A one-step, one-tube real-time RT-PCR based assay with an automated analysis for detection of SARS-CoV-2. Issue 7 (July 2020)

• Record Type: Journal Article
• Authors: Dharavath, Bhasker; Yadav, Neelima; Desai, Sanket; Sunder, Roma; Mishra, Rohit; Ketkar, Madhura; Bhanshe, Prasanna; Gupta, Anurodh; Redhu, Archana Kumari; Patkar, Nikhil; Dutt, Shilpee; Gupta, Sudeep; Dutt, Amit
• Is Part Of: Heliyon. Volume 6:Issue 7(2020)
• Publication Date: 2020-07
• Subjects: Infectious disease -- Microbial genomics -- Genomics -- Molecular biology -- Virology -- Diagnostics -- SARS-CoV-2 -- Real-time RT- PCR -- COVID-19 detection assay -- COVID qPCR analyzer -- Graphical user interface (GUI); Research -- Periodicals; Medical sciences -- Periodicals; Natural history -- Periodicals; Social sciences -- Periodicals; Earth sciences -- Periodicals; Physical sciences -- Periodicals; 507.2
• DOI: 10.1016/j.heliyon.2020.e04405
• Languages: English
• ISSNs: 2405-8440

Abstract

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