M gene targeted qRT-PCR approach for SARS-CoV-2 virus detection

Abstract

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection, and several qRT-PCR kits have been established targeting different genes of the virus. Due to the high mutation rate of these genes, false negative results arise thus complicating the interpretation of the diagnosis and increasing the need of alternative targets. In this study, an alternative approach for the detection of SARS-CoV-2 viral RNA targeting the membrane (M) gene of the virus using qRT-PCR was described. Performance evaluation of this newly developed in-house assay against commercial qRT-PCR kits was done using clinical oropharyngeal specimens of COVID-19 positive patients. The limit of detection was determined using successive dilutions of known copies of SARS-CoV-2 pseudovirus. The M gene based assay was able to detect a minimum of 100 copies of virus/mL indicating its capacity to detect low viral load. The assay showed comparable accuracy, sensitivity and specificity with commercially available kits while detecting all the variants efficiently. The study concluded that the in-house M gene based assay might be an effective alternative for the currently available commercial qRT-PCR kits.

Figure 1

A serial dilution of pseudovirus containing SARS-CoV-2 viral genome (10⁵, 10⁴, 10³, 10² and 10¹ copies/mL) was prepared. Represents continuous amplification of all replicates of gradient dilution particles of pseudovirus containing SARS-CoV-2 genome.

Figure 2

Differential dilutions of (15,000 copies/mL, 10,000 copies/mL, 100 copies/mL, 10 copies/mL) extracted variant reference materials were prepared. (A–D) Represent continuous amplification of Wuhan, UK, Brazil and African variant respectively.

Figure 3

Mutation rate of M gene of SARS-CoV-2 virus.