Isothermal RNA Amplification for the Detection of Viable Pathogenic Bacteria to Estimate the Salmonella Virulence for Causing Enteritis

Viable foodborne pathogens can cause intestinal infection and food poisoning. Herein, we reported an RNA assay allowing for sensitive (close to 1 CFU and 1% viable bacteria detectable) and rapid (within 2.5 h) detection of viable pathogenic bacteria by coupling isothermal RNA amplification (nucleic acid sequence-based amplification, NASBA) with a CRISPR/Cas13a system. NASBA allowed direct amplification of 16S rRNA extracted from viable S. enterica (RNAs degrade rapidly in dead bacteria), and the specificity of amplification was ensured using Cas13a/crRNA to recognize the amplicons. We used the CRISPR/Cas13-based NASBA assay (termed cNASBA assay) to investigate the in vivo colonization and intestinal infection of S. enterica in mice. We found that S. enterica was mainly colonized at the cecum, colon, and rectum, and the severity of enteritis caused by S. enterica was determined by the number of viable S. enterica rather than the total count of S. enterica. The cNASBA assay can quantify viable S. enterica and thus can improve the accuracy of virulence estimation compared to qPCR. It shows promise as a reliable tool for monitoring pathogen contamination and biosafety control.

Automated summary

We reported an RNA detection method that allows for rapid detection of viable pathogenic bacteria within 2.5 hours. This method involves the direct amplification of 16S rRNA from viable Salmonella enterica and uses Cas13a/crRNA to ensure the specificity of amplification. Using this method, we found that S. enterica mainly colonizes the cecum, colon, and rectum in mice, and the severity of enteritis is determined by the number of viable S. enterica. Compared to qPCR, this method can accurately estimate the virulence of the pathogen, showing promise as a tool for monitoring pathogen contamination and biosafety control.