Penggunaan Metode Quantitative Polymerase Chain Reaction (qPCR) untuk Deteksi Fragmen DNA Babi pada Produk Olahan Daging
Abstract
Using food ingredients and/or processed food products contaminated with pork, whether unintentionally or intentionally, has become a growing concern and issue. This condition encourages the development of an accurate method for specifically detecting the presence or absence of pork contamination. The research was carried out on two different samples: (1) fresh pork to provide an in-house positive control and (2) samples of pork-based processed meat products (floss, meatballs, corned beef, and sausages), tested using DNA markers. The use of samples originating from processed pork food is to determine the effect of the processing process on DNA fragments and the robustness of the extraction method for the detection process used. The research aimed to detect pork DNA fragments using the quantitative polymerase chain reaction (qPCR) method. The study stages were extracting fresh pork and processed products using an RNA extraction kit, DNA extraction kit, and salt extraction, as well as measuring the purity and concentration of DNA/RNA using a spectrophotometer. The RNA extract was converted into complementary DNA (cDNA), and the DNA extract was analyzed using qPCR with specific primers for pork DNA (Sus scrofa). The results showed that the concentration of RNA and DNA extracts was 71.1–296.025 ng/uL and of various purity. All processed meat product samples and in-house positive controls were amplified in the Ct range of 23–28 ng/uL. In this case, the meat processing had no effect on the DNA of the processed meat products analyzed, so DNA fragments could be detected. DNA qPCR was more time efficient than cDNA qPCR because it did not require an RNA reverse transcription step.
Keywords: beta actin, cycle threshold, fresh pork, pork DNA, qPCR
Downloads
References
Andriyani E, Fais NL, Muarifah S. 2019. Perkembangan penelitian metode deteksi kandungan babi untuk menjamin kehalalan produk pangan olahan. Journal of Islamic Studies and Humanities. 4(1): 104–126. https://doi.org/10.21580/jish.41.4888
Ardilla D, Taufik M, Tarigan DM, Thamrin M, Razali M, Siregar HS. 2018. Analisis lemak babi pada produk pangan olahan menggunakan spektroskopi UV-Vis. Jurnal Teknologi Pangan & Hasil Pertanian. 1(2): 111–116. https://doi.org/10.30596/agrintech.v1i2.2011
Cai Y, Li X, Lv R, Yang J, Li J, He Y, Pan L. 2014. Quantitative analysis of pork and chicken products by droplet digital PCR. BioMed Research International. 2014(2014): 810209. https://doi.org/10.1155/2014/810209
Canggih C, Fikriyah K, Yasin A. 2017. Inklusi pembayaran zakat di Indonesia. Jurnal Ekonomi dan Bisnis Islam. 3(1): 1–11. https://doi.org/10.20473/jebis.v3i1.3164
Cawthorn DM, Steinman HA, Witthuhn RC. 2011. Comparative study of different methods for the extraction of DNA from fish. Food Control. 22(2): 231–244. https://doi.org/10.1016/j.foodcont.2010.07.003
Coyle PV, Al Molawi NH, Kacem MABH, El Kahlout RA, Al Kuwari E, Al Khal A, Gillani I, Jeremijenko A, Saeb H, Al Thani M, Bertollini R, Rahim HFA, Chemaitelly H, Tang P, Latif AN, Al Kaabi S, Al Maslaman MARS, Morris BD, Al-Ansari N, Kaleeckal AH, Raddad LJA. 2022. Reporting of RTPCR cycle threshold (Ct) values during the first wave of COVID-19 in Qatar improved result interpretation in clinical and public health settings. Journal of Medical Microbiology. 71(5): 1–6. https://doi.org/10.1099/jmm.0.001499
Dastgheib S, Irajie C, Assaei R, Koohpeima F, Mokarram P. 2014. Optimization of RNA extraction from rat pancreatic tissue. Iranian Journal of Medical Science. 39(3): 282–288.
Dayanti FG, Djuminar A, Dermawan A, Tanta A. 2019. Perbandingan nilai pengukuran kuantitatif hasil ekstraksi DNA Salmonella typhi menggunakan metode boiling, NaOH, kit komersial. Jurnal Riset Kesehatan. 11(1): 350–357.
Hariyadi P. 2018. Keamanan Pangan: Tantangan Ganda Bagi Pembangunan Kesehatan Masyarakat dalam Prosiding Seminar Nasional & Diseminasi Hasil Pengabdian kepada Masyarakat Berbasis Riset.
Indrasti D, Mukhlisin MF, Darmawan N, Yuliana ND. 2022. Profil komponen volatil beberapa jenis satai menggunakan kromatografi gas. Jurnal Ilmu Pertanian Indonesia. 27(2): 199–215. https://doi.org/10.18343/jipi.27.2.199
Irwandi, Wardi ES, Dova S. 2020. Deteksi cemaran gen babi pada produk bakso sapi kemasan di kota Padang menggunakan metode PCR (polymerase chain reaction). Jurnal Akademi Farmasi Prayoga. 5(2): 10–21.
ISO. 2020. Molecular biomarker analysis—Detection of animal-derived materials in foodstuffs and feedstuffs by real-time PCR — Part 3: Porcine DNA detection method. ISO/TS 20224-3:2020.
