Optimization of Annealing Temperature and Agarose Concentration in Detecting the mecA Gene of Methicillin-Resistant Staphylococcus aureus (MRSA)
DOI:
https://doi.org/10.36590/jika.v8i1.1585Keywords:
Annealing, Agorasa, gen mecA, PCR, MRSAAbstract
Methicillin-resistant Staphylococcus aureus (MRSA) is associated with the expression of the mecA gene. This study aimed to determine the optimal annealing temperature and agarose concentration to achieve the best PCR product quality based on the evaluation of DNA band integrity, thickness, and resolution. DNA was extracted from five S. aureus samples to detect isolates containing the mecA gene. The experimental variations included annealing temperatures of 53°C, 55°C, 57°C, and 59°C, as well as agarose concentrations of 1% and 1.5%. Through densitometry measurements, the optimal mecA gene band thickness was achieved at an annealing temperature of 59°C. Furthermore, the use of a 1.5% agarose concentration provided the best DNA separation resolution compared to the 1% concentration. In conclusion, optimization of annealing temperature and agarose concentration parameters was crucial to ensure the accuracy of MRSA diagnosis in the laboratory .
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Algammal, A.M., Hetta, H.F., Elkelish, A., Alkhalifah, D.H.H., Hozzein, W.N., Batiha, G.E.S., Nahhas, N. El, Mabrok, M.A., 2020. Methicillin-Resistant Staphylococcus Aureus (MRSA): One Health Perspective Approach to The Bacterium Epidemiology, Virulence Factors, Antibiotic-Resistance, and Zoonotic Impact. Infection and Drug Resistance 13(1), 3255–3265. Https://Doi.Org/10.2147/IDR.S272733
Alghamdi, B.A., Al-Johani, I., Al-Shamrani, J.M., Alshamrani, H., Al-Otaibi, B.G., Almazmomi, K., Yusnoraini Yusof, N., 2023. Antimicrobial Resistance in Methicillin-Resistant Staphylococcus Aureus. Saudi Journal of Biological Sciences 30(4), 1–12. Https://Doi.Org/10.1016/J.Sjbs.2023.103604
Amanda, K., Sari, R., Apridamayanti, P., 2019. Optimasi Suhu Annealing Proses PCR Amplifikasi Gen Shv Bakteri Escherichia coli Pasien Ulkus Diabetik. Jurnal Mahasiswa Farmasi Fakultas Kedokteran UNTAN 4(1), 1–6.
Arslan, M., Tezcan, E., Camci, H., Avci, M.K., 2021. Effect of DNA Concentration on Band Intensity and Resolution in Agarose Gel Electrophoresis. Van Saglik Bilimleri Dergisi 14(3), 326–333. Https://Doi.Org/10.52976/Vansaglik.969547
Faisal, M., Sari, R., Apridamayanti, P., 2019. Optimasi Suhu Annealing Gen Meca Resistensi Antibiotik Amoksisilin dari Bakteri Staphylococcus Aureus pada Pasien Ulkus Diabetik. Jurnal Mahasiswa Farmasi Fakultas Kedokteran UNTAN 5, 1–12.
