Detection Of Pathogenic Microorganism Staphylococcus Aureus Based On CRISPR-Cas13a

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 bacterial detection method.In this study,we have taken full advantage of CRISPR-Cas13a system,namely,the crRNA programmability and Cas 13a "collateral effect" of promiscuous RNase activity upon target RNA recognition to develop a bacterial sensing strategy.We have named it as CCB-Detection(CRISPR-Casl3a based Bacterial Detection).Staphylococcus aureus(S.aureus)has been chosen as an example for detection.In short,four steps are carried out,which are simple extraction of genome DNA,specific gene amplification by PCR,in vitro transcription and the "collateral effect" cleavage of reporter RNA.As a proof-of-principle,our results have shown that as low as 100 attomolar of the target genomic DNA(gDNA)is successfully detected.The limit of detection(LOD)is 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 is less than 4.0 hours.This method has satisfactory selectivity for detecting S.aureus without interference from other bacteria.Furthermore,the practical applications of our proposed CCB-Detection for sensing S.aureus in real world samples with both known and unknown amounts bacteria(spiked ones and non-spiked ones)have also been realized.With proved reliability,sensitivity,specificity and simplicity,our proposed CCB-Detection could be extended and generalized for other bacterial detection,promising to be developed to meet the need in a wide range of areas such as food safety inspection,disease diagnosis,environment monitoring,etc.