SARS-CoV-2 Direct Real Time PCR without RNA Extraction: Appropriate Method or Not?

Spandan Chaudhary,Mayank Patadiya,Prathana Singhania,Shiv Patel,Juhi Patel,Nilay Dave,Ashish Hirpara,Pooja Chaudhary,Disha Patel,Ramachandiran Sivaramakrishnan,Tushar Sonagara,Ekta Jajodia,Kavisha Vyas,Neeraj Arora

doi:10.24018/ejmed.2022.4.1.1164

Abstract

Real time PCR (RT-PCR) detection method is the widely used for COVID-19 virus detection. This includes sample collection in viral transport medium (VTM), viral RNA extraction followed by detection of virus using fluorescence dye-based system using RT-PCR machine. Several studies have demonstrated a new method which replaces the extraction step by a simple method involving DTT and Proteinase-K and heat treatment. ICMR and few other governing bodies have approved such protocols but are they appropriate in clinical context? In present study, we tried to evaluate one such protocol by using ICMR and WHO approved COVID-19 detection protocol (of CoviPath™ COVID-19 RT-PCR Kit) by replacing RNA extraction step. We used 228 clinical COVID-19 samples for studying method which includes 176 positive (CT values from 14 to 23; 24 to 31 and 32 to 37 were considered as high, moderate and low positive respectively) and 52 negative nasopharyngeal and oropharyngeal swabs samples. We got 100% concordant results with negative samples and 92% concordant and 8% non-concordant results for positive samples. Non-concordant results are with low positive samples. Low level of positivity in the samples could indicate the initial/end stage of COVID-19 disease. If they are at the initial stage, they can be the potential carrier and spread the disease. Authors believe that direct methods can be used for screening bit not for diagnosis of COVID-19 disease.

Highlights

The SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is the causative agent of the global pandemic Coronavirus Disease (COVID-19)
We developed a method for direct RNA extraction from samples transported in viral transport medium (VTM) using two chemicals: Proteinase-K (PK) and dithiotheritol (DTT) and high temperature incubation
Though SARS-CoV-2 detection by reverse transcription–polymerase chain reaction (RT-PCR) is not a quantitative assay but cycle threshold (CT) values is considered for the therapy purpose and disease progress monitoring

Summary

Introduction

The SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is the causative agent of the global pandemic Coronavirus Disease (COVID-19). SARS-CoV-2 is an enveloped RNA virus of the betacoronavirus genus the family Coronaviridae, and the order Nidovirales [2]. Four subfamilies of coronaviruses are known: alpha, beta, gamma, and delta. SARS-CoV-2 was found to infect more human beings than either of its predecessors that include the SARS-CoV and the Middle East respiratory syndrome virus (MERS) [5]. The SARS-CoV-2 carries non-segmented positive strand RNA genome of 30 kb belonging to subgenus Sarbecoviruses. The viral contains four structural proteins and sixteen nonstructural proteins [6]. The four major structural genes encode the nucleocapsid protein (N), spike protein (S), membrane glycoprotein (M), and small membrane protein (SM) [3]

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