Gupta, N., Jais, M., Shrivastava, P.K., Sharma, A., 2022. Comparison of Different Phenotypic Methods of Detection of Methicillin-Resistant Staphylococcus Aureus with Polymerase Chain Reaction. Journal of Microbiology and Infectious Disease 12(3), 116–126. Https://Doi.Org/10.5799/Jmid.1176537
Idrees, M.M., Saeed, K., Shahid, M.A., Akhtar, M., Qammar, K., Hassan, J., Khaliq, T., Saeed, A., 2023. Prevalence of Meca- and Mecc-Associated Methicillin-Resistant Staphylococcus Aureus in Clinical Specimens, Punjab, Pakistan. Biomedicines 11(3), 1–11. Https://Doi.Org/10.3390/Biomedicines11030878
Kemalaputri, D.W., Jannah, S.N., Budiharjo, A., 2017. Deteksi MRSA (Methicillin-Resistant Staphylococcus aureus) pada Pasien Rumah Sakit dengan Metode MALDI-TOF MS dan Multiplex PCR. Jurnal Akademika Biologi 6(4), 51–61. https://ejournal3.undip.ac.id/index.php/biologi/article/view/19607
Khairullah, A.R., Rehman, S., Sudjarwo, S.A., Effendi, M.H., Ramandinianto, S.C., Gololodo, M.A., Widodo, A., Riwu, K.H.P., Kurniawati, D.A., 2022. Detection of Meca Gene and Methicillin-Resistant Staphylococcus Aureus (MRSA) Isolated from Milk and Risk Factors from Farms in Probolinggo, Indonesia. F1000 Research 11, 1–24. Https://Doi.Org/10.12688/F1000research.122225.1
Khehra, N., Padda, I.S., Zubair, M., 2025. Polymerase Chain Reaction (PCR) [WWW Document]. https://www.ncbi.nlm.nih.gov/books/NBK589663/. [Diakses Januari 2026].
Listiani, L., Dhanti, K.R., Kurniawan, K., Widodo, O.S.Y., 2023. Optimasi Suhu Annealing Gen Blaz dari Bakteri Methicillin-Resistant Staphylococcus Aureus (MRSA) pada Peralatan Medis. Borneo Journal of Medical Laboratory Technology 6(1), 420–425. Https://Doi.Org/10.33084/Bjmlt.V6i1.6083
Liu, Y., Zhang, J., Ji, Y., 2016. PCR-Based Approaches for The Detection of Clinical Methicillin-Resistant Staphylococcus Aureus. Open Microbiology Journal 14(10), 45–56. https://doi.org/10.2174/1874285801610010045
Lozano, C., Fernández-Fernández, R., Ruiz-Ripa, L., Gómez, P., Zarazaga, M., Torres, C., 2020. Human mecC-Carrying MRSA: Clinical Implications and Risk Factors. Microorganisms 8(10), 1–20. Https://Doi.Org/10.3390/Microorganisms8101615
Lucena-Aguilar, G., Sánchez-López, A.M., Barberán-Aceituno, C., Carrillo-Ávila, J.A., López-Guerrero, J.A., Aguilar-Quesada, R., 2016. DNA Source Selection for Downstream Applications Based on DNA Quality Indicators Analysis. Biopreservation and Biobank 14(4), 264–270. Https://Doi.Org/10.1089/Bio.2015.0064
Mandagi, S.C., Rumangkang, J.C., Prakarsa, C.P., Gobel, S.A. Van, Wawo, A.E., Kalalo, M.J., Edy, H.J., 2022. Potensi Senyawa Bioaktif dari Gedi Hijau (Abelmoschus Manihot) sebagai Inhibitor terhadap Bakteri Resisten Antibiotik. Pharmacon 11(2), 1417–1421. https://ejournal.unsrat.ac.id/v3/index.php/pharmacon/article/view/41730
[PAMKI] Perhimpunan Dokter Spesialis Mikrobiologi Klinik Indonesia., 2023. Pola Patogen dan Antibiogram di Indonesia Tahun 2022. Perhimpunan Dokter Spesialis Mikrobiologi Klinik Indonesia, Jakarta.
Prasetio, M, Barliana, M.I., 2016. Article Review: Gen Meca sebagai Faktor Munculnya Methicillin-Resistant Staphylococcus aureus (MRSA). Farmaka 14(3), 53–61. https://jurnal.unpad.ac.id/farmaka/article/view/10715
Rahman, I.W., Arfani, N., Rafika, R., Tadoda, J.V., 2023. Deteksi Bakteri MRSA Methicillin-Resistant Staphylococcus Aureus pada Sampel Darah Pasien Rawat Inap. Jurnal Ilmu Alam dan Lingkungan 14(1), 48–54. https://journal.unhas.ac.id/index.php/jai2/article/view/26212
Rocchetti, T.T., Martins, K.B., Martins, P.Y.F., Oliveira, R.A., Mondelli, A.L., Fortaleza, C.M.C.B., Cunha, M.D.L.R., 2018. Detection of The mecA Gene and Identification of Staphylococcus Directly from Blood Culture Bottles by Multiplex Polymerase Chain Reaction. Brazilian Journal of Infectious Disease 22(2), 99–105. Https://Doi.Org/10.1016/J.Bjid.2018.02.006
Seprianto, S., Wulansari, W., Wahyuni, F., Novianti, T., 2023. Optimization of The Annealing Temperature Specific Primers for Detection of Phytase Gene from Rhodotorula Mucilaginosa RG-PK20. Indonesian Journal of Biotechnology and Biodiversity 7(2), 72–81.
