The Use of Real-Time Polymerase Chain Reaction Combined with Specific-Species Primer for Analysis of Dog Meat DNA in Meatball

Abdul Rohman; Wiranti Sri Rahayu; Sudjadi Sudjadi; Sudibyo Martono

doi:10.22146/ijc.48930

The Use of Real-Time Polymerase Chain Reaction Combined with Specific-Species Primer for Analysis of Dog Meat DNA in Meatball

https://doi.org/10.22146/ijc.48930

Abdul Rohman^(1*), Wiranti Sri Rahayu⁽²⁾, Sudjadi Sudjadi⁽³⁾, Sudibyo Martono⁽⁴⁾

(1) Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(2) Faculty of Pharmacy, Universitas Muhammadiyah Purwokerto, Jl. Raya Dukuh Waluh, PO BOX 202 Purwokerto 53182, Central Java, Indonesia
(3) Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(4) Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(*) Corresponding Author

Abstract

The presence of dog meat is a crucial issue because dog meat is non-halal meat for Muslims. The objective of this study was to design and validate species-specific primer for the identification of dog meat DNA in meatball using real-time polymerase chain reaction (real-time PCR). The specific primer targeting mitochondrial cytochrome c oxidase subunit 1 (CO1) was validated. The specific primers used were designed using Integrated DNA Technologies (IDT) software and subjected to NCBI BLAST procedure. The candidate primers were tested for specificity study using several DNAs from fresh meat of pork, chicken, beef, lamb, and rat. The method was also validated by determining several parameters of linearity, sensitivity, precision, and efficiency. The results showed that primer could amplify specifically DNA target at an optimized annealing temperature of 56.6 °C. The limit of detection (LoD) obtained was 5 ng DNA, corresponding to 2.5% of dog meat in a meatball. The repeatability evaluation, expressed with relative standard deviation (RSD), and efficiency value was in the acceptable range (RSD < 25% and efficiency (90–105%). This method was successfully used for the analysis of marketed samples. Real-time PCR can be used as a standard method in halal authentication analysis through DNA analysis.

Keywords

dog meat; meatball; real-time polymerase chain reaction; halal authentication

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References

[1] Hargin, K.D., 1996, Authenticity issues in meat and meat products, Meat Sci., 43 (Suppl. 1), 277–289.

[2] 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, Int. J. Food Prop.,15 (1), 134–144.

[3] Purnomo, H., and Rahardiyan, D., 2008, Indonesian traditional meatball, Int. Food Res. J., 15 (2), 101–108.

[4] Rahman, M.M., Ali, M.E., Hamid, S.B.A., Mustafa, S., Hashim, U., and Hanapi, U.K., 2014, Polymerase chain reaction assay targeting cytochrome b gene for the detection of dog meat adulteration in meatball formulation, Meat Sci., 97 (4), 404–409.

[5] BBC newsbeat, 2017, The countries where people still eat cats and dogs for dinner, http://www.bbc.co.uk/newsbeat/article/39577557/the-countries-where-people-still-eat-cats-and-dogs-for-dinner, accessed on 15 January 2017.

[6] Khattak, J.Z.K., Mir, A., Anwar, Z., Wahedi, H.M., Abbas, G., Khattak, H.Z.K., and Ismatullah, H., 2011, Concept of halal food and biotechnology, Adv. J. Food Sci. Technol., 3 (5), 385–389.

[7] Regenstein, J.M., Chaudry, M.M., and Regenstein, C.E., 2003, The kosher and halal food laws, Compr. Rev. Food Sci. Food Saf., 2 (3), 111–127.

[8] Mursyidi, A., 2013, The role of analytical chemistry in halal certification, J. Food Pharm. Sci., 1, 1–4.

[9] Mansor, T.S.T., Che Man, Y.B., and Shuhaimi, M., 2012, Employment of differential scanning calorimetry in detecting lard adulteration in virgin coconut oil, J. Am. Oil Chem. Soc., 89 (3), 485–496.

[10] Nurjuliana, M., Che Man, Y.B., Mat Hashim, D., and Mohamed, A.K.S., 2011, Rapid identiﬁcation of pork for halal authentication using the electronic nose and gas chromatography-mass spectrometer with headspace analyzer, Meat Sci., 88 (4), 638–644.

[11] Indrasti, D., Che Man, Y.B., Mustafa, S., and Hashim, D.M., 2010, Lard detection based on fatty acids profile using comprehensive gas chromatography hyphenated with time-of-flight mass spectrometry, Food Chem., 122 (4), 1273–1277.

[12] Rohman, A., Triyana, K., Sismindari, and Erwanto, Y., 2012, Differentiation of lard and other animal fats based on triacylglycerols composition and principal component analysis, Int. Food Res. J., 19 (2), 475–479.