Fitrianingsih, Tasse AM, Astuti F, Rahayu N, Sani LOA, Sutopo D, Abad M. 2022. Teknologi pembuatan abon daging sapi untuk meningkatkan pendapatan hasil ternak pasca-Covid-19 di Desa Kiaea Kabupaten Konawe Selatan. Dalam: Prossiding Seminar Nasional Inovasi dan Teknologi Peternakan II: Optimasi Integrated Farming System Berbasis Teknologi Peternakan dalam Menunjang Pemenuhan Protein Hewani di Era New Normal. Kendari, 19 November 2022.
Kesmen Z, Gulluce A, Sahin F, Yetim H. 2009. Identification of meat species by TaqMan-based real-time PCR assay. Meat Science. 82: 444–449. https://doi.org/10.1016/j.meatsci.2009.02.019
Kuswandi B, Gani AA, Ahmad M. 2017. Immuno strip test for detection of pork adulteration in cooked meatballs. Food Bioscience. 19: 1–6. https://doi.org/10.1016/j.fbio.2017.05.001
Lam S, Kommadath A, Campos ÓL, Prieto N, Aalhus J, Juárez M, Dugan MER, Vahmani P. 2021. Evaluation of RNA quality and functional transcriptome of beef longissimus thoracis over time post-mortem. PloS ONE. 16(5): 1–14. https://doi.org/10.1371/journal.pone.0251868
Mariyani, Sismindari, Rumiyati. 2021. Validasi metode real-time polymerase chain reaction untuk deteksi DNA babi (Sus scrofa domestica) dan celeng (Sus barbatus) pada sosis sapi. Jurnal Ilmiah Indonesia. 6(8): 3925–2940
Mustaqimah DN, Septiani T, Roswien AP. 2020. Deteksi DNA babi pada produk sosis menggunakan real time-polymerase chain reaction (RT-PCR). 3(2): 106–111.
Nakyinsige K, Che Man Y, Sazili AQ. 2012. Halal authenticity issues in meat and meat products. Meat Science. 91(3): 207–214. https://doi.org/10.1016/j.meatsci.2012.02.015
Nygard AB, Jørgensen CB, Cirera S, Fredholm M. 2007. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Molecular Biology. 8: 67. https://doi.org/10.1186/1471-2199-8-67
Orbayinah S, Hari W, Adam H, Sismindari S, Abdul R. 2019. Application of real-time polymerase chain reaction using species specific primer targeting on mitochondrial cytochrome-b gene for analysis of pork in meatball products. Journal of Advanced Veterinary and Animal Research. 6(2): 260–265. https://doi.org/10.5455/javar.2019.f342
Pagani S, Maglio M, Sicuro L, Fini M, Giavaresi G, Brogini S. 2023. RNA extraction from cartilage: issues, methods, tips. International Journal of Molecular Sciences. 24: 1–22. https://doi.org/10.3390/ijms24032120
Panglipur PE, Sulandari L. 2014. Pengaruh jumlah salad oil dan CMC (carboxymethyl cellulose) terhadap sifat organoleptik kornet daging sapi. E-Journal Boga. 3(1): 160–165.
Rahmania YL, Widayat, Agustini TW, Suzery M, Albaari AN. 2021. Pengukuran kandungan DNA babi dalam berbagai produk pangan dengan metode real time polymerase chain reaction (RT-PCR). Indonesia Journal of Halal. 3(2): 129–133.
Safarrida A, Dewantoro A, Rahmasari D, Nuraeni U, Susmiarni RD, Apriori D, Damacena R, Amin MM. 2023. Verifikasi metode deteksi porcine berdasarkan SNI ISO/TS20224-3:2020 pada matriks gelatin. Jurnal Standardisasi. 25(2): 7988. https://doi.org/10.31153/js.v25i2.993
SNI. 2022. Analisis biomarker molekuler-deteksi bahan turunan hewan pada bahan pangan dan bahan pakan menggunakan real-time PCR-Bagian 3: Metode deteksi DNA babi. SNI-ISO/TS 20224-3:2020.
Stefanova P, Taseva M, Georgieva T, Gotcheva V, Angelov A. 2013. A modified CTAB method for DNA extraction from soybean and meat products. Biotechnology & Biotechnological Equipment. 27(3): 3803–3810. https://doi.org/10.5504/BBEQ.2013.0026
Waluyo S, Malau J, Raekiansyah M, Yulian E, Hardiman I. 2019. In silico analysis of actin gene as a candidate for DNA non-halal detection base on real-time PCR. Indonesian Journal of Halal Research. 3(2): 70–74.
Widayat, Agustini TW, Suzery M, Al-Baarri AN, Putri SR, Kurdianto. 2019. Real time-polymerase chain reaction (RT-PCR) sebagai alat deteksi DNA babi dalam beberapa produk non-pangan. Indonesian Journal of Halal. 2(1): 26–33. https://doi.org/10.14710/halal.v2i1.5361
Yalçınkaya B, Yumbul E, Mozioğlu E, Akgoz M. 2017. Comparison of DNA extraction methods for meat analysis. Food Chemistry. 221: 1253–1257. https://doi.org/10.1016/j.foodchem.2016.11.032
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
This journal is published under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License. Authors who publish with this journal agree to the following terms: Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. NonCommercial — You may not use the material for commercial purposes.
View JIPI StatsCounter
