Abstract
Latar Belakang: Deteksi dan identifikasi Escherichia coli yang akurat sangat penting dalam penelitian mikrobiologi dan diagnostik. Tujuan: Penelitian ini bertujuan untuk merancang dan memvalidasi primer spesifik untuk gen 16S rRNA pada Escherichia coli menggunakan NCBI Primer Blast serta mengevaluasi efektivitas pemurnian ganda. Metode: Primer Pair 2 (Forward:5’-CCTTAGGGCCTCTTGCCATC-3’,Reverse:5’-GAGCGTCAGTCTTCGTCCAG-3’) dipilih berdasarkan panjang oligonukleotida, Tm, kandungan GC, posisi target, dimer ujung 3’, dan self-complementarity. Pemurnian ganda diterapkan untuk memperoleh DNA berkualitas tinggi guna memastikan amplifikasi PCR yang optimal. Hasil: PCR menghasilkan produk 553 bp yang dikonfirmasi dengan elektroforesis gel agarosa 2%. Pseudomonas aeruginosa sebagai kontrol tidak menunjukkan amplifikasi, mengonfirmasi spesifisitas Primer Pair 2. Kesimpulan: Primer Pair 2 efektif untuk amplifikasi spesifik gen 16S rRNA Escherichia coli, sehingga direkomendasikan untuk meningkatkan spesifisitas dan akurasi deteksi dalam penelitian selanjutnya.References
Adema, C.M. (2021) ‘Sticky Problems: Extraction Of Nucleic Acids From Molluscs’, Philosophical Transactions of the Royal Society B: Biological Sciences, 376(1825), p. 20200162. Available at: https://doi.org/10.1098/rstb.2020.0162.
Branquinho, M.R., Ferreira, R.T.B. and Cardarelli-Leite, P. (2012) ‘Use Of Real-Time PCR To Evaluate Two DNA Extraction Methods From Food’, Food Science and Technology, 32(1), pp. 112–118. Available at: https://doi.org/10.1590/S0101-20612012005000012.
Ehtisham, M. et al. (2016) ‘Polymerase Chain Reaction (PCR): Back to Basics’, Indian Journal of Contemporary Dentistry, 4(2), p. 30. Available at: https://doi.org/10.5958/2320-5962.2016.00030.9.
Elfadadny, A. et al. (2024) ‘Antimicrobial Resistance Of Pseudomonas Aeruginosa: Navigating Clinical Impacts, Current Resistance Trends, And Innovations In Breaking Therapies’, Frontiers in Microbiology, 15(1). Available at: https://doi.org/10.3389/fmicb.2024.1374466.
Fakih, T.M., Wijaya, S. and Priani, S.E. (2021) ‘Desain Primer Gen 12S sRNA dari DNA Mitrokondria Babi (Sus Scrofa) secara In Silico sebagai Kandidat Primer dalam Analisis Molekuler Kehalalan Produk’, Jurnal Sains Farmasi & Klinis, 8(3), p. 316. Available at: https://doi.org/10.25077/jsfk.8.3.316-322.2021.
Fitri, L.E. et al. (2022) ‘Malaria Diagnostic Update: From Conventional To Advanced Method’, Journal of Clinical Laboratory Analysis, 36(4), pp. 1–14. Available at: https://doi.org/10.1002/jcla.24314.
Gautam, A. (2022) DNA and RNA Isolation Techniques for Non-Experts. Cham: Springer International Publishing. Available at: https://doi.org/10.1007/978-3-030-94230-4.
Handoyo, D. and Rudiretna, A. (2000) ‘Prinsip Umum Dan Pelaksanaan Polymerase Chain Reaction (PCR) [General Principles And Implementation Of Polymerase Chain Reaction]’, Unitas, 9(1), pp. 1–13.
Hawkins, J.L. et al. (2019) ‘The Effect of Previous Life Cycle Phase on the Growth Kinetics, Morphology, and Antibiotic Resistance of Salmonella Typhimurium DT104 in Brain Heart Infusion and Ground Chicken Extract’, Frontiers in Microbiology, 10. Available at: https://doi.org/10.3389/fmicb.2019.01043.
Hidayati, N. et al. (2021) ‘Bacillus Tequilensis Isolated From Fermented Intestine Of Holothuria Scabra Produces Fibrinolytic Protease With Thrombolysis Activity’, IOP Conference Series: Earth and Environmental Science, 707(1), p. 012008. Available at: https://doi.org/10.1088/1755-1315/707/1/012008.
Janda, J.M. and Abbott, S.L. (2007) ‘16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls’, Journal of Clinical Microbiology, 45(9), pp. 2761–2764. Available at: https://doi.org/10.1128/JCM.01228-07.
Jeong, K.H., Lee, Y.S. and K., B.H. (2021) ‘Polymerase Chain Reaction Apparatus and Polymerase Chain Reaction Method Using Same’.
Kartikasari, A.M. et al. (2019) ‘Isolasi dan Identifikasi Bakteri Escherichia Coli Kontaminan Pada Daging Ayam Broiler Di Rumah Potong Ayam Kabupaten Lamongan’, Jurnal Medik Veteriner, 2(1), pp. 66–71. Available at: https://doi.org/10.20473/jmv.vol2.iss1.2019.66-71.
Khehra, N., Padda, I.S. and Swift, C.J. (2025) Polymerase Chain Reaction (PCR), StatPearls. StatPearls [Internet]. Available at: http://www.ncbi.nlm.nih.gov/pubmed/1304285 (Accessed: 18 February 2025).
Lear, G. et al. (2018) ‘Methods For The Extraction, Storage, Amplification And Sequencing Of DNA From Environmental Samples’, New Zealand Journal of Ecology, 42(1), pp. 1–50. Available at: https://doi.org/10.20417/nzjecol.42.9.
Lesiani, B.R. et al. (2023) ‘Analysis of Purity and Concentration Escherichia Coli DNA by Boiling Method Isolation with Addition of Proteinase-K and RNase’, Indonesian Journal of Medical Laboratory Science and Technology, 5(2), pp. 160–171. Available at: https://doi.org/10.33086/ijmlst.v5i2.4773.
Maitriani, L.K.B., Wirajana, I.N. and Yowani, S.C. (2015) ‘Desain Primer Untuk Amplifikasi Fragmen GEN inhA ISOLAT 134 Multidrug Resistance Tuberculosis (Mdr-Tb) Dengan Metode Polymerase Chain Reaction’, Cakra Kimia (Indonesian E-Journal of Applied Chemistry), 3(2), pp. 89–96. Available at: www.ncbi.nlm.nih.gov.
McCance, D.J. (1996) ‘DNA Viruses: DNA Extraction, Purification, and Characterization’, in Virology Methods Manual. New York: Academic Press, pp. 191–230. Available at: https://doi.org/10.1016/B978-012465330-6/50011-7.
Moon, Y.-J., Lee, S.-Y. and Oh, S.-W. (2022) ‘A Review of Isothermal Amplification Methods and Food-Origin Inhibitors against Detecting Food-Borne Pathogens’, Foods, 11(3), p. 322. Available at: https://doi.org/10.3390/foods11030322.
Moynihan, M.A. (2021) ‘DNA Extraction With CTAB And Chloroform: Isoamyl Alcohol V1’. Available at: https://doi.org/10.17504/protocols.io.14egn7b9yv5d/v1.
This work is licensed under a Creative Commons Attribution 4.0 International License.
Copyright (c) 2025 Jurnal Wiyata: Penelitian Sains dan Kesehatan
