Wild Boar-Specific PCR Assay and Sequence Analysis Based on Mitochondrial Cytochrome-B Gene for Halal Authentication Studies

Ganea Qorry Aina; Abdul Rohman; Yuny Erwanto

doi:10.22146/ijc.42552

Wild Boar-Specific PCR Assay and Sequence Analysis Based on Mitochondrial Cytochrome-B Gene for Halal Authentication Studies

https://doi.org/10.22146/ijc.42552

Ganea Qorry Aina⁽¹⁾, Abdul Rohman^(2*), Yuny Erwanto⁽³⁾

(1) Department of Health Analyst, Polteknik Kesehatan Kementerian Kesehatan Kalimantan Timur, Jl. Kurnia Makmur No. 64, Samarinda, Kalimantan Timur, Indonesia Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(2) Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia Research Center of Halal Products, Gadjah Mada University, Yogyakarta, 55281, Indonesia
(3) Division of Animal Products Technology, Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna No. 3, Bulaksumur, Yogyakarta 55281, Indonesia Research Centre of Halal Products, Universitas Gadjah Mada, Jl. Kaliurang Km 4, Sekip, Yogyakarta 55281, Indonesia
(*) Corresponding Author

Abstract

Wild boar meat (WBM) is non-halal meat widely abused in Indonesia. The most common case is mixing beef with WBM either in raw or processed foods. Therefore, it is necessary to develop a detection method of WBM contamination. The objective of this study was to employ polymerase chain reaction (PCR) and sequence analysis using species specific primer (SSP) targeting on wild boar mitochondrial cytochrome-b (CYTBWB2-wb) gene for the identification of WBM in a meatball. The specificity of primer was tested, and the amplicon size was confirmed with conventional PCR and agarose electrophoresis. The base sequences were analyzed using GeneStudio software and subjected to BLAST using NCBI. CYTBWB2-wb primer was also used to test the reference meatballs made from beef and WBM using real-time PCR. The result showed that CYTBWB2-wb amplified wild boar Cyt-B mt-DNA gene specifically. The amplicon size was 194 base pair (bp) with a similarity of 93–98% toward gen Cyt-B mt-DNA of several wild boar types. The primer is able to detect WBM on the reference meatballs up to 0.1% wt/wt with efficiency value of 108.0% and coefficient of determination (R²) of 0.970. The CYTBWB2-wb primer proved to be specific and could be used as a standard method to identify the presence of WBM contamination in meatball products for halal authentication studies.

Keywords

CYTBWB2-wb primer; meatball; halal authentication; sequencing; wild boar

Full Text:

Full Text PDF

References

[1] Rohman, A., 2019, The employment of Fourier transform infrared spectroscopy coupled with chemometrics techniques for traceability and authentication of meat and meat products, J. Adv. Vet. Anim. Res., 6 (1), 9-17.

[2] Ali, M.E., Hashim, U., Mustofa, S., Che Man, Y.B., Dhahi, T.S., Kashif, M., Uddin, M.K., and Abd Hamid, S.B., 2012, Analysis of pork adulteration in commercial meatballs targeting porcine-specific mitochondrial cytochrome B gene by TaqMan probe real-time polymerase chain reaction, Meat Sci., 91 (4), 454–459.

[3] Guntarti, 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.

[4] Mursyidi, A., 2013, The role of chemical analysis in the halal authentication of food and pharmaceutical products, J. Food Pharm. Sci, 1, 1–4.

[5] Guntarti, A., Martono, S., Yuswanto, A., and Rohman, A., 2015, FTIR spectroscopy in combination with chemometrics for analysis of wild boar meat in meatball formulation, Asian J. Biochem., 10, 165–172.

[6] Munir, S., and ur Rahman, S., 2018, Optimization of countercurrent immunoelectrophoresis and agar gel immunodiffusion tests for the comparative detection of horse and donkey meat, MSM, 2 (1), 14–17.

[7] Necidová, L., Renčová, E., and Svoboda, I., 2002, Counter immunoelectrophoresis: A simple method for the detection of species-specific muscle proteins in heat-processed products, Vet. Med.–Czech, 47 (5), 143–147.

