CLEVER assay: A visual and rapid RNA extraction-free detection of SARS-CoV-2 based on CRISPR-Cas integrated RT-LAMP technology.

AIM: The current scenario of COVID-19 pandemic has presented an almost insurmountable challenge even for the most sophisticated hospitals equipped with modern biomedical technology. There is an urgency to develop simple, fast and highly accurate methods for the rapid identification and isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. To address the ongoing challenge, the present study offers a CLEVER assay (CRISPR-Cas integrated RT-LAMP Easy, Visual and Extraction-free RNA) which will allow RNA extraction-free method to visually diagnose COVID-19. RNA extraction is a major hurdle in preventing rapid and large-scale screening of samples particularly in low-resource regions because of the logistics and costs involved. METHOD AND RESULT: Herein, the visual SARS-CoV-2 detection method consists of RNA extraction-free method directly utilizing the patient's nasopharyngeal and oropharyngeal samples for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Additionally, the assay also utilizes the integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12-based system using different guide RNAs of N, E and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readout-based dip sticks with unaided eye (~100 min). Overall, the clinical sensitivity and specificity of the CLEVER assay were 89.6% and 100%, respectively. CONCLUSION: Together, our CLEVER assay offers a point-of-care tool with no equipment dependency and minimum technical expertise requirement for COVID-19 diagnosis. SIGNIFICANCE AND IMPACT OF THE STUDY: To address the challenges associated with COVID-19 diagnosis, we need a faster, direct and more versatile detection method for an efficient epidemiological management of the COVID-19 outbreak. The present study involves developing a method for detection of SARS-CoV-2 in human body without RNA isolation step that can visually be detected with unaided eye. Taken together, our assay offers to overcome one major defect of the prior art, that is, RNA extraction step, which could limit the deployment of the previous assays in a testing site having limited lab infrastructure.

Diagnostic efficiency of RT-LAMP integrated CRISPR-Cas technique for COVID-19: A systematic review and meta-analysis.

To address the challenges associated with COVID-19 diagnosis, we need a faster, direct, and more versatile detection method for efficient epidemiological management of the COVID-19 pandemic. RT-qPCR (reverse transcription quantitative real-time Polymerase Chain Reaction) although the most popular diagnostic method suffers from a major drawback of equipment dependency and trained molecular biologists that limits rapid and large-scale screening, particularly in low resource regions. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a feasible alternative for RT-qPCR; however, it also suffers from the drawback of false-positive issues. Recently, RT-LAMP has been integrated with the CRISPR-Cas technique to take care of the problems associated with RT-LAMP for COVID-19 diagnosis. In this study, a meta-analysis was conducted using three scientific databases considering the PRISMA guidelines to assess the diagnostic efficiency of RT-LAMP integrated CRISPR-Cas technology. Out of a total of 1286 studies on COVID-19, we identified 15 articles that met our eligibility criteria of using simultaneous RT-LAMP and CRISPR-Cas technique. Our meta-analysis of the included studies revealed that most of the studies were conducted in the USA with the N gene as the most common target and fluorescence-based detection method. The meta-analysis results of all included studies have further revealed a pooled sensitivity value of higher than 85% and a pooled specificity value of 80% with the confidence interval of 95%, respectively, as revealed from the forest plot and SROC curve. The accuracy rate of included studies was also calculated which varied from 77.4% to 100%. Furthermore, the precision of included studies varied from 75% to 100%. Lastly, a quality assessment of bias and applicability was performed based on QUADAS-2. Taken together, combined RT-LAMP and CRISPR-Cas technique could be a potential alternative to RT-qPCR particularly in low resource regions having a high demand for rapid testing.

An Inventory of Diagnostic Tools for Detection of COVID-19.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by SARS-COV-2 has afflicted millions of lives globally and disrupted almost all the activities of mankind. Under such pressing circumstances when no effective therapeutics are available, a fast and accurate diagnosis of the coronavirus is the only way out to limit the transmission. Since the inception of COVID-19, the demand for diagnostic tests has increased day by day and RT-PCR is the commonly used screening test that is not only time-consuming but requires sophisticated resources. To address the increasing rate of spread of COVID-19, there is an urgent need for more diagnostic tools as the research on vaccines is still at a rudimentary level. This review summarizes an inventory of the diverse and currently available diagnostic methods based on nucleic acid and serology along with some of those working on novel principles viz. CRISPR, biosensors, and NGS. Additionally, accessible diagnostic kits that are already approved by the US and European authorities for the diagnosis of COVID-19 are also suggested that will help in selecting the most effective tests under the given scenario. Taken together, this review will pave way for further strengthening the research on the rapid and safer diagnostics of SARS-COV-2.