In Situ Cas12a-Based Allele-Specific PCR for Imaging Single-Nucleotide Variations in Foodborne Pathogenic Bacteria

Anal Chem. 2024 Feb 6;96(5):2032-2040. doi: 10.1021/acs.analchem.3c04532. Epub 2024 Jan 26.

Abstract

In situ profiling of single-nucleotide variations (SNVs) can elucidate drug-resistant genotypes with single-cell resolution. The capacity to directly "see" genetic information is crucial for investigating the relationship between mutated genes and phenotypes. Fluorescence in situ hybridization serves as a canonical tool for genetic imaging; however, it cannot detect subtle sequence alteration including SNVs. Herein, we develop an in situ Cas12a-based amplification refractory mutation system-PCR (ARMS-PCR) method that allows the visualization of SNVs related to quinolone resistance inside cells. The capacity of discriminating SNVs is enhanced by incorporating optimized mismatched bases in the allele-specific primers, thus allowing to specifically amplify quinolone-resistant related genes. After in situ ARMS-PCR, we employed a modified Cas12a/CRISPR RNA to tag the amplicon, thereby enabling specific binding of fluorophore-labeled DNA probes. The method allows to precisely quantify quinolone-resistant Salmonella enterica in the bacterial mixture. Utilizing this method, we investigated the survival competition capacity of quinolone-resistant and quinolone-sensitive bacteria toward antimicrobial peptides and indicated the enrichment of quinolone-resistant bacteria under colistin sulfate stress. The in situ Cas12a-based ARMS-PCR method holds the potential for profiling cellular phenotypes and gene regulation with single-nucleotide resolution at the single-cell level.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • CRISPR-Cas Systems / genetics
  • In Situ Hybridization, Fluorescence
  • Polymerase Chain Reaction
  • Quinolones* / pharmacology
  • Salmonella enterica* / genetics

Substances

  • Quinolones