Primer-probe set test for N1, N2, and RdRp genes from SARS-CoV-2 detection using multiplex RT-qPCR

COVID-19 pandemic originated in December 2019 due to SARS-CoV-2 virus infection. Standardized SARS-CoV-2 detection is examined by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) with N and RdRp genes as the main target. Local SARS-CoV-2 detection product is not readily available with only N2, RdRp, and Orf1b as gene targets. This study aims to optimize multiplex RT-qPCR with two pairs of primer set targeting N and RdRp genes with RPP30 gene as internal control. The study was done using several methods: Escherichia coli culture production and plasmid extraction, HepG2 cell RNA extraction, PCR, RT-PCR, RT-qPCR, and electrophoresis. The result of the earlier steps was successful plasmid and RNA extraction. PCR on plasmid targeting N and RdRp genes and RT-PCR on RNA targeting RPP30 gene giving results of bands appearance with the size around 100 bp after electrophoresis is done. RT-qPCR of two pairs of primer set generally giving positive results with the appearance of logarithmic graph on RT-qPCR plots. The conclusion of the study is RT-qPCR was done successfully using two sets of primer that targeting N and RdRp genes with RPP30 gene as internal control.