A one-step, one-tube real-time RT-PCR based assay with an automated analysis for detection of SARS-CoV-2

Bhasker Dharavath,Neelima Yadav,Sanket Desai,Roma Sunder,Rohit Mishra,Madhura Ketkar,Prasanna Bhanshe,Anurodh Gupta,Archana Kumari Redhu,Nikhil Patkar,Shilpee Dutt,Sudeep Gupta,Amit Dutt

doi:10.1016/j.heliyon.2020.e04405

Abstract

Early diagnosis of SARS-CoV-2 infected patients is essential to control the dynamics of the COVID-19 pandemic. We develop a rapid and accurate one-step multiplex TaqMan probe-based real-time RT-PCR assay, along with a computational tool to systematically analyse the data. Our assay could detect to a limit of 15 copies of SARS-CoV-2 transcripts—based on experiments performed by spiking total human RNA with in vitro synthesized viral transcripts. The assay was evaluated by performing 184 validations for the SARS-CoV-2 Nucleocapsid gene and human RNase P as an internal control reference gene with dilutions ranging from 1-100 ng for human RNA on a cohort of 26 clinical samples. 5 of 26 patients were confirmed to be infected with SARS-CoV-2, while 21 tested negative, consistent with the standards. The accuracy of the assay was found to be 100% sensitive and 100% specific based on the 26 clinical samples that need to be further verified using a large number of clinical samples. In summary, we present a rapid, easy to implement real-time PCR based assay with automated analysis using a novel COVID qPCR Analyzer tool with graphical user interface (GUI) to analyze the raw qRT-PCR data in an unbiased manner at a cost of under $3 per reaction and turnaround time of less than 2h, to enable in-house SARS-CoV-2 testing across laboratories.

Highlights

Diagnostics can play an important role in the containment of COVID19, enabling rapid implementation of control measures that limit the spread through case identification, isolation, and contact tracing
We describe a comparative account of qRT-PCR based detection of SARS-CoV-2 using SYBR Green I and Taqman probes
SYBR Green-based qRT-PCR protocol is comparatively economical, the nonspecific binding of the dye to DNA resulted in low specificity and sensitivity with limit of detectability of 150 copies of viral RNA molecules/ reaction, whereas TaqMan probe-based protocol could detect as low as 15 copies of viral RNA molecules/reaction suggesting higher sensitivity

Summary

Introduction

Diagnostics can play an important role in the containment of COVID19, enabling rapid implementation of control measures that limit the spread through case identification, isolation, and contact tracing (i.e., identifying people that may have come in contact with an infected patient). Apart from conventional RT-PCR and quantitative RT-PCR, various studies have reported modifications like nested rRT-PCR, RT-PCR with locked nucleic acid probes and RT-Loop Mediated isothermal Amplification (RT-LAMP) for detection of Coronaviruses [9, 10, 11], e.g. the RT-LAMP method for detection of MERS-CoV with sensitivity as high as ~3.4 copies of RNA/reaction, a microarray-based methodology to detect strains of coronaviruses simultaneously, CRISPR effector Cas based SHERLOCK for viral RNA detection with isothermal amplification, and more recently for the COVID-19 [9, 12, 13, 14] Despite such varied efforts to increase sensitivity and specificity of the viral diagnostics, there is still an unmet need for user-friendly and cost-effective detection method to combat the ongoing catastrophe created by SARS-CoV-2

Methods

Results

Conclusion