Direct Viral RNA Detection of SARS-CoV-2 and DENV in Inactivated Samples by Real-Time RT-qPCR: Implications for Diagnosis in Resource Limited Settings with Flavivirus Co-Circulation

Abstract

The RT-qPCR method remains the gold standard and first-line diagnostic method for the detection of SARS-CoV-2 and flaviviruses, especially in the early stage of viral infection. Rapid and accurate viral detection is a starting point in the containment of the COVID-19 pandemic and flavivirus outbreaks. However, the shortage of diagnostic reagents and supplies, especially in resource-limited countries that experience co-circulation of SARS-CoV-2 and flaviviruses, are limitations that may result in lesser availability of RT-qPCR-based diagnostic tests. In this study, the utility of RNA-free extraction methods was assessed for the direct detection of SARS-CoV-2 and DENV-2 in heat-inactivated or chemical-inactivated samples. The findings demonstrate that direct real-time RT-qPCR is a feasible option in comparison to conventional real-time RT-qPCR based on viral genome extraction-based methods. The utility of heat-inactivation and direct real-time RT-qPCR for SARS-CoV-2, DENV-2 viral RNA detection was demonstrated by using clinical samples of SARS-CoV-2 and DENV-2 and spiked cell culture samples of SARS-CoV-2 and DENV-2. This study provides a simple alternative workflow for flavivirus and SARS-CoV-2 detection that includes heat inactivation and viral RNA extraction-free protocols, with aims to reduce the risk of exposure during processing of SARS-CoV-2 biological specimens and to overcome the supply-chain bottleneck, particularly in resource limited settings with flavivirus co-circulation.

An improved RT-qPCR method for direct quantification of enveloped RNA viruses

“…A handful of direct (i.e. RNA extraction-free) RT-qPCR methods have been recently developed for SARS-CoV-2 virus detection [ 1 , 2 ]. As SARS-CoV-2 is a single-stranded positive-sense RNA (+ssRNA) enveloped virus [3] , these direct detection approaches rely on removing the lipid envelope, either by heat-inactivation or by use of detergents, such as Igepal [ 1 , 2 ], prior to amplification.…”

A review of current effective COVID-19 testing methods and quality control

“…A SARS-CoV-2 phylogenetic tree and other pathogenic CoVs (Hou et al 2020 ). B Overview schematic of the real-time RT-qPCR workflow (Mao et al 2021 ) …”

Novel Antiviral Efficacy of Hedyotis diffusa and Artemisia capillaris Extracts against Dengue Virus, Japanese Encephalitis Virus, and Zika Virus Infection and Immunoregulatory Cytokine Signatures

“…Over a third of the global population are at risk of flavivirus infection [ 1 , 2 , 3 ]. In the recent decade, flaviviruses have caused several global epidemics with high morbidity and mortality rates, in which a majority of these epidemics were caused by dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) [ 4 , 5 ].…”