Low cost and comprehensive pork detection in processed food products with a different food matrix


Fenny Aulia Sugiana(1), Henni Widyowati(2), Muhammad Ali Warisman(3), Suryani Suryani(4), Desriani Desriani(5*)

(1) 1. Research Center for Biotechnology, Indonesian Institute of Sciences 2. Department of Biochemistry, Bogor Agricultural University. Jl. Raya Darmaga, Kampus IPB, Bogor 16680, West Java, Indonesia. Tel : +62-51-8423 267
(2) Research Center for Biotechnology, Indonesian Institute of Sciences
(3) Research Center for Biotechnology, Indonesian Institute of Sciences
(4) Department of Biochemistry, Bogor Agricultural University. Jl. Raya Darmaga, Kampus IPB, Bogor 16680, West Java, Indonesia.
(5) Research Center for Biotechnology, Indonesian Institute of Sciences
(*) Corresponding Author


The adulteration of processed beef-based meat products with pork is a sensitive issue in Indonesia. In this study, we developed a detection method for the low cost identification of pork in processed meat products. We used the cost-efficient Taq DNA polymerase, DreamTaq Green PCR master mix (2x), and duplex PCR method to recognize pork simultaneously with 18S rRNA detection. A positive control containing a pork gene inserted into pGEM®-T easy was prepared, along with a negative control. The results of the duplex PCR were used to assess its specificity, detection limit, and its ability to recognize pork in processed meat products with a different food matrix. 18S rRNA detection was for confirming DNA integrity of DNA extracted from the processed food, while the positive control confirmed that the reagents were working well and the negative control confirmed a non-contamination problem. Following this, the duplex PCR was optimized and the optimum concentration primer for duplex PCR detection was found to be 3 µm for pork and 0.2 µm for 18S rRNA. As little as 3.125 ng of the DNA template could be used to detect whether a sample contained pork. Duplex PCR is a simple, fast, sensitive, specific, and low cost method of detecting pork in processed meat products.


18S rRNA; duplex PCR; low cost; pork; processed meat

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Sugiana et al.


Ali E, Razzak A, Bee S, Hamid A, Rahman M, Amin A, Raifana N, Rashid A. 2015. Multiplex PCR assay for the detection of five meat species forbidden in Islamic foods. Food Chem. 177:214–224. doi:10.1016/j.foodchem.2014.12.098.

Al-tamimi A, Ashhab Y. 2012. Identification of meat species and meat products by a Multiplex PCR method. Hebron: Palestine Polytechnic University.

Asensio L, Garcı T, Gonza I, Martin R. 2008. Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA). Food Control. 19:1–8. doi:10.1016/j.foodcont.2007.02.010.

Bo H, Xianrong M, Liyuan Z, Jinyue G, Shaowen L, Hui J. 2014. Development of a sensitive and specific multiplex PCR method for the simultaneous detection of chicken, duck and goose. Meat Sci. 101:90–94. doi:10.1016/j.meatsci.2014.11.007.

Dalmasso A, Fontanella E, Piatti P, Civera T, Rosati S, Bottero MT. 2004. A multiplex PCR assay for the identification of animal species in feedstuffs. Mol Cell Probes. 18:81–87. doi:10.1016/j.mcp.2003.09.006.

Desriani. 2014. Comparison study of three different polymerase amplifying breast cancer HER-2 gen for PCRbased diagnostic application purpose. In: Proceedings of the 3rd International Seminar on Chemistry. Jatinangor. p. 161–163

Desriani, Widyowati H. 2017. Construction of complete pork detection system on processed food with PCR and realtime PCR specific species approach. Paper presented in the International Symposium on Bioeconomics of Natural Resources Utilizations, Bogor

Fajardo V, Gonzalez I, Rojas M, Garcıa T, Martin R. 2010. A review of current methodologies for the authentication of meats from game animal species. Trends Food Sci Technol. 21:408–421. doi:10.1016/j.tifs.2010.06.002.

Ghovvati S, Nassiri MR, Mirhoseini SZ, Moussavi AH, Javadmanesh A. 2009. Fraud identification in industrial meat products by multiplex PCR assay. Food Control. 20:696–699. doi:10.1016/j.foodcont.2008.09.002.

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. Journal of Food and Nutrition Research. 3:392–398. doi:10.12691/jfnr-3-6-6.

Heininger A, Binder M, Ellinger A, Botzenhart K, Unertl K. 2003. DNAse pretreatment of master mix reagents improves the validity of universal 16s rRNA gene PCR results. 41:1763–1765. doi:10.1128/JCM.41.4.1763.

Hidayat AS, Siradj M. 1999. Sertifikasi halal dan sertifikasi non halal [Halal and non-halal certification]. Ahkam. 15:199–210.

