Tesis

Pengembangan Sistem Deteksi SARS-CoV-2 Berbasis Metode Loop Mediated Isothermal Amplification (LAMP) = Development of SARS-CoV-2 Detection System Based on Loop Mediated Isothermal Amplification (LAMP) Method.

Pandemi virus SARS-CoV-2 yang terjadi telah membuat kebutuhan untuk pemeriksaan massal deteksi virus menjadi meningkat. Saat ini pemeriksaan gold standard untuk mendeteksi virus SARS-CoV-2 adalah dengan metode reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) yang berbiaya mahal. Oleh karena itu dibutuhkan metode pemeriksaan alternatif yang lebih murah, cepat, dan mudah digunakan sebagai sistem deteksi virus. Metode Loop Mediated Isothermal Amplification (LAMP) dapat melakukan reaksi amplifikasi nukleotida pada suhu isothermal tanpa perlu mesin thermal cycler dan dapat diamati langsung dengan penambahan zat pewarna. Metode deteksi virus berbasis LAMP yang dikembangkan menggunakan desain primer yang menarget gen S dan ORF1ab ditambah pewarna Calcein-Mangan dan senyawa tambahan Guanidine Hydrochloride (GuHCl). Sistem deteksi dilakukan optimasi konsentrasi komponen reaksi, suhu dan template yang menggunakan plasmid DNA kontrol. Optimasi didapatkan dengan konsentrasi reaksi FIP/BIP 0,4 µM, F3/B3 0,2 µM, Loop F/P 0,4 µM, Calcein-Mangan 12 µM, dan GuHCl 40 mM. Sistem LAMP yang dikembangkan dapat mendeteksi sekuens gen target pada DNA kontrol hingga 1 copy number dalam waktu sekitar 1 jam pada inkubasi suhu 55 ºC. Meski begitu, sistem LAMP yang dikembangkan belum mapu mengamplifikasi RNA virus SARS-CoV-2 hasil ekstraksi sampel swab pasien. Hal ini disebabkan oleh enzim Bst 3.0 yang dipakai dalam sistem LAMP tidak memiliki kemampuan reverse transcriptase yang diharapkan.
Kata kunci: SARS-CoV-2, Loop Mediated Isothermal Amplification (LAMP), Polymerase Chain reaction (PCR), Deteksi Virus, Calcein-Mangan.


The SARS-CoV-2 virus pandemic has made the need for mass screening of virus detection increased. Currently, the gold standard test to detect SARS-CoV-2 virus is reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) method that costly. Therefore, an alternative method that is cheaper, faster, and easier to use as a virus detection system is needed. The Loop Mediated Isothermal Amplification (LAMP) method can perform nucleotide amplification reactions at isothermal temperatures without the need of thermal cycler machine and can be observed directly with the addition of coloring agents. The LAMP-based virus detection method development use primers design targeting the S and ORF1ab genes with Calcein-Manganese dyes and the additive compound Guanidine Hydrochloride (GuHCl). The detection system performed optimization of the concentration of reaction components, temperature and template using plasmid DNA control. Optimization was obtained with reaction concentration of FIP/BIP 0.4 μM, F3/B3 0.2 μM, Loop F/P 0.4 μM, Calcein-Manganese 12 μM, and GuHCl 40 mM. The developed LAMP system can detect target gene sequences in control DNA up to 1 copy number in about 1 hour at 55 ºC incubation temperature. Even so, the LAMP system developed still unable to amplify the RNA of the SARS-CoV-2 virus from the extraction of patient swab samples. This issue occurred due to the Bst 3.0 enzyme that used in the reaction did not have reverse transcriptase activity as expected.
Keywords: SARS-CoV-2, Loop Mediated Isothermal Amplification (LAMP), Polymerase Chain Reaction (PCR), Viral Detection, Calcein-Mangan.