An improved, simple and field‐deployable CRISPR‐Cas12a assay for the detection of SARS‐CoV‐2

• Published in: Journal of applied microbiology 2022-10, Vol.133 (4), p.2668-2677
• Main Authors: Misra, Chitra S., Rangu, Shyam S., Phulsundar, Ravindra D., Bindal, Gargi, Singh, Mandeep, Shashidhar, Ravindranath, Saha, Tushar K., Rao, Akkipeddi V. S. S. N., Rath, Devashish
• Format: Article
• Language: English
• Subjects: Amplification; Cleavage; Control equipment; CRISPR; Fluorescence; Instrumentation; Portability; Reagents; Ribonuclease P; Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Viral diseases; Viruses
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/jam.15737

Aims The RT‐PCR is the most popular confirmatory test for SARS‐CoV‐2. It is sensitive, but high instrumentation cost makes it difficult for use outside routine clinical setup. This has necessitated the development of alternative methods such as CRISPR‐based DETECTR method which uses lateral flow technology. Although accurate and sensitive, this method is limited by complex steps and recurrent cost of high‐quality lateral flow strips. The main goal of this study was to improve the Cas12a‐based SARS‐CoV‐2 DETECTR method and develop a portable and field‐deployable system to reduce the recurring consumable cost. Methods and results Specific regions of N and E genes from SARS‐CoV‐2 virus and human RNase P (internal control) were reverse transcribed (RT) and amplified by loop‐mediated isothermal amplification (LAMP). The amplified products were detected by a Cas12a‐based trans‐cleavage reaction that generated a fluorescent signal which could be easily visualized by naked eye. Detection of internal control, RNase P gene was improved and optimized by redesigning RT‐LAMP primers. A number of steps were reduced by combining the reagents related to the detection of Cas12a trans‐cleavage reaction into a single ready‐to‐use mix. A portable, cost‐effective battery‐operated instrument, CRISPR‐CUBE was developed to run the assay and visualize the outcome. The method and instrument were validated using both contrived and patient samples. Conclusions The simplified CRISPR‐based SARS‐CoV‐2 detection and instrument developed in this study, along with improved design for internal control detection allows for easier, more definitive viral detection requiring only reagents, consumables and the battery operable CRISPR‐CUBE. Significance and impact of study Significant improvement in Cas12 method, coupled with simple visualization of end point makes the method and instrument deployable at the point‐of‐care (POC) for SARS‐CoV‐2 detection, without any recurrent cost for the lateral flow strips which is used in other POC methods.