Deteksi SARS-COV-2 dengan metode colorimetric saliva-based RT-LAMP menggunakan miniPCR
Kata Kunci:
COVID-19, RT-LAMP, saliva, mini PCR, colorimetricAbstrak
Coronavirus disease 2019 (COVID-19) adalah penyakit yang disebabkan oleh virus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) yang pertama kali terjadi di China dan dikonfirmasi menjadi pandemi global oleh World Health Organization (WHO). Transmisi virus COVID-19 menyebar sangat cepat, untuk mengatasi hal tersebut diperlukan peningkatan dan perkembangan metode deteksi yang lebih efektif. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) merupakan gold standard yang masih digunakan untuk metode deteksi virus COVID-19. Kelemahan dalam metode ini yaitu peralatan yang mahal dan waktu pengerjaan yang lama. Selain itu pengambilan sampel swab mengakibatkan ketidaknyamanan bagi pasien. Terdapat metode alternatif deteksi virus SARS-CoV-2 yaitu saliva based RT-LAMP. Adapun tujuan penelitian ini adalah untuk mengetahui kemampuan metode colorimetric saliva based RT-LAMP untuk deteksi virus SARS-CoV-2 yang menyebar di Indonesia. Pada penelitian ini digunakan 5 sampel saliva pasien positif COVID-19 yang berasal dari RS PKU Muhammadiyah Kota Yogyakarta. Setelah sampel terkumpul, virus diinaktivasi menggunakan heating block pada suhu 95oC selama 5 menit dan ditambahkan larutan PBS sebanyak 400μl. Kemudian hasil ekstraksi diamplifikasi menggunakan miniPCR pada suhu 65°C selama 30 menit. Visualisasi hasil reaksi tersebut dideteksi secara colorimetric menggunakan phenol red dan dikonfirmasi menggunakan elektroforesis. Manfaat dari penelitian ini adalah dapat memberikan metode alternatif secara molekuler dalam deteksi SARS-CoV- 2 berbasis saliva di Indonesia dan memberikan keuntungan kepada pasien karena metode colorimetric saliva based RT-LAMP dapat mengurangi mengurangi ketidaknyamanan saat pengambilan sampel, membutuhkan waktu yang cepat dan biaya yang lebih murah. Pengujian metode saliva based RT-LAMP dengan visualisasi phenol red belum mampu untuk mendeteksi virus SARS-CoV-2 secara efektif dan efisien karena phenol red yang kurang kompatibel sebagai pewarna atau sudah rusak akibat penyimpanan yang terlalu lama. Namun berdasarkan hasil visualisasi elektroforesis metode saliva based RT-LAMP efektif mendeteksi virus SARS-CoV-2 menggunakan thermal cycler yaitu pada suhu 65°C dengan waktu 30 menit.
Referensi
Aoki, M. N., de Oliveira Coelho, B., Góes, L. G. B., Minoprio, P., Durigon, E. L., Morello, L. G., Marchini, F. K., Riediger, I. N., do Carmo Debur, M., Nakaya, H. I., & Blanes, L. (2021). The diagnostic sensitivity of SARS-CoV-2 LAMP depends on color interpretation and viral load.Scientific Report,11(1), 1–10. https://doi.org/10.1038/s41598-021-88506-y
Cevik, M., Kuppalli, K., Kindrachuk, J., & Peiris, M. (2020). Virology , transmission , and pathogenesis of SARS-CoV-2. 2019, 1–6.
Corman V. M., Landt O., Kaiser M., Molenkamp R., Meijer A., Chu D. K., Bleicker T., Brünink S., Schneider J., Schmidt M. L., Mulders D. G., Haagmans B. L., van der Veer B., van den Brink S., Wijsman L., Goderski G., Romette J. L., Ellis J., Zambon M., Peiris M., Goossens H., Reusken C., Koopmans M. P., & Drosten C. (2020). Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020 Jan;25(3):2000045. doi: 10.2807/1560-7917.ES.2020.25.3.2000045.
