CRISPR-Cas13a based bacterial detection platform: Sensing pathogen Staphylococcus aureus in food samples

Abstract

The severity of foodborne diseases caused by foods contaminated by pathogens or their toxins creates an urgent need for the development of specific and sensitive method for detection of bacteria. In this study, taking advantages of CRISPR-Cas13a system, namely, the crRNA programmability and Cas13a "collateral effect" of promiscuous RNase activity upon target RNA recognition, we developed a bacterial sensing strategy with the name of CCB-Detection (CRISPR-Cas13a based Bacterial Detection). Staphylococcus aureus (S. aureus) was chosen as a model bacteria for validating the performance of CCB-Detection. Specifically, four steps were carried out: 1) simple extraction of genome DNA; 2) specific gene amplification by PCR; 3) in vitro transcription; and 4) the "collateral effect" cleavage of reporter RNA to report the analyte signal. It was observed that CCB-Detection was capable to successfully detect the target genomic DNA (gDNA) as low as 10⁰ aM. The limit of detection (LOD) was 1 CFU/mL with a dynamic detection range of S. aureus from 10⁰ to 10⁷ CFU/mL. The entire sample-to-answer time for this biosensor was less than 4 h. CCB-Detection demonstrated satisfactory selectivity for S. aureus without interference from other bacteria. Furthermore, CCB-Detection was successfully applied for sensing S. aureus in real food samples with both known and unknown amounts bacteria (spiked ones and non-spiked ones) and its performance is comparable to the conventional culture-based counting method but with short assay time and high sensitivity. With desirable reliability, sensitivity, specificity and simplicity, herein proposed CCB-Detection could be extended and generalised for other bacterial detection, and has great potential to be used in a wide range of applications such as food safety inspection, disease diagnosis, environment monitoring, etc.

Keywords: Bacterial detection; CRISPR-Cas13a biosensing; Collateral RNA cleavage; Food safety; Sensitivity and specificity; Staphylococcus aureus.