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

Abstract

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 <i>S. enterica</i> (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 <i>in vivo</i> colonization and intestinal infection of <i>S. enterica</i> in mice. We found that <i>S. enterica</i> was mainly colonized at the cecum, colon, and rectum, and the severity of enteritis caused by <i>S. enterica</i> was determined by the number of viable <i>S. enterica</i> rather than the total count of <i>S. enterica</i>. The cNASBA assay can quantify viable <i>S. enterica</i> 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.