Key to isolation of infected patients is detection of the virus at the earliest and hence the need to expand the diagnostic scale in the country multifold. CSIR labs are well equipped with the technology and know-how of Coronavirus diagnostic tests and also possess expertise in developing newer and rapid diagnostics.
Diagnostic Testing of Coronavirus Samples: CSIR is deeply involved in the testing of human samples for the presence of coronavirus infection using the RT-PCR test. Several of its laboratories are engaged in the work:
- Laboratories already testing: CSIR-CCMB, CSIR-IIIM, CSIR-IMTECH, CSIR-NBRI, CSIR-CFTRI, CSIR-CDRI, CSIR-IHBT, CSIR-IITR, CSIR-NEERI, CSIR-IGIB, CSIR-CLRI,CSIR-IIP and CSIR-NEIST
- Laboratories Supporting State Governments with RT-PCR machines: CSIR-NIIST and CSIR-NIO
- The total number of tests done by CSIR labs (as on 20 November, 2021) are given below:
Sr. No Name of Lab No. of Samples Tested 1 CSIR-IIIM 94429 2 CSIR-IMTECH 8821 3 CSIR-IHBT 182095 4 CSIR-NEERI 68309 5 CSIR-IITR 383916 6 CSIR-CCMB 86521 7 CSIR-CDRI 121778 8 CSIR-CLRI 79773 9 CSIR-IGIB 16660 10 CSIR-NEIST 37442 11 CSIR-IIP 28147 12 CSIR-NBRI 266582 13 CSIR-CFTRI 549439 Total 1923912
Training of Human Resource for Testing: CSIR is engaged in training of human resource for conducting coronavirus testing. More than 180 people (outside CSIR) trained so far.
- Countrywide Deployment of Indigenous Truenat PCR Testing Handheld device Truenat developed under CSIR-NMITLI has been approved for testing of the coronavirus at country level. It can capture data from the installed machines and feed the central database for surveillance purposes. About 1000 devices are being used across the country for coronavirus testing. The cost per test is about Rs.1200 as against Rs.4500/test of RT-PCR test.
- Diagnostics Validation Centre: Several Indian companies are coming up with new diagnostic kits that need to be validated by the government before accepting them and putting them to use. CSIR-CCMB is the only non-ICMR designated Validation Center and CSIR-IGIB is ready to be a Validation Centre.
Development of new, faster more sensitive and cheaper diagnostic test: CRISPR/ Cas based paper diagnostic test (FELUDA) A CRISPR/ Cas -based paper diagnostic test has been developed by CSIR. The FELUDA methodology has been developed in CSIR-IGIB for detecting single nucleotide variants in RNA or DNA or more broadly detecting any DNA or RNA fragment, without the need for sequencing. The principle of discrimination is derived from the natural property of the enzyme being used for the invention, Francisella novicida Cas9 (FnCas9) which shows a very low binding affinity to mismatched substrates. For COVID-19 detection, RNA is isolated from body fluids containing the inactivated virus, converted to DNA, and further amplified to increase the number of copies. The amplification is done using custom generated primers resulting in DNA products labeled with biotin ligand. Upon incubation of a CRISPR complex (labeled with a Fluorescein amidite group) with these DNA products, the Cas9 protein interacts with the biotin-labeled DNA only if there is a CoV-2 sequence present. The complex formed is then applied on a paper strip whose chemistry is defined by the production of the test line where the Cas9: DNA complex gets deposited giving rise to a visible band. The presence of this line shows that the patient sample is positive for COVID-19.
- Currently, the technology is being regularly validated both at CSIR-IGIB and NCDC on a large number of patient samples to increase the robustness of detection, and tie-ups with industrial partners have been initiated for scaling up for deployment. Tata Sons have agreed to support this endeavor. The kit is expected to be 10 times cheaper than the current method of testing, and would work in most pathological labs of the country without the need of expensive and sophisticated equipment.