[8] Erwanto, Y., Abidin, M., Rohman, A., and Sismindari, S., 2011, PCR-RFLP using BseDI enzyme for pork authentication in sausage and nugget products, Media Peternakan, 34, 14–18.

[9] Murugaiah, C., Noor, Z.M., 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.

[10] Raharjo, T.J., Pratama, G.A., Nuryanti, I., and Pratiwi, R., 2019, Forgery detection beef with mice meat (Mus musculus) in meatballs using real-time polymerase chain reaction (Real Time PCR) primer specific for a target mitochondrial DNA ND-1 gen, Indones. J. Chem., 19 (1), 86–96.

[11] Rohman, A., Himawati, A., Triyana, K., Sismindari, and Fatimah, S., 2017, Identification of pork in beef meatballs using Fourier transform infrared spectrophotometry and real-time polymerase chain reaction, Int. J. Food Prop., 20 (3), 654–661.

[12] Lockley, A.K., and Bardsley, R.G., 2000, DNA-based methods for food authentication, Trends Food Sci. Technol., 11(2), 67–77.

[13] Aina, G.Q., Rohman, A., and Erwanto, Y., 2019, The employment of q-PCR using specific primer targeting on mitochondrial cytochrome-b gene for identification of wild boar meat in meatball samples, J. Adv. Vet. Anim. Res., Accepted.

[14] Anonymous, 2005, ISO 21569:2005, Foodstuffs–Methods of analysis for the detection of genetically modified organisms and derived products–Qualitative nucleic acid based methods. https://www.iso.org/obp/ui/#iso:std:iso:21569:ed-1:v1:en, accessed on 3 December, 2018.

[15] Mane, B.G., Mendiratta, S.K., Tiwari, A.K., and Narayan, R., 2013, Sequence analysis of mitochondrial 16S rRNA gene to identify meat species, J. Appl. Anim. Res., 41 (1), 77–81.

[16] Mutalib, S.A., Nazri, W.S.W., Shahimi, S., Yaakob, N., Sani, N.A., Abdullah, A., Badji, A.S., and Ghani, M.A., 2012, Comparison between pork and wild boar meat (Sus scrofa) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), Sains Malays., 41 (2), 199–204.

[17] Arini, R.L., Ramadhani, D., Pebriyanti, N.W., Sismindari, and Rohman, A., 2018, The use of species-specific primer targeting on D-loop mitochondrial for identification of wild boar meat in meatball formulation, J. Adv. Vet. Anim. Res., 5 (3), 361–368.

[18] Parkanyi, V., Ondruska, L., Vasicek, D., and Slamecka, J., 2014, Multilevel D-loop PCR identification of hunting game, Appl. Transl. Genomics, 3 (1), 1–7.

[19] Pereira, F., Carneiro, J., and Amorim, A., 2008, Identification of species with DNA-based technology: current progress and challenges, Recent Pat. DNA Gene Sequences, 2 (3), 187–199.

[20] Farag, M.R., Alagawany, M., El-Hack, M.E.A., Tiwari, R., and Dhama, K., 2015, Identification of different animal species in meat and meat products – Trends and advances, Adv. Anim. Vet. Sci., 3 (6), 334–346.

[21] Maede, D., 2006, A strategy for molecular species detection in meat and meat products by PCR-RFLP and DNA sequencing using mitochondrial and chromosomal genetic sequences, Eur. Food Res. Technol., 224 (2), 209–217.

[22] Olson, N.D., and Morrow, J.B., 2012, DNA extract characterization process for microbial detection methods development and validation, BMC Res. Notes, 5, 668.

[23] Chudori, Y.N.C., 2017, Desain dan studi performa metode probe TaqMan real-time polymerase chain reaction spesifik terhadap gen ATPase 6 babi (Sus scrofa), Undergraduate Thesis, Universitas Gadjah Mada, Yogyakarta.

[24] Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A., and Arnheim, N., 1985, Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science, 230 (4732), 1350–1354.

[25] Kumar, A., Kumar, R.R., Sharma, B.D., Gokulakrishnan, P., Mendiratta, S.K., and Sharma, D., 2015, Identification of species origin of meat and meat products on the DNA basis: A review, Crit. Rev. Food Sci. Nutr., 55 (10), 1340–1351.

DOI: https://doi.org/10.22146/ijc.42552