Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2

Abstract

At the end of 2019, a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused a pandemic that persists to date and has resulted in more than 6.2 million deaths. In the last couple of years, researchers have made great efforts to develop a diagnostic technique that maintains high levels of sensitivity and specificity, since an accurate and early diagnosis is required to minimize the prevalence of SARS-CoV-2 infection. In this context, CRISPR-Cas systems are proposed as promising tools for development as diagnostic techniques due to their high specificity, highlighting that Cas13 endonuclease discriminates single nucleotide changes and displays collateral activity against single-stranded RNA molecules. With the aim of improving the sensitivity of diagnosis, this technology is usually combined with isothermal preamplification reactions (SHERLOCK, DETECTR). Based on this, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP)-CRISPR-Cas13a method for SARS-CoV-2 virus detection in nasopharyngeal samples without using RNA extraction that exhibits 100% specificity and 83% sensitivity, as well as a positive predictive value (PPV) of 100% and negative predictive values (NPVs) of 100%, 81%, 79.1%, and 66.7% for cycle threshold (C_T) values of <20, 20 to 30, >30 and overall, respectively. IMPORTANCE The coronavirus disease 2019 (COVID-19) crisis has driven the development of innovative molecular diagnosis methods, including CRISPR-Cas technology. In this work, we performed a protocol, working with RNA extraction kit-free samples and using RT-LAMP-CRISPR-Cas13a technology; our results place this method at the forefront of rapid and specific diagnostic methods for COVID-19 due to the high specificity (100%), sensitivity (83%), PPVs (100%), and NPVs (81% for high viral loads) obtained with clinical samples.

Keywords: COVID-19; CRISPR-Cas; CRISPR-Cas13; RT-LAMP; SARS-CoV-2; diagnosis.

FIG 1

Workflow of the novel developed and optimized protocol for infectious disease diagnosis based on CRISPR-Cas13a technology.

FIG 2

LOD assay for SARS-CoV-2 detection with the N2 gene as the target using serial dilutions (1:10) from two samples with different C_T values.

FIG 3

(A) Test strips (left) for SARS-CoV-2 detection using samples with C_T values ranging from 13 to 38 and negative samples as negative controls, with numerical results (right) for each interval of C_T values (<20, 20 to 30, and >30). (B) Results obtained using the N2 gene for SARS-CoV-2 detection. (C) Table containing the specificity, sensitivity, PPV, and NPV RT-LAMP-CRISPR-Cas13a technique values obtained by processing the data in Fig. 4B.

FIG 4

(A) ROC curve for RT-LAMP-CRISPR-Cas13a technology. (B) Scatterplot of two groups, false-negative and true-positive detections with RT-LAMP-CRISPR-Cas13a, versus the C_T values of the respective samples.