Dos Santos, C. A., de Oliveira, K. G., Mendes, G. M., Silva, L. C., de Souza, M. N., Estrela, P. F. N., Guimarães, R. A., Silveira-Lacerda, E. P., & Duarte, G. R. M (2021). Detection of SARS-CoV-2 in Saliva by RT-LAMP During Worker Screening in Brazil, Including Pre-Symptom Carriers. Journal of the Brazilian Chemical Society,32(11), 2071–2077. https://doi.org/10.21577/0103- 5053.20210098.
Gonza´lez., Mendoza-Ramos J. L., Pedroza S. C., Cuellar-Monterrubio A. A., Ma´rquezIpiña A. R., Lira-Serhan D. (2019). Validation of use of the miniPCR thermocycler for Ebola and Zika virus detection. Ansumana R, editor. PLoS One. Public Library of Science; 2019; 14: e0215642. https://doi.org/10.1371/journal.pone.0215642. PMID: 31071117.
Griesemer, S. B., Van Slyke, G., Ehrbar, D., Strle, K., Yildirim, T., Centurioni, D. A., Walsh, A. C., Chang, A. K., Waxman, M. J., & St. George, K. (2021). Evaluation of specimen types and saliva stabilization solutions for SARS-CoV-2 testing. Journal of Clinical Microbiology, 59(5), 1–13. https://doi.org/10.1128/JCM.01418-20
Huang, W. E., Lim, B., Hsu, C. C., Xiong, D., Wu, W., Yu, Y., Jia, H., Wang, Y., Zeng, Y., Ji, M., Chang, H., Zhang, X., Wang, H., & Cui, Z. (2020). RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2. Microbial Biotechnology, 13(4), 950–961. doi: 10.1111/1751-7915.13586. https://pubmed.ncbi.nlm.nih.gov/32333644/.
Jamal A. J., Mozafarihashjin M., Coomes E., Powis J., Li A. X., Paterson A., Anceva-Sami S., Barati S., Crowl G., Faheem A., Farooqi L., Khan S., Prost K., Poutanen S., Taylor M., Yip L., Zhong X. Z., McGeer A. J., & Mubareka S.; Toronto Invasive Bacterial Diseases Network COVID-19 Investigators. (2021). Sensitivity of Nasopharyngeal Swabs and Saliva for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2. Clin Infect Dis. 2021 Mar 15;72(6):1064-1066. doi: 10.1093/cid/ciaa848. PMID: 32584972; PMCID: PMC7337630.
Janíková, M., Hodosy, J., Boor, P., Klempa, B., & Celec, P. (2021). Loop-mediated isothermal amplification to detect SARS-CoV-2 in saliva.Microbial Biotechnology,14(1), 307–316. https:// doi.org/10.1111/1751-7915.13737.
Jaroenram W., Chatnuntawech I., Kampeera J., Pengpanich S., Leaungwutiwong P., Tondee B., Sirithammajak S., Suvannakad R., Khumwan P., Dangtip S., Arunrut N., Bantuchai S., Nguitragool W., Wongwaroran S., Khanchaitit P., Sattabongkot J., Teerapittayanon S., & Kiatpathomchai W. (2022). One-step colorimetric isothermal detection of COVID-19 with AI-assisted automated result analysis: A platform model for future emerging point-of-care RNA/DNA disease diagnosis. Talanta. 2022 Nov 1;249:123375. doi: 10.1016/j.talanta.2022.123375. Epub 2022 Mar 10. PMID: 35738204; PMCID: PMC9404558.
Kemenkes. 2021. Infeksi emerging : Situasi Terkini perkembangan covid 19. https://infeksiemerging.kemkes.go.id/. Diakses pada tanggal 29 November 2021.