Hossain MAM, Ali E, Bee S, Hamid A, Mustafa S, Nasir M, Desa M, Zaidul ISM. 2016. Double gene targeting multiplex polymerase chain reaction–restriction fragment length polymorphism assay discriminates beef, buffalo, and pork substitution in Frankfurter products. doi:10.1021/acs.jafc.6b02224.

Irine. 2013. Penggunaan gen sitokrom β (cyt β) dalam identifikasi spesies anjing, kucing, dan harimau untuk menjamin keaslian produk pangan dan obat [Using cytochrome β (cyt β) gene for species identification of dog, cat, and tiger to ensure food and drugs authenticity] [Master’s thesis]. [Bogor]: Bogor Agricultural University.

Jain S, Brahmbhait M, Rank D, Joshi C, Solank J. 2007. Use of cytochrome b gene variability in detecting meat species by multiplex PCR assay. Indian J Anim Sci. 77:880–881.

Joko T, Kusumandari N, Hartono S. 2011. Optimasi metode PCR untuk deteksi Pectobacterium carotovorum, penyebab penyakit busuk lunak anggrek [Optimization of PCR method for the detection of Pectobacterium carotovorum, a causal agent of soft-rot disease on orchid]. Jurnal Perlindungan Tanaman Indonesia. 17:54–59.

Kim M, Kim H. 2017. Species identification of commercial jerky products in food and feed using direct pentaplex PCR assay. Food Control. 78:1–6. doi:10.1016/j.foodcont.2017.02.027.

Kim S-H, Huang T-S, Seymour TA, Wei C-I, Kempf SC, Bridgman CR, Momcilovic D, Clemens RA, An H. 2005. Development of immunoassay for detection of meat and bone meal in animal feed. Journal of Food Protection. 68:1860–1865. doi:10.4315/0362-028X-68.9.1860.

Kitpipit T, Sittichan K, Thanakiatkrai P. 2014. Direct-multiplex PCR assay for meat species identification in food products. Food Chemistry. 163:77–82. doi:10.1016/j.foodchem.2014.04.062.

Krishnan S, Musa C, Omar C, Zahran I, Syazwan N, Alyaa S. 2017. The awareness of Gen Z’s toward halal food industry. Management. 7:44–47.

Kundave VR, Ram H, Rafqi SI, Garg R, Tiwari AK, Banerjee PS. 2017. Comparative evaluation of microscopy and pcr assay for detection of Theileria annulata infection in ruminants. Journal of Animal Research. 7:699. doi:10.5958/2277-940X.2017.00107.3.

Lupan I, Ianc MB, Ochiş C, Popescu O. 2013. The evidence of contaminant bacterial DNA in several commercial Taq polymerases. Romanian Biotechnological Letters. 18:8007–8012.

Markoulatos P, Siafakas N, Moncany M. 2002. Multiplex polymerase chain reaction: A practical approach. Journal of Clinical Laboratory Analysis. 16:47–51. doi:10.1002/jcla.2058.

Marniemi J, Parkki MG. 1975. Radiochemical assay of glutathione S-epoxide transferase and its enhancement by phenobarbital in rat liver in vivo. Biochem Pharmacol. 24:1569–1572.

Martín I, García T, Fajardo V, Rojas M, Pegels N, Hernández PE, González I, Martín R. 2009. SYBR-Green real-time PCR approach for the detection and quantification of pig DNA in feedstuffs. Meat Science. 82:252–259. doi:10.1016/j.meatsci.2009.01.023.

Matsunaga T, Chikuni K, Tanabe R, Muroya S, Shibata K, Yamada J, Shinmura Y. 1999. A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Science. 51:143–148. doi:10.1016/S0309-1740(98)00112-0.

Montowska M, Pospiech E. 2007. Species identification of meat by electrophoretic methods. Acta Sci Pol, Technol Aliment. 6:5--16.

Nguyen TT, Van Giau V, Vo TK. 2016. Multiplex PCR for simultaneous identification of E. coli O157:H7, Salmonella spp. and L. monocytogenes in food. 3 Biotech. 6. doi:10.1007/s13205-016-0523-6.

Parada JL, Magasanik B. 1975. Expression of the hut operons of Salmonella typhimurium in Klebsiella aerogenes and in Escherichia coli. J Bacteriol. 124:1263–1268.

Przybylski W, Żelechowska E, Czauderna M, Jaworska D, Kalicka K, Wereszka K. 2017. Protein profile and physicochemical characteristics of meat of lambs fed diets supplemented with rapeseed oil, fish oil, carnosic acid, and different chemical forms of selenium. Archives Animal Breeding. 60:105–118. doi:10.5194/aab-60-105-2017.

Soares S, Amaral JS, Oliveira MBPP, Mafra I. 2013. A SYBR Green real-time PCR assay to detect and quantify pork meat in processed poultry meat products. Meat Science. 94:115–120. doi:10.1016/j.meatsci.2012.12.012.

DOI: https://doi.org/10.22146/ijbiotech.32372

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