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

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.

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.

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.

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.