Development of Cas13a-based Assays for Detection and Gyrase A Determination.

BACKGROUND

is one of the most common bacterial sexually transmitted infections. The emergence of antimicrobial-resistant is an urgent public health threat. Currently, diagnosis of infection requires expensive laboratory infrastructure, while antimicrobial susceptibility determination requires bacterial culture, both of which are infeasible in low-resource areas where prevalence is highest. Recent advances in molecular diagnostics, such as Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) using CRISPR-Cas13a and isothermal amplification, have the potential to provide low-cost detection of pathogen and antimicrobial resistance.

METHODS AND RESULTS

We designed and optimized RNA guides and primer-sets for SHERLOCK assays capable of detecting via the A gene and of predicting ciprofloxacin susceptibility via a single mutation in the gyrase A ( A) gene. We evaluated their performance using both synthetic DNA and purified isolates. For A, we created both a fluorescence-based assay and lateral flow assay using a biotinylated FAM reporter. Both methods demonstrated sensitive detection of 14 isolates and no cross-reactivity with 3 non-gonococcal isolates. For A, we created a fluorescence-based assay that correctly distinguished between 20 purified isolates with phenotypic ciprofloxacin resistance and 3 with phenotypic susceptibility. We confirmed the A genotype predictions from the fluorescence-based assay with DNA sequencing, which showed 100% concordance for the isolates studied.

CONCLUSION

We report the development of Cas13a-based SHERLOCK assays that detect and differentiate ciprofloxacin-resistant isolates from ciprofloxacin-susceptible isolates.