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

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

Heliyon. 2020 Jul 7;6(7):e04405. doi: 10.1016/j.heliyon.2020.e04405. eCollection 2020 Jul.

Authors

Bhasker Dharavath^{1

2}, Neelima Yadav^{1

2}, Sanket Desai^{1

2}, Roma Sunder¹, Rohit Mishra¹, Madhura Ketkar^{3

2}, Prasanna Bhanshe^{4

2}, Anurodh Gupta^{4

2}, Archana Kumari Redhu¹, Nikhil Patkar^{4

2}, Shilpee Dutt^{3

2}, Sudeep Gupta^{5

2}, Amit Dutt^{1

2

6}

Affiliations

¹ Integrated Cancer Genomics Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Kharghar, Navi Mumbai, Maharashtra, 410210, India.
² Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai, Maharashtra, 400094, India.
³ Shilpee Dutt Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Kharghar, Navi Mumbai, Maharashtra, 410210, India.
⁴ Haematopathology Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Kharghar, Navi Mumbai, Maharashtra, 410210, India.
⁵ Department of Medical Oncology, Advanced Centre for Treatment, Research, and Education in Cancer, Kharghar, Navi Mumbai, Maharashtra, 410210, India.
⁶ Adjunct Faculty, Institute of Advanced Virology, Kerala State Council for Science, Technology and Environment, Govt of Kerala, Thonnakkal, Kerala, 695317, India.

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.

Keywords: COVID qPCR analyzer; COVID-19 detection assay; Diagnostics; Genomics; Graphical user interface (GUI); Infectious disease; Microbial genomics; Molecular biology; Real-time RT- PCR; SARS-CoV-2; Virology.