Kobayashi, G. S.; Brito, L. A.; Moreira, D.d. P.; Suzuki, A. M.; Hsia, G. S. P.; Pimentel, L. F.; de Paiva, A. P. B.; Dias, C. R.; Lourenço, N. C. V.; Oliveira, B.A.; et al. (2021). A Novel Saliva RT-LAMP Workflow for Rapid Identification of COVID-19 Cases and Restraining Viral Spread. Diagnostics 2021, 11, 1400. https://doi.org/10.3390/diagnostics11081400
Lamb LE, Bartolone SN, Ward E, Chancellor MB. Rapid detection of novel coronavirus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by reverse transcription-loop-mediated isothermal amplification. PLoS One. 2020 Jun 12;15(6):e0234682. doi: 10.1371/journal.pone.0234682. PMID: 32530929; PMCID: PMC7292379.
Lu, R., Zhao, X., Li, J., Niu, P., Yang, B., Wu, H., Wang, W., Song, H., Huang, B., Zhu, N., Bi, Y., Ma, X., Zhan, F., Wang, L., Hu, T., Zhou, H., Hu, Z., Zhou, W., Zhao, L., & Tan, W. (2020). Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. The Lancet, 395(10224), 565–574. https://doi.org/10.1016/S0140-6736(20)30251-8.
Li J., Hu X., Wang X., Yang J., Zhang L., Deng Q., Zhang X., Wang Z., Hou T., & Li S. (2021). A novel One-pot rapid diagnostic technology for COVID-19. Anal Chim Acta. 2021 Apr 15;1154 : 338310. doi: 10.1016/j.aca.2021.338310. Epub 2021 Feb 11. PMID: 33736798; PMCID: PMC7877206.
Matic, N., Lawson, T., Ritchie, G., Stefanovic, A., Leung, V., Champagne, S., Romney, MG, & Lowe, C. F. (2021). Pengujian molekuler otomatis air liur untuk SARS-CoV-2 deteksi.Mikrobiologi Diagnostik dan Penyakit Menular,100(1),1–10. https://doi.org/10.1016/j.diagmicrobio.2021.115324
Mautner L., Christin K, B., Heike M, H., Wolfram V., Patrick G., Ute E., Nikolaus A., Andreas S., Melanie P., Ottmar G., Ulrich B., Lars W., Ingrid H., & Armin B. (2020). Rapid Point of Care Detection of SARS‑CoV‑2 Using Reverse Transcription Loop‑Mediated Isothermal Amplification (RT‑LAMP). Virology Journal, 17:160. https://doi.org/10.1186/s12985-020-01435-6.
Morais O. M., Azevedo Alves M. R., & Fernandes P. A. D. C. (2022). Impact of Thermal Pretreatment of Saliva on the RT-PCR Detection of SARS-CoV-2. Adv Virol. 2022 Jun 1;2022:7442907. doi: 10.1155/2022/7442907. PMID: 35693127; PMCID: PMC9177321.
Mukama O., Nie C., Habimana J. D., Meng X., Ting Y., Songwe F., A. l. Farga A., Mugisha S., Rwibasira P., Zhang Y., & Zeng L. (2020). Synergetic performance of isothermal amplification techniques and lateral flow approach for nucleic acid diagnostics. Anal Biochem. 2020 Jul 1;600:113762. doi: 10.1016/j.ab.2020.113762. Epub 2020 May 5. PMID: 32387190.
Nidom, R. v, Indrasari, S., Normalina, I., Nidom, A. N., Afifah, B., Dewi, L., Putra, A. K., Ansori, A. N. M., Kusala, M. K. J., & Alamudi, M. Y. (2021). Phylogenetic and full-length genome mutation analysis of SARS-CoV-2 in Indonesia prior to COVID-19 vaccination program in 2021. Bulletin of the National Research Centre, 45(1), 1–9. doi: 10.1186/s42269-021-00657-0. https://pmc.ncbi.nlm.nih.gov/articles/PMC8606223/.
