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 methods for detection of bacteria. In this study, taking advantages of the 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 bacterium 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 of successfully detecting 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 of 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 generalized for other bacterial detection and has great potential to be used in a wide range of applications, such as food safety inspection, disease diagnosis, and environment monitoring.

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CRISPR-Cas13a-Based Bacterial Detection Platform: Sensing Pathogen Staphylococcus aureus in Food Samples

  • Long Ma,
  • Yaru Li,
  • Shuli Man

摘要

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 methods for detection of bacteria. In this study, taking advantages of the 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 bacterium 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 of successfully detecting 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 of 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 generalized for other bacterial detection and has great potential to be used in a wide range of applications, such as food safety inspection, disease diagnosis, and environment monitoring.