Sumedha Guha, Techno India University
The outbreak of the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) towards the latter part of 2019 had brought mankind to a screeching halt for some time. The global pandemic that followed in its wake is something that every nation of the world is still grappling hard to recover from. There have been 4,39,65,951 reported cases of SARS-CoV-2 infection with 1.17 million deaths worldwide due to the virus as of October 2020. Human to human transmission from infected individuals with no visible to mild symptoms has been reported. To facilitate early intervention and reduce disease transmission risk, a rapid, accessible and accurate detection method of SARS-CoV-2 is the need of the hour.
Quantitative reverse transcription-polymerase chain reaction (qRTPCR) has been considered as the ‘gold standard’ and is the most commonly used detection method for SARS-CoV-2. Although the efficiency of qRTPCR is undoubted, carrying out the procedure requires professional personnel, long operation time and expensive equipment. And it is these very reasons that render this method impractical for large-scale point-of-care (POC) diagnostic applications. In such a backdrop, clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease-based methods with properties of ultrasensitive, cheaper and portable diagnostic tests for the assessment of suspected cases, could help advance the diagnosis of SARS-CoV-2.
Rui Wang and the team have proposed a one-pot visual SARS-CoV-2 detection system named “opvCRISPR” by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12a nuclease mediated cleavage in a single reaction system for visual detection of SARS-CoV-2. The diagnostic accuracy and reliability of the opv-CRISPR have been validated with 50 SARS-CoV-2 potentially infected clinical samples. SARS-CoV-2 RNA templates (extracted from a respiratory swab) are amplified by RT-LAMP followed by mixing with Cas12a reagents for cleaving. Once the Cas12a nuclease is activated by recognising DNA target, it splits the fluorescent-tagged ssDNA reporter indiscriminately, generating a fluorescent signal visible to the naked eye under blue light.
This method enables detection at the nearly single-molecule level and the whole detection process could be accomplished in just 45 minutes. The most advantageous aspect of opvCRISPR as a diagnostic tool for SARS-CoV-2 detection is the minimum equipment required to operate the protocol. It requires pipettes, reagent tubes, a thermos block and blue light – all of which could be integrated into a 60 × 50 cm² suitcase, thus providing a great potential to enable point-of-care testing outside of the clinical diagnostic laboratory, such as airports, local emergency departments and clinics and other locations.
Also read: Significance of Clinical correlations of SARS-CoV-2 antibody responses
Read more on https://www.sciencedirect.com/science/article/pii/S0956566320307533?via%3Dihub
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