. When the method was applied for the analysis of commercial meat samples (n = 66), the results showed that seven samples contained non-declared meat components. The developed assay has the potential to monitor routine meat species identification from raw and cooked meat products and the method can also be implemented in food-testing laboratories.
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References
Cawthorn D, Steinman HA, Hoffman LC (2013) A high incidence of species substitution and mislabelling detected in meat products sold in South Africa. Food Control 32:440–449
Ali ME, Razzak MA, Hamid SBA, Rahman MM, Amin MA, Rashid NRA (2015) Multiplex PCR assay for the detection of five meat species forbidden in Islamic foods. Food Chem 177:214–224
Premanandh J (2013) Horse meat scandal: a wake-up call for regulatory authorities. Food Control 34:568–569
Zhao L, Hua MZ, Li S, Liu J, Zheng W, Lu X (2019) Identification of donkey meat in foods using species-specific PCR combined with lateral flow immunoassay. RSC Adv 9:26552–26558
Montowska M, Pospiech E (2013) 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 Chem 136:1461–1469
Kumar A, Kumar RR, Sharma BD, Gokulakrishnan P, Mendiratta SK, Sharma D (2015) Identification of species origin of meat and meat products on the DNA basis: a review. Crit Rev Food Sci Nutr 55:1340–1351
Jiang H, Wang W, Zhuang H, Yoon SC, Yang Y, Zhao X (2019) Hyperspectral imaging for a rapid detection and visualization of duck meat adulteration in beef. Food Anal Methods 12:2205–2215
Hsieh YH, Ofori JA (2014) Detection of horse meat contamination in raw and heat-processed meat products. J Agric Food Chem 62:12536–12544
Zvereva EA, Kovalev LI, Ivanov AV, Kovaleva MA, Zherdev AV, Shishkin SS, Dzantiev BB (2015) Enzyme immunoassay and proteomic characterization of troponin I as a marker of mammalian muscle compounds in raw meat and some meat products. Meat Sci 105:46–52
Wang W, Wang X, Zhang Q, Liu Z, Zhou X, Liu B (2020) A multiplex PCR method for detection of five animal species in processed meat products using novel species-specific nuclear DNA sequences. Eur Food Res Technol 246:1351–1360
Hossain MAM, Ali ME, Sultana S, Asing BSQ, Kader MA, Rahman MA (2017) Quantitative tetraplex real-time polymerase chain reaction assay with TaqMan probes discriminates cattle, buffalo, and porcine materials in food chain. J Agric Food Chem 65:3975–3985
Dalsecco LS, Palhares RM, Oliveira PC, Teixeira LV, Drummond MG, de Oliveira DAA (2018) A fast and reliable real-time PCR method for detection of ten animal species in meat products. J Food Sci 83:258–265
Kim MJ, Kim HY (2018) Direct duplex real-time loop mediated isothermal amplification assay for the simultaneous detection of cow and goat species origin of milk and yogurt products for filed use. Food Chem 246:26–31
Ahamad MNU, Hossain MAM, Uddin SMK, Sultana S, Nizar NN, Bonny SQ, Johan MR, Ali ME (2019) Tetraplex real-time PCR with TaqMan probes for discriminatory detection of cat, rabbit, rat and squirrel DNA in food products. Eur Food Res Technol 245:2183–2194
Hsieh MK, Shih PY, Wei CF, Vickroy TW, Chou CC (2016) Detection of undeclared animal by-products in commercial canine canned foods: comparative analyses by ELISA and PCR-RFLP coupled with slab gel electrophoresis or capillary gel electrophoresis. J Sci Food Agric 96:1659–1665
Meira L, Costa J, Villa C, Ramos F, Oliveira MBPP, Mafra I (2017) EvaGreen real-time PCR to determine horse meat adulteration in processed foods. LWT Food Sci Technol 75:408–416
Mohamad NA, Mustafa S, Khairil Mokhtar NF, El Sheikha AF (2018) Molecular beacon-based real-time PCR method for detection of porcine DNA in gelatin and gelatin capsules. J Sci Food Agric 98:4570–4577
Xu R, Wei S, Zhou G, Ren J, Liu Z, Tang S, Cheung PCK, 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
Dooley JJ, Paine KE, Garrett SD, Brown HM (2004) Detection of meat species using TaqMan real-time PCR assays. Meat Sci 68:431–438
Khazani NA, Noor NZ, Yean CY, Hasan H, Suraiya S, Mohamad S (2017) A thermostabilized, one-step PCR assay for simultaneous detection of Klebsiella pneumonia and Haemophilus influenzae. J Trop Med. https://doi.org/10.1155/2017/7210849
Arunrut N, Kiatpathomchai W, Ananchaipattana C (2018) Multiplex PCR assay and lyophilization for detection of Salmonella spp., Staphylococcus aureus and Bacillus cereus in pork products. Food Sci Biotechnol 27:867–875
Balakrishna K, Sreerohini S, Parida M (2019) Ready-to-use single tube quadruplex PCR for differential identification of mutton, chicken, pork and beef in processed meat samples. Food Addit Contam Part A 36:1435–1444
ENGL-European Network of GMO Laboratories (2015) Definition of minimum performance requirements for analytical methods for GMO testing. JRC Technical Report, JRC95544 http://gmocrl.jrc.ec.europa.eu/doc/MPR%20Report%20Application%2020_10_2015.pdf
Sharma GK, Mahajan S, Das B, Ranjan R, Kanani A, Sanyal A, Pattnaik B (2014) Comparison of stabilizers for development of a lyophilised multiplex reverse-transcription PCR mixture for rapid detection of foot and mouth disease virus serotypes. Rev Sci Tech 33:859–867
Crowe LM, Reid DS, Crowe JH (1996) Is trehalose special for preserving dry biomaterials? Biophys J 71:2087–2093
Qu S, Shi Q, Zhou L, Guo Z, Zhou D, Zhai J, Yang R (2010) Ambient stable quantitative PCR reagents for the detection of Yersinia pestis. PLOS Negl Trop Dis 4:e629
Rombach M, Kosse D, Faltin B, Wadle S, Roth G, Zengerle R, von Stetten F (2014) Real-time stability testing of air-dried primers and fluorogenic hydrolysis probes stabilized by trehalose and xanthan. Biotechniques 57:151–155
Cheng X, He W, Huang F, Huang M, Zhou G (2014) Multiplex real-time PCR for the identification and quantification of DNA from duck, pig and chicken in Chinese blood curds. Food Res Int 60:30–37
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
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
Prusakova OV, Glukhova XA, Afanas’eva GV, Trizna YA, Nazarova LF, Beletsky IP (2018) A simple and sensitive two-tube multiplex PCR assay for simultaneous detection of ten meat species. Meat Sci 137:34–40
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The authors thank Director, DFRL for his keen interest in this study.
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Konduru, B., Sagi, S. & Parida, M. Dry reagent-based multiplex real-time PCR assays for specific identification of chicken, mutton, beef and pork in raw and processed meat products. Eur Food Res Technol 247, 737–746 (2021). https://doi.org/10.1007/s00217-020-03662-1
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DOI: https://doi.org/10.1007/s00217-020-03662-1