iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2

Abstract

The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.

The diagnostic accuracy of nucleic acid point-of-care test for human coronavirus: A systematic review and meta-analysis

“…On the contrary, only a fifth (n = 5 out of 25) of studies that used purified RNA reported less than 90% sensitivity. Among the six studies that used purified RNA samples but has less than 90% sensitivity or specificity [34, 35, 42, 43, 45, 53], three studies have unusual QUADAS-2 risks and concerns, comparing other included studies. Hong et al [35] and Poon et al [43] had high risk of reference standard bias and concern of standard applicability.…”

“…About half of all studies (n = 23 out of 43) have ln(DOR) below that of the pooled data. Eighteen of these studies are those with sensitivity or specificity no more than 90% [26-29, 31, 34, 35, 40, 42, 43, 50, 52, 53, 56, 57, 60, 61, 66]. The other five studies have low ln(DOR) despite having 100% sensitivity and 100% specificity [36, 44, 48, 51, 67].…”

Diagnostics of COVID-19 based on CRISPR-Cas coupled to Isothermal Amplification: A Comparative Analysis and Update

“…It is important to consider that the intellectual property behind the RPA method belongs to TwistDx Inc [49], whereas the patent of the LAMP technology has recently expired [50]. Yet, homemade isothermal LAMP enzymes used in the method iScan achieved a significant analytical sensitivity of 10 viral copies per reaction and 100 % of clinical specificity and sensitivity [51], indicating that homemade production of isothermal enzymes perform effectively, efficiently and could reduce costs.…”

“…Also, RT-RPA can be run in parallel (one-pot test) with CRISPR-Cas systems from mesophilic microorganisms. For RT-LAMP, the only way to circumvent the temperature limitation and to develop one-pot methods is using temperature-resistant EnAs-Cas12a (works at 60 ºC) or thermostable AapCas12b, which run simultaneously with the RT-LAMP at 59-62 ºC [26,40,51,52].…”

Emerging diagnostic tools for detection of COVID-19 and perspective

“…The Cas13 can be used in point of care setting and it would be low cost when compared to traditional lateral flow assay as it doesn’t require antibody conjugates for the detection (Rauch et al 2020 ). Another effective use of CRISPR Cas 12 was seen by integrating into a lateral flow system named as iSCAN having potential to be used as effective management system for point of care detection(Ali et al 2020 ). AIOD CRISPR assay, made use of a pair of crRNAs to initiate the detection using CRISPR-Cas12a for improved sensitivity.…”