Development of DNA Marker of Growth Hormone Intron for the Detection of Contaminated Porcine DNA in Halal Food and Food Products by PCR Method
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Abstract
Pork contamination in food and food products is a major concern affecting consumer confidence in halal foods. This work aims to develop a DNA marker for detecting contaminated porcine DNA in halal food and food products using the Polymerase Chain Reaction (PCR) method to amplify growth hormone (GH) intron, 105 base pairs, in which other standardized methods are limited in their sensitivities and long testing process. When validating the developed DNA marker, 4 meat sources: beef (Bos taurus), pork (Sus scrofa domesticus), wild boar (Sus scrofa), and chicken (Gallus gallus), 1 plant starch: maize (Zea mays), and 5 pork-based products: fragrance pork floss, Vietnamese sausage, ham, pork sausage, and Chinese sausage were examined. The PCR results showed positive given a 105-bp DNA band on pork, wild boar, and all pork-based products, but no amplified products in other tested samples except for beef and mixed beef samples, where two off-target DNA bands were present at approximately 270 and 1,100 bp. The PCR sensitivity was additionally conducted using beef-pork binary admixtures in different pork percentages (w/w) at 50, 25, 5, 1, and 0.1 (25, 12.5, 2.5, 0.5, and 0.05 ng, respectively). The results showed high sensitivity, detecting as low as 0.1% (0.05 ng). The PCR sensitivity was also performed using a mixture of bovine and porcine DNAs in various percentages (v/v) of porcine DNA: 50, 25, 5, 1, 0.1, and 0.01 (25, 12.5, 2.5, 0.5, 0.05, and 0.005 ng, respectively). The results enabled the detection of as little as 0.01% (0.005 ng) of porcine DNA. The pork derivatives in 5 halal foods were also investigated. The PCR gave positive results in 2 halal foods. Thus, the DNA marker and PCR method developed in this study are highly sensitive, easy to use, and can apply to detecting porcine DNA presence in halal foods and food products.
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References
Montiel-Sosa, J.F., Ruiz-Pesini, E., Montoya, J., Roncalés, P., López-pérez M.J. and Pérez-Martos, A. (2000). Direct and highly species specific detection of pork meat and fat in meat product by PCR amplification of mitochondrial DNA. Journal of Agricultural and Food Chemistry. 48: 2829-2832.
Lubis, H.N., Mohd-Naim, N.F., Alizul, N.N. and Ahmed, M.U. (2016). From market to food plate: Current trusted technology and innovations in halal food analysis. Trends in Food Science & Technology. 58: 55-68. doi: 10.1016/j.tifs.2016.10.024.
Ren, Y., Li, X., Liu, Y., Yang, L., Cai, Y., Quan, S. and Chen, S. (2017). A novel quantitative real-time PCR method for identification and quantification of mammalian and poultry species in foods. Food Control. 76: 42-51. doi: 10.1016/j.foodcont.2017.01.003.
National Statistical Office, Ministry of Digital Economy and Society. (2017). Statistical Yearbook Thailand 2017. [Online] Available from http://service.nso.go.th/ nso/nsopublish/pubs/e-book/SYB-2021/ index.html. [Accessed June 30, 2022] (in Thai).
Jones, S.J. and Patterson, R.L.S. (1985). Double-antibody ELISA for detection of trace amounts of pig meat in raw meat mixtures. Meat Science. 15: 1-13.
Dincer, B., Spearow, J.L., Cassens, R.G. and Greaser, M.L. (1987). The effect of curing and cooking on the detection of species origin of meat products by competitive and indirect ELISA techniques. Meat Science. 20: 253-267.
Paiva-Cavalcanti, M., Regis-da-Sival, C.G. and Gomes, Y.M. (2010). Comparison of real-time PCR and conventional PCR for detection of Leishmania (Leishmania) infantum infection: a mini-review. The Journal of Venomous Animals and Toxins including Tropical Diseases. 16(4): 537-542.
Zdeňková, K., Akhatova, D., Fialová, E., Krupa, O., Kubica, L., Lencová, S. and Demnerová, K. (2018). Detection of meat adulteration: use of efficient and routine-suited multiplex polymerase chain reaction-based methods for species authentication and quantification in meat products. Journal of Food and Nutrition Research. 57(4): 351-362.
Carrel, A.L. and Allen, D.B. (2000). Effects of growth hormone on body composition and bone metabolism. Endocrine. 12(2): 163-172. doi:10.1385/ENDO:12:2:163.
Ma, Q., Liu, S.F., Zhuang, Z.M., Lin, L., Sun, Z.Z., Liu, C.L., Ma, H., Su, Y.Q. and Tang, Q.S. (2012). Genomic structure, polymorphism and expression analysis of the growth hormone (GH) gene in female and male Half-smooth tongue sole (Cynoglossus semilaevis). Gene. 493(1): 92-104.
Meeker, N.D., Hutchinson, S.A., Ho, L. and Trede, N.S. (2007). Method for isolation of PCR-ready genomic DNA from zebrafish tissues. Huntsman Cancer Institute. 43: 610-614.
Ni, J., You, F., Xu, J., Xu, D., Wen, A., Wu, Z., Xu, Y. and Zhang, P. (2012). Single nucleotide polymorphisms in intron 1 and intron 2 of Larimichthys crocea growth hormone gene are correlated with growth traits. Chinese Journal of Oceanology and Limnology. 30(2): 279-285.
Meyer, R., Candrian, U. and Luthy, J. (1994). Detection of pork in heated meat products by the polymerase chain reaction. Journal of AOAC International. 77: 617-622.
Intalaw, S., Poopat, L. and Poopat, T. (2012). Identification of Sus domestica’s DNA by analysis of the mitochondrial cytochrome b gene. Forensic medicine Journal 4(3): 304-311. (in Thai)
Che Man, Y.B., Mustafa, S., Mokhtar, N.F.K., Nordin, R. and Sazili, A.Q. (2012). Porcine-specific polymerase chain reaction assay based on mitochondrial D-loop gene for identification of pork in raw meat. International Journal of Food Properties. 15: 134-144.
Che Man, Y.B., Aida, A.A., Raha, A.R. and Son, R. (2007). Identification of pork derivatives in food products by species-specific polymerase chain reaction (PCR) for halal verification. Food Control. 18: 885-889.
Jitnupong, W., Rungmee, S., Penchan, N. and Thalungraung, W. (2011). Pork detection method by real time PCR. In Kasetsart University, (ed). Proceedings of 49th Kasetsart University Annual Conference: Agro-Industry, pp 665-672. February 1-4, 2011. Bangkok, Thailand (in Thai).
Schrader, C., Schielke, A., Ellerbroek, L. and Johne, R. (2012). PCR inhibitors – occurrence, properties and removal. Journal of Applied Microbiology. 113(5): 1014-1026. doi:10.1111/j.1365-2672. 2012.05384.x.
Chen, X., Lu, L, Xiong, X., Xiong, X. and Liu, Y. (2020). Development of a real-time PCR assay for the identification and quantification of bovine ingredient in processed meat products. Scientific Report. 10:2052. doi: 10.1038/s41598-020-59010-6.