Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19

Abstract

The recent outbreak of human infections caused by SARS-CoV-2, the third zoonotic coronavirus has raised great public health concern globally. Rapid and accurate diagnosis of this novel pathogen posts great challenges not only clinically but also technologically. Metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methodologies. However, each has their own limitations. In this study, we developed an isothermal, CRISPR-based diagnostic for COVID-19 with near single-copy sensitivity. The diagnostic performances of all three technology platforms were also compared. Our study aimed to provide more insights into the molecular detection of SARS-CoV-2, and also to present a novel diagnostic option for this new emerging virus.

A Review on Applicable and Available Paraclinical Methods for Diagnosis of Coronavirus Disease-19

“…9,10 Today, RT-PCR and mNGS are two common molecular diagnostic approaches for 2019-nCov. 11 In RT, cDNA is synthesized from the extracted RNAs from the samples and then, in the presence of the designed and targeted primers for specific sequences (like 2019-nCov specific sequence), amplification of the designated sequence is accomplished by thermocycler. 12 In order to confirm that the amplified products are the targeted sequence, gel electrophoresis or sequencing methods could be performed.…”

“…thermocycler) for the isothermal molecular methods. 11 CRISPR-based method and loopmediated isothermal amplification (LAMP) are two isothermal molecular techniques.…”

“…Although the main aim of CRISPR is gene editing, the cleavage activities of Cas nuclease in rule-less and irregular manner results in the use of CRISPR/Cas technique in nucleic acid detection. 11 Because of the global concern about the need for specific, sensitive and rapid diagnostic techniques, tremendous attention has been attracted by CRISPR-nCov, as this technique could detect as few as 10 RNA copies/µL of 2019-nCov just within 40 minutes. 11,26 The sensitivity and speed of the technique will be increased by applying machine learning designing.…”

An Update on Molecular Diagnostics for COVID-19

“…It has exhibited limit of detection in 10 fM of RNA target. This technique is much advanced than CRISPR COVID (Hou et al, 2020) as previously mentioned in this manuscript, which provides qualitative results using isothermal amplification. The sensitivity of other CRISPR based tests have been taken into consideration while developing this assay.…”

“…Many techniques can be exploited to make rapid and affordable testing available to common people that include Point of Care (POC) also known as bed side testing. POC testing is developing as a portable and promising tool in pathogen diagnosis (Yeh et al, 2017), a major example is the discovery of CRISPR COVID that has been shown to deliver comparable specificity and sensitivity as that of RT-PCR and sequencing based meta genomic diagnostic approaches (Hou et al, 2020). Moreover, lateral flow assay (LFA) is one of the latest POC techniques, which works on the principle of fluid flow, and it is employed in immune assays targeting protein, DNA or RNA based samples (Tang et al, 2017).…”