Optimization of Annealing Temperature and Primer Concentration for Detection of Mycobacterium Tuberculosis Isoniazid Resistance Real-Time PCR Method
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Abstract
Indonesia has the second highest number of TB cases in the world according to the WHO report in 2022. Eradication of the National TB Program is difficult due to the presence of Mycobacterium tuberculosis (M. Tb) bacteria that are resistant to various anti-tuberculosis drugs (OAT) known as Multidrug- Resistant Tuberculosis (MDR-TB). Therefor, currently, a nucleic acid-based diagnostic kit has been developed that is designed to directly detect the presence of resistance to INH with mutations of the KatG gene codon 315 (S315T, S315N, S315I, S315R, S315G, R463L) and rifampicin sensitive M. Tb using the Real-Time PCR method. PCR components that are important for development include annealing temperature and primer concentration. This study aims to determine the optimum annealing temperature and primer concentration. The type of research is the Quasi Experiment. The treatment in this study is by adding each primer that varies in concentration, namely 200nM-400nM while varying the annealing temperature with a range of 52-62°C according to the Tm of each primer. The data obtained from this research are Ct values from the target genes S315T, S315N, S315I, S315R, S315G, R463L and M. Tb Rifampicin sensitive.
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References
I. Sari DP. MENGETAHUI, MENGENALI, MENCEGAH DAN MENGOBATI PENYAKIT TUBERKULOSIS (TB). KAMI MENGABDI. 2022;2(1). doi:10.52447/km.v2i1.6504
II. WHO. Global Tuberculosis Report 2022.; 2022. http://apps.who.int/bookorders.
III. Sandegren L, Groenheit R, Koivula T, et al. Genomic stability over 9 years of an isoniazid resistant mycobacterium tuberculosis outbreak strain in Sweden. PLoS One. 2011;6(1). doi:10.1371/journal.pone.0016647
IV. Lina Rosilawati M. Deteksi Mycobacterium tuberculosis dan resistensinya terhadap rifampisin dengan metode nested polymerase chain reaction (PCR) dan sequencing. Universa Medicina. 2007;26(1).
V. Herrera-León L, Molina T, Saíz P, Sáez-Nieto JA, Jiménez MS. New multiplex PCR for rapid detection of isoniazid-resistant Mycobacterium tuberculosis clinical isolates. Antimicrob Agents Chemother. 2005;49(1). doi:10.1128/AAC.49.1.144-147.2005
VI. Weninggalih AureliaA. DESAIN PRIMER DAN PROBE UNTUK DETEKSI MUTASI GEN KatG Mycobacterium Tuberculosis SECARA IN SILICO. Jurusan Teknologi Laboratorium Medis; 2023.
VII. Yuenleni. LANGKAH-LANGKAH OPTIMASI PCR ISSN 2655 4887 ( Print ), ISSN 2655 1624 ( Online ) ISSN 2655 4887 ( Print ), ISSN 2655 1624 ( Online ). indonesian journal of laboratory. 2019;1(3).
VIII. Kartika AI. Optimasi Annealing Temperature Primer mRNA RECK dengan Metode One Step qRT-PCR. Jurnal Labora Medika. 2018;2(1).
IX. Yusuf ZK, Pengajar S, Kesehatan J, Fikk M. POLYMERASE CHAIN REACTION (PCR).
X. Ludyasari A, Susilowati R, Abidin HM. Pengaruh Suhu Annealing Pada Program PCR Terhadap Keberhasilan Amplifikasi DNA Udang Jari (Metapenaeus elegans De Man, 1907) Laguna Segara Anakan, Cilacap, Jawa Tengah. Universitas Islam Maulana Malik Ibrahim. 2016;1(12).
XI. Amanda K, Sari R, Apridamayanti P. Optimasi Suhu Annealing Proses PCR Amplifikasi Gen shv Bakteri Escherichia coli Pasien Ulkus Diabetik. Jurnal Mahasiswa Farmasi Fakultas Kedokteran UNTAN. 2019;4(1).
XII. Rinanda T. KAJIAN MOLEKULER MEKANISME RESISTENSI MYCOBACTERIUM TUBERCULOSIS. Jurnal Kedokteran Syiah Kuala. 2015;15(3):162-167. https://jurnal.unsyiah.ac.id/JKS/article/view/3666
XIII. Handoyo D, Rudiretna A. Prinsip umum dan pelaksanaan Polymerase Chain Reaction (PCR). Unitas. 2001;9(1).
XIV. Inada Y, Tsunoda T, Tanimura H. New quantitative determination of Candida albicans by PCR and identification of Candida species by nested PCR in fungemia. Japanese Journal of Chemotherapy. 2001;49(1).
XV. Shrestha NK, Tuohy MJ, Hall GS, Reischl U, Gordon SM, Procop GW. Detection and Differentiation of Mycobacterium tuberculosis and Nontuberculous Mycobacterial Isolates by Real-Time PCR. J Clin Microbiol. 2003;41(11). doi:10.1128/JCM.41.11.5121-5126.2003
XVI. Borah P. Primer Designing for PCR.; 2011. www.sciencevision.in
XVII. Rahmadhan D, Sari R, Apridamayanti P. Pengaruh suhu annealing terhadap amplifikasi gen tem menggunakan primer dengan %GC rendah. Jurnal Mahasiswa Farmasi Fakultas Kedokteran UNTAN. 2019;4(1).
XVIII. Maksum IP, Suhaili S, Amalia R, Kamara DS, Rachman SD, Rachman RW. PCR Multipleks untuk Identifikasi Mycobacterium tuberculosis Resisten terhadap Isoniazid dan Rifampisin pada Galur Lokal Balai Laboratorium Kesehatan Provinsi Jawa Barat. Jurnal Kimia VALENSI. 2018;4(2). doi:10.15408/jkv.v4i2.7226
XIX. Cao B, Mijiti X, Deng L Le, et al. Genetic characterization conferred co-resistance to isoniazid and ethionamide in mycobacterium tuberculosis isolates from Southern Xinjiang, China. Infect Drug Resist. 2023;16. doi:10.2147/IDR.S407525
XX. Biorad_Life Science Research. Life Science Research. 2006;6.
XXI. Raymaekers M, Smets R, Maes B, Cartuyvels R. Checklist for optimization and validation of real-time PCR assays. J Clin Lab Anal. 2009;23(3):145-151. doi:10.1002/jcla.20307