Using its expertise in genome sequencing and analysis pipeline of genome sequence data, the Delhi-based CSIR lab Institute of Genomics and Integrative Biology (IGIB) has successfully developed a unique pool of primers and kits to be used in RT-PCR testing of SARS-CoV-2 virus. The most distinguishing aspect of the work carried by a team led by Dr. Sridhar Sivasubbu and Dr. Vinod Scaria at IGIB that was developing primers that will not be affected by mutations seen in SARS-CoV-2 variants. This may allow the primers to detect any new SARS-CoV-2 variants that might emerge immaterial of the novel mutations that the variants might have. The primers developed in a way future-proofs the ability to detect without fail any new SARS-CoV-2 variants that may emerge.
Many mutations
The Omicron variant, for instance, has 32 mutations in the spike protein alone. None of these mutations were seen in other variants that emerged earlier — the Alpha, Beta, Gamma and Delta. Each new variant that emerges develops a unique set of mutations that makes it more transmissible and/or causes severe disease.
RT-PCR tests currently available contain primers for two or more genes. The gene targets selected could be a combination of S gene, N gene and E- gene.
Based on over 40,000 high-quality SARS-CoV-2 virus genome sequence data available in the public domain including a significant large number of genome sequences from India, Dr. Sivasubbu’s team, which includes two PhD students — Paras Sehgal and Gyan Ranjan, was able to identify nearly 80,000 unique variants. The high number of variants seen is because SARS-CoV-2 is a rapidly mutating virus and tends to collect mutations during infection. The SARS-CoV-2 is naturally endowed to collect about one mutation every 10-14 days.
“Though the SARS-CoV-2 is a rapidly mutating virus, we found that not all regions of the virus were accumulating mutations at the same rate. There are some regions of the virus which collect mutations at a fast rate while some other regions tend to collect mutations at a slow rate,” says Dr. Sivasubbu.
“We at IGIB already had an algorithm that allowed us to look at the conserved regions that were not prone to accumulating mutations.. This allowed us to analyse which regions of the virus are conserved and which were not,” says Dr. Sivasubbu.
Conserved region of a virus contains genes which are very essential and hence either do not undergo any mutations or such mutations happen rarely.
In parallel, the IGIB researchers had also collected information about primers used in commercial and non-commercial tests across the world. “Once we had the mutation map of the SARS-CoV-2 virus, and by using the information gathered by studying the primers used across the world, we found that a majority of primers designed and used were overlapping with the regions of the virus collecting significant number of mutations,” he says.
Strategic problem
“The primers widely in use have a small strategic problem. We realised that at some point in the future, the existing primers used in RT-PCR test kits for detecting SARS-CoV-2 virus would not be able to bind properly to the genome leading to erroneous results,” Dr. Sivasubbu says. The primers were tested to detect all the SARS-CoV-2 variants — the Alpha, Beta, Gamma, Delta and Omicron. Based on the knowledge of the conserved regions in the virus, the team developed a pool of primers that would be future-proof. Each RT-PCR test uses at least two gene targets and each gene target requires a pair of primers. The pool of primers developed by IGIB will allow them to be used in various combinations.
“The pool of primers has been developed to target regions of the virus which are unlikely to undergo mutations. So the primers developed for RT-PCR tests will perform very well when new variants emerge,” says Dr. Anurag Agrawal, Director of IGIB.
The development of the mutation map of SARS-CoV-2 virus and the pool of primers were done in collaboration with Siemens Healthcare Private Limited. The technology has been transferred to the company and the product tested and approved by ICMR and launched on January 25 this year.
Siemens will be manufacturing the tests (IMDX SARS-CoV-2 Multiplex ORF1ab/N gene) at its new plant at Vadodara. According to the company spokesperson, the tests have 100% sensitivity and specificity. The plant has a capacity to manufacture 12 million tests per month. “The company will strive to make it affordable and accessible,” the spokesperson said.
Used worldwide
The project began in May–June 2020 when Illumina launched its new Covidseq Next-Generation Sequencing for SARS-CoV-2 virus and the first global field validation was carried out in India at IGIB. Nearly 1,700 genome sequence samples from seven States were sequenced at IGIB using Covidseq Next-Generation Sequencing as part of the field validation. Once the field validation by IGIB was completed, countries across the world started using Illumina’s new Covidseq genome sequencing protocol. By the end of December 2020, a large number of SARS-CoV-2 genomes were sequenced across the world, including 7,000 from India. Of the 7,000 genomes sequenced in India, 50% were exclusively by IGIB in collaboration with other entities in India.
