An accurate amplification-free CRISPR/Cas12a-based assay for GES β-lactamase detection

Abstract

Objective

Guiana-Extended-Spectrum (GES) β-lactamases belong to the minor class A β-lactamases and are probably underdiagnosed due to a lack of specific diagnostic tests. There is therefore an urgent need to develop new molecular diagnostic tools that will be able to fill the gap in the detection of rare β-lactamases. Here, we propose an optimized, amplification-free CRISPR/Cas12a-based assay for the accurate detection of GES β-lactamases and we validate its application with clinical isolates (Graphic abstract). Based on the results of examination of 79 standard collection, the proposed assay exhibited 100% sensitivity and specificity, as well as 100% positive and negative predictive values in less than 1.5 hours.

Methods

We optimized the CRISPR/Cas12a method by harnessing a multiplex crRNA strategy, a highly efficient DNA reporter (TTATT-5C) and the Murine RNase Inhibitor to prevent crRNA degradation.

Results

Our yielded limits of detection of 1 ng/µL and 3 ng/µL in Enterobacterales and Pseudomonas aeruginosa, respectively. The observed difference is due to the location of the bla_GES gene. The gene occurs in a chromosomal integron present only in one to three copies in P. aeruginosa, whereas it occurs in plasmids present in multiple copies in Enterobacterales.

Conclusions

The proposed method could be established as a routine diagnostic tool in clinical microbiology laboratories to fill the gap in availability of commercial diagnostic tests for GES β-lactamases.