Development of Cas13a-based assays for Neisseria gonorrhoeae detection and gyrase A determination

Abstract

ABSTRACT Neisseria gonorrhoeae is one of the most common bacterial sexually transmitted infections. The emergence of antimicrobial-resistant N. gonorrhoeae is an urgent public health threat. Currently, the diagnosis of N. gonorrhoeae infection requires expensive laboratory infrastructure, while antimicrobial susceptibility determination requires bacterial culture, both of which are infeasible in low-resource areas where the prevalence of infection 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. We designed and optimized RNA guides and primer sets for SHERLOCK assays capable of detecting N. gonorrhoeae via the porA gene and of predicting ciprofloxacin susceptibility via a single mutation in the gyrase A (gyrA) gene. We evaluated their performance using both synthetic DNA and purified N. gonorrhoeae isolates. For porA, we created both a fluorescence-based assay and lateral flow assay using a biotinylated fluorescein reporter. Both methods demonstrated sensitive detection of 14 N. gonorrhoeae isolates and no cross-reactivity with 3 non-gonococcal Neisseria isolates. For gyrA, we created a fluorescence-based assay that correctly distinguished between 20 purified N. gonorrhoeae isolates with phenotypic ciprofloxacin resistance and 3 with phenotypic susceptibility. We confirmed the gyrA genotype predictions from the fluorescence-based assay with DNA sequencing, which showed 100% concordance for the isolates studied. We report the development of Cas13a-based SHERLOCK assays that detect N. gonorrhoeae and differentiate ciprofloxacin-resistant isolates from ciprofloxacin-susceptible isolates. IMPORTANCE Neisseria gonorrhoeae, the cause of gonorrhea, disproportionately affects resource-limited settings. Such areas, however, lack the technical capabilities for diagnosing the infection. The consequences of poor or absent diagnostics include increased disease morbidity, which, for gonorrhea, includes an increased risk for HIV infection, infertility, and neonatal blindness, as well as an overuse of antibiotics that contributes to the emergence of antibiotic resistance. We used a novel CRISPR-based technology to develop a rapid test that does not require laboratory infrastructure for both diagnosing gonorrhea and predicting whether ciprofloxacin can be used in its treatment, a one-time oral pill. With further development, that diagnostic test may be of use in low-resource settings.

Keywords

• Neisseria gonorrhoeae
• antimicrobial resistance
• diagnostics
• CRISPR