A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection

Abstract

With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable.

Early and long term antibody kinetics of asymptomatic and mild disease COVID-19 patients

“…Real-time quantitative reverse transcriptase polymerase chain reactions (qRT-PCRs) were performed using the TaqPath Covid-19 RT-PCR Kit (A48067; ThermoFisher Scientific, Massachusetts, USA), a fast, highly sensitive, multiplex and robust RT-qPCR assay for the detection of SARS-CoV-2 28 . Nucleic acids were isolated according to manufacturer’s instructions.…”

Review of Current COVID-19 Diagnostics and Opportunities for Further Development

“…These allow simultaneous detection of two or more target sequences via specific fluorescent-labeled probes (65). For instance, the FDA emergency use authorized Abbott RealTime SARS-CoV-2 assay is a dual target RT-PCR assay that detects RdRp and N genes; the TaqPath TM COVID-19 Combo Kit by Life Technologies (Thermo Fisher Scientific, Inc.) employs quantitative recognition of ORF1ab, N, and S genes simultaneously (125,126). However, the CDC and WHO recommend separating internal control reactions as opposed to multiplexing them in the same PCR reaction with SARS-CoV-2 target genes because relatively high levels of human RNase P RNA compared to SARS-CoV-2 viral RNA may reduce sensitivity of SARS-CoV-2 target genes when multiplexed in one reaction (63).…”

Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2

“…Additionally, RNA-dependent RNA polymerase (RdRP) gene encodes an enzyme responsible for viral replication. These indispensable genes are the main target for the molecular detection of SARS-CoV-2 [12,[26][27][28][29]. Kakhki et al [30] PLOS ONE reported that ORF8 gene is a possible target for detecting COVID-19 and it is different from reported genes (RdRP, E and N genes), and ORF8 is fully specific to COVID-19 and has no cross-reactivity with other types of coronaviruses.…”