OptiGene. (2020). LAMP User Guide-Mastermixes & Assay Optimisation.
Priatni, D., Alifuddin, M., & Djokosetiyanto, D. (2007). Effect of Heating at Various Temperatures for 30 Minutes on Pathogenicity of White Spot Syndrome Virus (WSSV) in Tiger Prawn (Penaeus monodon Fabr.). Jurnal Akuakultur Indonesia, 5(1), 5. https://doi.org/10.19027/jai.5.5-12.
Susilo, A., Rumende, C. M., Pitoyo, C. W., Santoso, W. D., Yulianti, M., Sinto, R., Yunihastuti, E. (2020). Coronavirus Disease 2019 : Tinjauan Literatur Terkini. Jurnal Penyakit Dalam Indonesia, 7(1), 45–67. doi: 10.7454/jpdi.v7i1.415. https://scholarhub.ui.ac.id/jpdi/vol7/iss1/8/.
Tanner, N. A., Zhang, Y., & Evans, T. C. (2015). Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. BioTechniques, 58(2), 59–68. https://doi.org/10.2144/000114253.
To, K. K.-W., Tsang, O. T.-Y., Yip, C. C.-Y., Chan, K.-H., Wu, T.-C., Chan, J. M.-C., Leung, W.-S., Chik, T. S.-H., Choi, C. Y.-C., Kandamby, D. H., Lung, D. C., Tam, A. R., Poon, R. W.-S., Fung, A. Y.-F., Hung, I. F.-N., Cheng, V. C.-C., Chan, J. F.-W., & Yuen, K.-Y. (2020). Consistent Detection of 2019. Novel Coronavirus in Saliva. Clinical Infectious Diseases, 71(15), 841–843 https://doi.org/10.1093/cid/ciaa149.
Wang, Q., Zhang, Y., Wu, L., Niu, S., Song, C., Zhang, Z., Lu, G., Qiao, C., Hu, Y., Yuen, K. Y., Wang, Q., Zhou, H., Yan, J., & Qi, J. (2020). Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2. Cell, 181(4), 894-904.e9. https://doi.org/10.1016/j.cell.2020.03.045
World Health Organization. (2020). WHO Coronavirus disease 2019 (COVID-19) dashboard. https.//covid19.who.int/. Diakses pada tanggal 29 November 2021.
Wu S., Liu X., Ye S., Liu J., Zheng W., Dong X., & Yin X. (2021). Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination. Heliyon. 2021 Apr;7(4):e06886. doi: 10.1016/j.heliyon.2021.e06886. Epub 2021 Apr 21. PMID: 33903853; PMCID: PMC8059943.
Xu, J., Zhao, S., Teng, T., Abdalla, A. E., Zhu, W., Xie, L., Wang, Y., & Guo, X.(2020). Systematic Comparison of Two Animal-to-Human Transmitted Human Coronaviruses: SARS-CoV-2 and SARS-CoV. Viruses, 12(2), 244. https://doi.org/10.3390/v12020244.
Yamazaki W, Matsumura Y, Thongchankaew-Seo U, Yamazaki Y, Nagao M. Development of a point-of-care test to detect SARS-CoV-2 from saliva which combines a simple RNA extraction method with colorimetric reverse transcription loop-mediated isothermal amplification detection. J Clin Virol. 2021 Mar;136:104760. doi: 10.1016/j.jcv.2021.104760. Epub 2021 Feb 11. PMID: 33610926; PMCID: PMC7877809.
Yu L, Wu S, Hao X, Dong X, Mao L, Pelechano V, Chen WH, Yin X. Rapid Detection of COVID-19 Coronavirus Using a Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) Diagnostic Platform. Clin Chem. 2020 Jul 1;66(7):975-977. doi: 10.1093/clinchem/hvaa102. PMID: 32315390; PMCID: PMC7188121.
