key: cord-294483-mozabpcs
authors: Choudhary, Manohar Lal; Vipat, Veena; Jadhav, Sheetal; Basu, Atanu; Cherian, Sarah; Abraham, Priya; Potdar, Varsha A.
title: Development of in vitro transcribed RNA as positive control for laboratory diagnosis of SARS-CoV-2 in India
date: 2020-04-28
journal: Indian J Med Res
DOI: 10.4103/ijmr.ijmr_671_20
sha: 
doc_id: 294483
cord_uid: mozabpcs

nan

Using whole-genome sequence of the first Indian COVID-19 case, forward primers with T7 promoter tag at the 5´ end, were designed to amplify full-length E gene, N gene and partial RdRp and ORF 1b regions. Gene-specific PCR was carried out to amplify the desired PCR product. Amplicons were purified using Qiagen direct PCR purification kit (Qiagen, Hilden, Germany). IVT was synthesized using T7 Riboprobe (Promega, USA) as per the kit protocol. Ten-fold serial dilutions of each transcribed RNA products were tested with respective gene primer probe sets for specific detection and limit of detection.

Gene-specific desired amplification was also observed in conventional RT-PCR (E: 550 bp, N: 1254 bp, RdRP: 344, ORF: 388) (Fig.1) . Further, the IVT RNA of each gene was serially diluted 10-fold (10 1 to 10 10 ), and the performance was tested with genespecific primer probe by real-time RT-PCR. All the transcribed RNA showed amplification with specific primer probe. The limit of detection for E gene was 10 6 yielding a cycle threshold (Ct) at cycle 29, RdRP (p1) was 10 5 with 27 Ct, RdRp (p2) was 10 6 with 29 Ct and ORF 1b10 6 with 28 Ct, whereas N gene showed 10 3 with 25 Ct ( Fig. 2A-E) .

When the assay was first set up at the National Influenza Centre of ICMR-NIV, Pune, the IVT RNA for E and SARS coronavirus Frankfurt 1 strain were received from EVAg. The real-time PCR screening assay (E gene) was also established at the 13 VRDLs as part of ICMR's efforts to expand testing to VRDLs closer to major airports 3 . However, due to screening of low number of samples, the repeated use of positive controls was made. The supply of IVT RNA as positive control for E gene from EVAg was limited and had non-consistent performance when diluted further. In addition, the control for RdRp assay was from a SARS coronavirus Frankfurt 1 isolate, which yielded weak signal with RdRp Wuhan-specific probe. This necessitated the development of an indigenous IVT RNA for E and RdRp. In addition, majority of the WHO screening protocols (5 of 6) are based on N gene targeting different nucleotide positions and require multiple specific positive controls 4 . Hence, an IVT RNA was designed for entire N gene which would be compatible for multiple protocols.

We demonstrated the successful use of IVT RNA for N gene recommended in various protocols available on WHO site. The partial RdRp IVT RNA worked well with both the RdRp probes described in Charité, Berlin, Germany 5 , especially Wuhan-specific RdRp probe 2, which could be used as confirmatory test. All the IVT RNA had good yield and performed well with specific primer probe.

In conclusion, gene-specific IVT RNA was synthesized for all the gene targets used in real time PCR. These IVTs were used effectively by all VRDLs as positive control. Successful establishment of diagnostic system including in-house positive control was beneficial to provide timely diagnosis and accelerate clinical management and isolation of SARS-CoV-2 patients and to control further spread. 

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia

Establishment of a network of laboratories for managing epidemics and natural calamities (VRDL)

Laboratory testing for 2019 novel coronavirus (2019-nCoV) in suspected human cases. WHO; 2020

Coronavirus disease (COVID-19) technical guidance: Laboratory testing for 2019-nCoV in humans. WHO; 2020