[13] Rohman, A., Sismindari, Erwanto, Y., and Che Man, Y.B., 2011, Analysis of pork adulteration in beef meatball using Fourier transform infrared (FTIR) spectroscopy, Meat Sci., 88 (1), 91–95.

[14] Fadzillah, N.A., Che Man, Y.B., Rohman, A., Rosman, A.S., Ismail, A., Mustafa, S., and Khatib, A., 2015, Detection of butter adulteration with lard by employing ¹H-NMR spectroscopy and multivariate data analysis, J. Oleo Sci., 64 (7), 697–703.

[15] Maryam, S., Sismindari, Raharjo, T.J., Sudjadi, and Rohman, A., 2016, Determination of porcine contamination in laboratory prepared dendeng using mitochondrial D-loop686 and cyt b gene primers by real time polymerase chain reaction, Int. J. Food Prop., 19 (1), 187–195.

[16] Chung, H.H, 2018, Real-time polymerase chain reaction (RT-PCR) for the authentication of raw meat, Int. Food Res. J., 25 (2), 632–638.

[17] Ballin, N.Z., Vogensen, F.K., and Karlsson, A.H., 2009, Species determination – Can we detect and quantify meat adulteration?, Meat Sci., 83 (2), 165–174.

[18] Navarro, E., Serrano-Heras, G., Castaño, M.J., and Solera, J., 2015, Real-time PCR detection chemistry, Clin. Chim. Acta, 439, 231–250.

[19] Rodríguez, M.A., García, T., González, I., Hernández, P.E., and Martín, R., 2005, TaqMan real-time PCR for detection and quantitation of pork in meat mixtures, Meat Sci., 70 (1), 113–120.

[20] Kesmen, Z., Gulluce, A., Sahin, F., and Yetim, H., 2009, Identiﬁcation of meat species by TaqMan-based real-time PCR assay, Meat Sci., 82 (4), 444–449.

[21] Kesmen, Z., Güllüce, A., Yilmaz, M.T., Yetiman, A.E., and Yetim, H., 2014, Taqman-based duplex real-time polymerase chain reaction approach for the detection and quantification of donkey and pork adulterations in raw and heat-processed meats, Int. J. Food Prop., 17 (3), 629–638.

[22] Rahmawati, Sismindari, Raharjo, T.J., Sudjadi, and Rohman, A., 2016, Analysis of pork contamination in abon using mitochondrial D-loop22 primers using real-time polymerase chain reaction method, Int. Food Res. J., 23 (1), 370–374.

[23] Guntarty, A., Martono, S., Yuswanto, A., and Rohman, A., 2017, Analysis of beef meatball adulteration with wild boar meat using real-time polymerase chain reaction, Int. Food Res. J., 24 (6), 2451–2455.

[24] Widyasari, Y.I., Sudjadi, and Rohman, A., 2015, Detection of rat meat adulteration in meat ball formulations employing real time PCR, Asian J. Animal Sci., 9 (6), 460–465.

[25] Ali, M.E., Rahman, M.M., Hamid, S.B.A., Mustafa, S., Bhassu, S., and Hashim, U., 2014, Canine-specific PCR assay targeting cytochrome b gene for the detection of dog meat adulteration in commercial frankfurters, Food Anal. Methods, 7 (1), 234–241.

[26] Manalu, H.Y., Sismindari, and Rohman, A., 2019, The use of primer-specific targeting on mitochondrial cytochrome b combined with real-time polymerase chain reaction for the analysis of dog meat in meatballs, Trop. Life Sci. Res., 30 (3), 1–14.

[27] Sudjadi, Wardani, H.S., Sepminarti, T., and Rohman, A., 2016, Analysis of porcine gelatin DNA in a commercial capsule shell using real-time polymerase chain reaction for halal authentication, Int. J. Food Prop., 19 (9), 2127–2134.

[28] Perestam, A.T., Fujisaki, K.K., Nava, O., and Hellberg, R.S., 2017, Comparison of real-time PCR and ELISA-based methods for the detection of beef and pork in processed meat products, Food Control, 71, 346–352.

[29] Safdar, M., Junejo, Y., Arman, K., and Abasiyanik, M.F., 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 (2), 296–300.

[30] Murugaiah, C., Noor M.Z., Mastakim M., Bilung L.M., Selamat J., and Radu S., 2009, Meat species identification and halal authentication analysis using mitochondrial DNA, Meat Sci., 83 (1), 57–61.

[31] Soares, S., Amaral, J.S., Oliveira, M.B.P.P., and Mafra, I., 2013, A SYBR Green real-time PCR assay to detect and quantify pork meat in processed poultry meat products, Meat Sci., 94 (1), 115–120.

DOI: https://doi.org/10.22146/ijc.48930