- Development of RT-LAMP diagnostic kit:CSIR-IIIM has partnered with Reliance Industries Limited (RIL) to develop and scale-up a new Reverse Transcriptase-Loop Mediated Isothermal Amplification (RT-LAMP) based COVID-19 diagnostic kit for which a formal MOU has also been signed between CSIR-IIIM, Jammu and RIL. The COVID-19 RT-LAMP test is a nucleic acid-based test carried out from nasal/throat swab samples from patients. The test recipe has been developed and successfully demonstrated using synthetic templates. It is a rapid (45-60 min), cost-effective and accurate test. It has been tested with a small number of patientsí samples and validating the kit on more patient samples is planned and will be done together with RIL.
Pooled Sample Testing: To further increase the pace and scope of testing, CSIR has developed methodology for pooled sample testing. Diagnostic assays using quantitative Polymerase Chain Reaction most commonly process patient samples one by one. While this is usually an effective and reliable method, the current efforts against the COVID-19 pandemic demand more efficient measures. Diagnostic assays can be scaled up by the method of High-Throughput qPCR via sample pooling.
Pooling, the action of combining multiple samples into one tube, is most effective when the chance of positive detection of the target, SARS-CoV-2 RNA, is low. In such cases, large groups of samples can be conclusively classified as negative with a single test, with no need to individually test every sample. However, different frequencies of the target-product presence in the samples, require different pool/batch sizes for optimal results.
- At present, CSIR-CCMB is pooling 5 samples into one tube for diagnosis. CSIR-IMTech has also started the pooling of samples.
Large scale sample testing using Next Generation Sequencing (NGS): Next-generation sequencing (NGS) allows for analyzing a large number of viral sequences from infected patients, presenting novel prospects for studying the structure of viral populations, and understanding virus evolution and epidemiology.
- CSIR and Syngene have come together to use NGS for testing of a large number of samples. Using sample pooling and barcoding, it is possible to do sample testing of about 20,000 samples within 3 days. CSIR and Syngene are agreeing to implement this strategy.
Nested PCR for efficient testing: Nested polymerase chain reaction (Nested PCR) is a modification of polymerase chain reaction intended to reduce non-specific binding in products due to the amplification of unexpected primer binding sites.
- CSIR-CCMB is working to develop nested PCR for use in sample testing as it is a more efficient method of testing.
- Innovation in Testing: CSIR has formed a team of scientists to bring in innovations in testing to make it more cost-effective, fast, and accurate. A method for doing the RT-PCR Test without the step of RNA extraction has been developed by CSIR-CCMB. Sampling is through the dry swab method. Using a dry swab, the new method has shown 100% concordance with a normal RT-PCR test. The test will save costly reagents and VDM and can be positioned as a Screening Test. It will be validated independently at CSIR-IMTECH and CSIR-CCMB will approach ICMR for approval.
Serology based Diagnostic Tests: Antibody tests are faster, cheaper and can be tested at point of care and can be carried out on large scale and indicate exposure of virus in the population. These techniques hold much value for ramping up the country's capacity in COVID-19 testing
- CSIR-CCMB and CSIR-IICB are working towards developing these tests.
Scale Up of Resources for Diagnostic Tests: Reverse transcriptase needed for RT-PCR based assay is being produced in large quantities. It is feared that country will face a shortage of this crucial enzyme for pursuing the testing of coronavirus.
- CSIR-IICT has optimized a large-scale production of Reverse Transcriptase a key enzyme in running the RT-PCR diagnostic tests which would be sufficient for lakhs of tests.
- Shipping Container-based Diagnostic Labs: To increase the testing capacity in the country, along with HP, Shipping Container-based Diagnostic Labs (Self Contained, -ve pressure, TrueNat MicroPCR, etc.) have been developed and are being deployed in Delhi (2 nos) and Chennai (2 nos).
- Bio-repositories for SARS-CoV-2: Three CSIR labs, CSIR-CCMB in Hyderabad, CSIR-IGIB in Delhi and CSIR-IMTech Chandigarh have been designated bio-repositories for SARS-CoV-2 viral samples. This will help in the development of indigenous diagnostics, therapeutics and vaccines and research to better understand the COVID19 disease in the Indian scenario.
Testing and Kit Validation at CSIR-CCMB