Establishment of A Rapid RNA Detection Method for Listeria monocytogenes Based on CRISPR-Cas13
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Abstract:
In order to realize the rapid detection of Listeria monocytogenes contaminated in food, a homogeneous RNA detection technology based on the CRISPR-Cas system and Broccoli aptamer was constructed in this study. Cas 13 was combined with the crRNA anchor sequence to form crRNA-Cas13 complex as a recognition element. The presence of target RNA activated the non-specific RNase activity of Cas13, and the light-up RNA aptamer Broccoli was used as the signal probe to monitor the activation state of crRNA-Cas13. There is a linear relationship between the change of fluorescence value and the concentration of Listeria monocytogenes, which is used to detect Listeria monocytogenes. The process for identification and detection of Listeria monocytogenes can be completed within 30 minutes with a detection limit of 148 CFU/mL. This method has good detection specificity for bacteria and can distinguish E. coli, S. enterica, Salmonella typhimurium and B. cereus. The recovery rate was 95.15%~97.99% in the milk model samples spiked with Listeria monocytogenes. Therefore, this method has good sensitivity and specificity, and can directly target the pathogen RNAs without reverse transcription, PCR amplification and nucleic acid labeling, which simplifies the experimental process and is of great of significance for realizing on-site detection and biosafety control of Listeria monocytogenes in food.