Setyawati, R., Zubaidah, S., 2021. Optimasi Konsentrasi Primer dan Suhu Annealing dalam Mendeteksi Gen Leptin pada Sapi Peranakan Ongole (PO) Menggunakan Polymerase Chain Reaction (PCR). Indonesian Journal of Laboratory 4(1), 36–40. Https://Doi.Org/10.22146/Ijl.V4i1.65550
Sugireng, S., Rosdarni, R., 2020. Deteksi MRSA (Methicillin-Resistant Staphylococcus aureus) dengan Metode PCR pada Pasien Ulkus Diabetikum. Prosiding Seminar Nasional Biologi di Era Pandemi COVID-19 6(1), 31–35. https://journal.uin-alauddin.ac.id/index.php/psb/article/view/15232
Sundari, S., Priadi, B., 2019. Teknik Isolasi dan Elektroforesis DNA Ikan Tapah. Buletin Teknik Litkayasa Akuakultur 17(2), 87–90. https://ejournal-balitbang.kkp.go.id/index.php/btla/article/view/8608
Triani, N., 2020. Isolasi DNA Tanaman Jeruk dengan Menggunakan Metode CTAB (Cetyl Trimethyl Ammonium Bromide). Jurnal Teknologi Terapan 3(2), 221–226. Https://Doi.Org/10.33379/Gtech.V3i2.419
Trisia, A., Philyria, R., Toemon, A.N., 2018. Uji Aktivitas Antibakteri Ekstrak Etanol Daun Kalanduyung (Guazuma Ulmifolia Lam.) terhadap Pertumbuhan Staphylococcus Aureus dengan Metode Difusi Cakram (Kirby-Bauer). Anterior Jurnal 17(2), 136–143. Https://Doi.Org/10.33084/Anterior.V17i2.12
Ume, E.J., Waworuntu, O.A., Homenta, H., 2025. Identifikasi Methicillin – Resistant Staphylococcus Aureus (MRSA) pada Tenaga Kesehatan di Ruang Intensive Care Unit (ICU) Rumah Sakit DR. J.H Awaloei Manado. Jurnal Ilmiah Indonesia 5(4), 1393–1400. Https://Doi.Org/10.59141/Cerdika.V5i4.2468
Violita, V., Achyar, A., Zulyusri, Z., Atifah, Y., Putri, D.H., Nabilah, R., 2024. Primer Design and Optimization of Annealing Temperature for Gene Amplification GSTL2 on Rice. Al-Kauniyah Jurnal Biologi 17(2), 377–386. Https://Doi.Org/10.15408/Kauniyah.V17i2.32859
Wacnik, K., Rao, V.A., Chen, X., Lafage, L., Pazos, M., Booth, S., Vollmer, W., Hobbs, J.K., Lewis, R.J., Foster, S.J., 2022. Penicillin-Binding Protein 1 (PBP1) of Staphylococcus Aureus Has Multiple Essential Functions in Cell Division. Mbio 13, 1–19. Https://Doi.Org/10.1128/Mbio.00669-22
Yuenleni, Y., 2019. Langkah-Langkah Optimasi PCR. Indonesian Journal of Laboratory 1(3), 51–56. Https://Doi.Org/10.22146/Ijl.V1i3.48723
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Copyright (c) 2026 Muhammad Abdullah Alhakim, Kurnia Ritma Dhanti, Kurniawan, Arif Mulyanto
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