Development of In Vitro qPCR-based Diagnostic Tests for the Detection of Viral Nucleic Acid Sequences in Biological Samples

Abstract

In 2019, the virus SARS-COV-2 caused a worldwide pandemic necessitating rapid and accurate diagnostic tests. RT-qPCR testing of nasopharyngeal swab samples is considered the gold standard diagnostic technique. The envelope (E1) gene is a viral conserved genes that is considered a suitable target for an amplification-based diagnosis. Standard RT-qPCR requires RNA purification, which is both a time and resource-consuming. This study used in vitro transcribed E1 RNA to assess transcript pull down as a simple, cost-effective alternative technique to RNA purification. Different cDNA primers, reagents, and temperatures were tested to determine the optimal RT-qPCR reaction conditions. A synthetic saliva cell (SSC) solution with eukaryotic cells (LAMA78 cell) and RNA transcript was used to determine reaction efficiency. This study revealed that the use of the E1 qPCR primer in the reverse transcription step increases RT-qPCR efficiency compared to a cDNA biotin primer. Pull down of E1 gene transcript decreases reaction efficiency.