Development and validation of cost-effective SYBR Green-based RT-qPCR and its evaluation in a sample pooling strategy for detecting SARS-CoV-2 infection in the Indonesian setting

Abstract Coronavirus disease 2019 or COVID-19 is caused by SARS-CoV-2 infection and it was first recorded in Wuhan, China, in December 2019. The gold standard method for detecting SARS-CoV-2 is TaqMan probe-based real-time polymerase chain reaction (RT-qPCR). Since this technique is expensive due to reliance on the use of fluorogenic probes, a cost-effective diagnostic test as an alternative approach for RT-qPCR diagnosis is necessary, especially in low- and middle-income countries with limited resources. In this study, we developed and validated an inexpensive SYBR Green-based RT-qPCR method to detect SARS-CoV-2. Primers targeting a conserved and vital region of the N genes of SARS-CoV-2 were designed and selected for further analysis. In-silico study was performed to analyse the compatibility of the selected primer pair with 500 Indonesian SARS-CoV-2 genome sequences available from the GISAID database, including Delta and Omicron variants. We determined the linearity of our new assay using serial dilution of SARS-CoV-2 RNA from clinical samples with known virus concentration. The assay was then evaluated using clinically relevant samples in comparison to a commercial TaqMan-based in vitro diagnostic test kit. Finally, we applied the assay in sample pooling strategies for SARS-CoV-2 detection. Our results showed that the SYBR Green-based RT-qPCR methods were sensitive and reliable as an alternative cost-effective assay for SARS-CoV-2 detection. In addition, it could be used in sample pooling strategies for mass screening testing or epidemiological surveillance purposes in the COVID-19 post-pandemic era.