RAPID PCRâ€“BASED DETECTION OPTIMIZATION OF PORCINE DNA IN GELATIN CAPSULE SHELL
Objective: This study was conducted to optimize the genomic deoxyribonucleic acid (DNA) based molecular detection of gelatin derived from porcine by performing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and duplex PCR method employing cyt B gene.
Methods: Optimization was carried out for DNA extraction, PCR conditions, and the sensitivity of the PCR-RFLP method. Due to the very low DNA trace in gelatin after the various manufacturing process, the extraction was optimized to obtain sufficient DNA which was visible on the agarose gel. PCR-RFLP was carried out using universal primers and BsaJI restriction enzyme, and duplex PCR was carried out using two sets of porcine-specific primers. Porcine and bovine DNA were mixed in various concentration to confirm sensitivity of both methods, i.e. 100%, 50%, 10%, 1%, 0.5%, 0.1%, 0.05%, and 0.01%
Results: Both methods, PCR-RFLP, and Duplex PCR, were able to detect as low as 0.01% porcine DNA, indicated by the presence of porcine DNA amplicon bands (131 bp and 228 bp for PCR-RFLP, 212 bp and 398 bp for duplex PCR). Although DNA bands presented in low intensity, identification of porcine and bovine species and estimation of DNA quantities were possible.
Conclusion: Both conventional PCR methods, i.e. PCR-RFLP and Duplex PCR, were sensitive, specific, and suitable as a rapid initial detection method for molecular detection of porcine in gelatin capsule shells.
2. Karim AA, Bhat R. Fish gelatin: properties, challenges, and prospects as an alternative to mammalian gelatins. Food Hydrocolloids 2009;23:563â€“76.
3. Badan Pusat Statistik [Internet]. Sensus Penduduk 2010â€“Penduduk Menurut Kelompok Umur dan Agama yang Dianut. Available from: http://sp2010.bps.go.id/index.php/ site/tabel? searchtabel=Penduduk+Menurut+Kelompok+Umur+dan+Agama+yang+Dianutandtid=320andsearch-wilayah=Indonesiaandwid=0000000000andlang=id. [Last accessed on 29 Jan 2017]
4. Rohman A, Che Man Y, Hashim P, Ismail A. FTIR spectroscopy combined with chemometrics for analysis of lard adulteration in some vegetable oils Espectroscopia FTIR combinada con quimiometrÃa para el anÃ¡lisis de adulteraciÃ³n con grasa de cerdo de aceites vegetales. CyTA J Food 2011;9:96-101.
5. Hermanto S, Fatimah W. Differentiation of bovine and porcine gelatin based on spectroscopic and electrophoretic analysis. J Food Pharm Sci 2013;1:68-73.
6. Nemati M, Oveisi M, Abdollahi H, Sabzevari O. Differentiation of bovine and porcine gelatins using principal component analysis. J Pharm Biomed Anal 2004;34:485-92.
7. Zhang G, Liu T, Wang Q, Chen L, Lei J, Luo J, et al. Mass spectrometric detection of marker peptides in tryptic digests of gelatin: a new method to differentiate between bovine and porcine gelatin. Food Hydrocolloids 2009;23:2001-7.
8. Venien A, Levieux D. Differentiation of bovine from porcine gelatines using polyclonal anti-peptide antibodies in indirect and competitive indirect ELISA. J Pharm Biomed Anal 2005;39:418-24.
9. Shabani H, Mehdizadeh M, Mousavi S, Dezfouli E, Solgi T, Khodaverdi M, et al. Halal authenticity of gelatin using species-specific PCR. Food Chem 2015;184:203-6.
10. Mutalib SA, Muin NM, Abdullah A, Hassan O, Aida W, Mustapha W, et al. Sensitivity of polymerase chain reaction (PCR)-southern hybridization and conventional PCR analysis for Halal authentication of gelatin capsules. LWT--Food Sci Technol 2015;63:714â€“9.
11. Aida A, Che Man Y, Wong C, Raha A, Son R. Analysis of raw meats and fats of pigs using polymerase chain reaction for halal authentication. Meat Sci 2005;69:47-52.
12. Malik A, Sutantyo M, Hapsari I, Sinurat A, Purwati E, Jufri M, et al. Simultaneous identification and verification of gelatin type in capsule shells by electrophoresis and polymerase chain reaction. J Pharm Invest 2016;46:475-85.
13. Nikzad J, Shahhosseini S, Tabarzad M, Nafissi-Varcheh N, Torshabi M. Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for halal authentication. Daru J Pharm Sci 2017;25:1-11.
14. Unajak S, Meesawat P, Anyamaneeratch K. Identification of species (meat and blood samples) using nested-PCR analysis of mitochondrial DNA. Afr J Biotechnol 2011;10:5670â€“6.
15. Kocher TD, Thomas WK, Meyer A, Edward SV, Paabo S, Villablanca FX. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proceedings National Academy Sci USA 1898; 86:6196â€“200.
16. Lahiff S, Glennon M, Oâ€™Brien L, Lyng J, Smith T, Maher M, et al. Species-specific PCR for the identification of ovine, porcine and chicken species in meat and bone meal (MBM). Mol Cell Probes 2001;15:27-35.
17. Srivastava G, Rajput N, Jadav K, Shrivastav A, Joshi H. Single nucleotide markers of D-loop for identification of indian wild pig (sus scrofa cristatus). Vet World 2015;8:532-6.
18. Uniprot [Internet]. MT-ATP8-ATP synthase protein 8-Homo sapiens (Human)-MT-ATP8 gene and protein. Available from: https://www.uniprot.org/uniprot/P03928. [Last accessed on 16 Jul 2017].
19. Nuraini H, Primasari A, Andreas E, Sumantri C. The use of cytochrome b gene as a specific marker of the rat meat (Rattus Norvegicus) on meat and meat products. Media Peternakan 2012;35:15-20.
20. Gil Arriortua M, Salona Bordas M, Caine L, Pinheiro F, de Pancorbo M. Cytochrome B as A useful tool for the identification of blowflies of forensic interest (diptera, calliphoridae). Forensic Sci Int 2013;228:132-6.
21. Goodwin W, Linarce A, Hadi S. An introduction to forensic genetics. 1st ed. Chicester: John Wiley and Sons; 2007.
22. Desjardins P, Conklin D. Nanodrop microvolume quantitation of nucleic acids. J Visualized Exp 2010;45:2565.
23. Toyobo [Internet]. KOD-plus-neo: instruction manual KOD-plus-neo 1109. Available from: http://www.toyobo-global.com/seihin/xr/lifescience/products/pcr_016.html. [Last accessed on 28 Aug 2018].
24. Murugaiah C, Noor Z, Mastakim M, Bilung L, Selamat J, Radu S. Meat species identification and halal authentication analysis using mitochondrial DNA. Meat Sci 2009;83:57-61.
25. Poritz M, Ririe K. Getting things backwards to prevent primer dimers. J Mol Diagn 2014;16:159-62.
26. Kainz P. The PCR plateau phaseâ€“towards an understanding of its limitations. biochim biophys acta. Gene Struct Expression 2000;1494:23-7.