Sensitive Detection of a Single-Nucleotide Polymorphism in Foodborne Pathogens Using CRISPR/Cas12a-Signaling ARMS-PCR

Abstract

Salmonella infection, particularly that caused by drug-resistant strain, has become a worldwide public health issue. Herein, we presented a CRISPR/Cas12a-signaling ARMS-PCR assay, termed cARMS, capable of sensitively detecting drug-resistant Salmonella enterica (S. enterica) involving single-nucleotide polymorphism (SNP). Owing to the dual-recognition processes, i.e., allele-specific primed polymerization and CRISPR/Cas12 binding, the cARMS assay yielded a high sensitivity for detecting SNP down to ∼0.5%. We used the cARMS assay to investigate the adaptation of SNP-involved drug-resistant S. enterica to salt stress. It was found that the mutants exhibited stronger adaptation to salt stress, indicating the potential risk of using high salt content as a sterilization strategy. The results verified the feasibility of the cARMS assay in controlling SNP-involved bacteria-associated biosafety.

CRISPR-based nucleic acid diagnostics for pathogens

“…(A) Schematic of Cas12a-based electrochemical biosensor for the detection of mecA gene of MRSA [ 125 ]. (B) Schematic of the cARMS assay for the detection of SNP-involved drug-resistant Salmonella [ 126 ]. PCR primers designed with artificial mutations improve the capacity for discriminating single-nucleotide variation.…”

“…Cas12a-signaling amplification refractory mutation system (ARMS)-PCR, called cARMS assay was capable of specific differentiation of SNP-involved Salmonella via adopting an allele-specific primer with artificial mismatch ( Fig. 9 B) [ 126 ]. The dual-recognition processes — allele-specific primed polymerization and Cas12a/crRNA hybridization — conferred single-nucleotide discrimination capacity.…”

Antibiotic resistance mechanism and diagnosis of common foodborne pathogens based on genotypic and phenotypic biomarkers

“…Yang et al. (2022) have recently introduced an innovative approach to detect single nucleotide polymorphism (SNP) in the gyrA gene of Salmonella , which is closely linked to fluoroquinolone resistance. The method employed amplification refractory mutation system polymerase chain reaction (ARMS‐PCR) in conjunction with the CRISPR and Cas12a system.…”

“…To improve the sensitivity and specificity of SNP detection, the proteins, including DNA damage repair proteins, ligase, CRISPR-associated proteins (Figure 6a), and Argonaute (Figure 6b), have been proposed to be combined with an amplification strategy (Song et al, 2019). Yang et al (2022) have recently introduced an innovative approach to detect single nucleotide polymorphism (SNP) in the gyrA gene of Salmonella, which is closely linked to fluoroquinolone resistance. The method employed amplification refractory mutation system polymerase chain reaction (ARMS-PCR) in conjunction with the CRISPR and Cas12a system.…”

CRISPR-based nucleic acid diagnostics for pathogens

“…Cas12asignaling amplification refractory mutation system (ARMS)-PCR, called cARMS assay was capable of specific differentiation of SNP-involved Salmonella via adopting an allele-specific primer with artificial mismatch (Fig. 8B) [121]. The dual-recognition processes -allele-specific primed polymerization and Cas12a/crRNA hybridization -conferred the single-nucleotide discrimination capacity.…”