DEVELOPMENT OF INACTIVATED VIRAL TRANSPORT MEDIUM (VTM) TO SUPPORT SARS-COV-2 DIAGNOSTIC TESTING IN INDONESIA

• Main Author: Rahmani, Silmi
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/72616
• Institution: Institut Teknologi Bandung

COVID-19 pandemic created an urgent needed for diagnostic test components for the detection of SARS-CoV-2, including viral transport medium (VTM), a medium that stores virus samples in clinical specimens. However, VTM without the ability to inactivate the infectious of viable SARSCoV- 2, is known to endanger personnel involved in COVID-19 diagnostic detection. Chaotropic agent, such as guanidium thiocyanate, applied together with anionic detergents; chelators; buffers; and surfactants in an inactive VTM (iVTM) formulation are reported to be able to completely inactivate viruses and protect polynucleotides from RNAase degradation. This research study aimed to determine the best iVTM formulation (pH 4 and pH 6), evaluate the stability of viral RNA in iVTM for 30 days in two storage conditions (at 4oC and 25-28oC), and validating iVTM for RT-PCR based detection of SARS-CoV-2 compared to Jun Nou®VTM (commercial VTM). In vitro inactivation test used Indonesian SARS-CoV-2 isolate (variant B1) as a virus model. SARS-CoV-2 was cultivated on Vero E6 cells with an MOI (Multiplicity of Infection) of 0,01. Virus infectivity after the inactivation process was determined by the TCID50 (50% Tissue Culture Infective Dose) method. The viral titer was calculated according to Reed & Muench formula. Viral RNA for stability test was determined by multiplex RT-qPCR that targets the RdRp and Helicase genes of the SARSCoV- 2. However, viral RNA for validity test targets the E, N, and ORf1b genes of the SARS-CoV- 2. This study was carried out in a type II BSC (Biosafety Cabinet) with a level of safety of Biosafety Level 3 (BSL 3) at the Biotechnology Research Center, National Research and Innovation Agency (BRIN). The results demonstrate that the iVTM pH 6 is the best formulation compared to iVTM pH 4. The stability test results showed that iVTM pH 6 could maintained SARS-CoV-2 RNA for up to 30 days at both 4?C and 25-28?C (room temperature) storage conditions. The inactivation efficacy assay showed that iVTM pH 6 could eliminate the infectivity of high concentrations of SARS-CoV- 2 (reducing virus titer for more than 4.5 log10) for 5, 10, and 30-minute incubation time. In addition, long-term stability tests on iVTM pH 6 for 4 months (at 25-28oC) showed clarity, good stability in pH, no changes in colour solution, and free from bacteria and fungi viability. The validation test result showed that iVTM pH 6 could detect 20 positive cases of suspected COVID-19 patients from 20 clinical specimens tested through the RT-qPCR. In conclusion, we conclusively revealed that iVTM pH 6 securely handles SARS-CoV-2 specimens within 5 minutes, ensures the integrity of viral RNA during storage, and preseve RNA in the medium for 30 days at room temperature. These promising findings encourage iVTM's future application as a viral inactivation and stabilization media for RT-PCR-based detection of SARS-CoV-2 to support COVID-19 testing in Indonesia.