Molecular Diagnosis of COVID-19: An Update and Review

Abstract

SARS-CoV-2 belongs to genus Betacoronavirus subgenus Sarbecovirus of the family Coronaviridae, which originated as a global public health problem. The disease caused is termed as Coronavirus Disease (COVID-19). The virus spread in more than 213 countries and territories all over the world and disease was declared as a pandemic. SARS-CoV-2 is an enveloped positive-sense single- stranded ribonucleic acid (ssRNA) virus. Severe infection and high-mortality are seen in patients with comorbid conditions like diabetes, hypertension, cancer, old age, malnutrition, children, and pregnancy. In India, strategy for testing of COVID-19 has been framed and revised over the course of time by the Indian Council of Medical Research (ICMR), Department of Health Research (DHR), Ministry of Health and Family Welfare (MOHFW), Government of India. ICMR has created a network of viral research and diagnostic laboratories (VRDLs) all over the country, and COVID-19 testing has also been undertaken by ICMR. AIIMS Jodhpur is a regional level VRDL, mentoring 38 government and private laboratories for molecular diagnosis of COVID-19 in the states of Rajasthan and Gujarat. Currently, real-time reverse transcription polymerase chain reaction (rRT-PCR) assay is used all over the country for diagnosis of COVID-19. It uses the TaqMan fluorogenic probe-based chemistry and 5′- nuclease activity of Taq DNA polymerase. It targets specific genes like the RdRp gene, HKU-Orf1ab gene, E gene, and N gene. Apart from rRT-PCR, other isothermal nucleic acid test (NAT)-like transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), etc. are emerging diagnostic tools to detect COVID-19. Cepheid Xpert Xpress SARS- CoV-2 test and Truenat betaCoV are point-of-care molecular assays which gives results in less than 1 hour, and helps to provide rapid and accurate results. Automated molecular assays like Cobas SARS-CoV-2 and Multiplex NAAT BioFire Respiratory Panel 2.1 (RP2.1) are newer techniques to curb the disease. Sherlock CRISPR SARS-CoV-2 kit is a highly specific and sensitive assay developed to diagnose COVID-19. rRT-PCR has been combined with techniques like bead hybridization, digital droplet PCR, microarray, etc. for improving the correct diagnosis. Recent molecular assays are the future pillars for containment of COVID-19 outbreaks all over the world.

Dynamics ofORF1abandNGene among hospitalized COVID-19 positive cohorts: A hospital based retrospective study

“…Meril COVID-19 One-step RTPCR Kit is approved by Drug Controller General of India (DCGI), Food and Drug Administration (FDA) and Foundation for Innovative New Diagnostics (FIND), Govt. Of India as RT-PCR molecular test that can be used in patient care settings [19]. Only single kit was considered for the experiment due kit to kit variations and issue of efficiency.…”

“…Nucleocapsid, a type of structural protein of SARS-CoV-2 is encoded by 908 nucleotides N gene at the near 3’ end and it’s a potential target for the detection of virus as recommended by CDC, United States [7, 17]. It is always advisable to use two molecular targets at a time to avoid potential cross- reaction with other endemic coronaviruses as well as potential genetic drift of SARS-CoV-2 [18, 19]. Studies from many countries reveal that investigators adopting two target strategy like Berlin Institute of Virology, Berlin and German Centre for Infection Research (DZIF), Berlin, Germany used RdRp and E gene [16]; National Institute for Viral Disease Control, China CDC used ORF1ab and N gene [20]; CDC, United States used two loci of N gene ( N1 and N2 ) [17].…”

Summary of the Detection Kits for SARS-CoV-2 Approved by the National Medical Products Administration of China and Their Application for Diagnosis of COVID-19

“…Currently, the most widely approved tests for the diagnosis of COVID-19 are based on PCR (Arun Krishnan et al 2020;Hu et al 2020;James and Alwneh 2020;Meo et al 2020;Oliveira et al 2020;Priyadarshi et al 2020;Shen et al 2020;Xu T et al 2020;. Two different strategies are used for PCRbased assays: rRT-PCR and isothermal amplification PCR.…”

“… 2020 ; James and Alwneh 2020 ; Meo et al . 2020 ; Oliveira et al 2020 ; Priyadarshi et al 2020 ; Shen et al . 2020 ; Wang H et al .…”

“…These three categories of detection kits play complementary roles in the management of the COVID-19 pandemic. SARS-CoV-2 viral RNA and antigens can be identified in SARS-CoV-2-infected individuals during the acute phase of infection, while serological tests subsequently identify individuals who have developed antibodies to the virus, which can be used for close contact tracing and monitoring of the immune status of individuals and groups over time (Arun Krishnan et al 2020;Hu et al 2020;James and Alwneh 2020;Meo et al 2020;Oliveira et al 2020;Priyadarshi et al 2020;Shen et al 2020;Xu T et al 2020).…”