An EXPAR-CRISPR/Cas12a Assay for Rapid Detection of Salmonella

Abstract

Salmonella is considered as one of the primary pathogens associated with foodborne diseases globally. The effective treatment of these illnesses depends on the rapid and accurate identification of this organism. Traditional culture methods, however, necessitate extended testing periods, while many alternative techniques often lack precision. This research presents an innovative detection system that employs CRISPR-Cas12a for the detection of Salmonella. The detection system specifically targets the yfiR gene, which is amplified through isothermal exponential amplification (EXPAR). Target DNA hybridizes with the hairpin probe to form the DNA strand. The DNA strand was nicked to generate a nick by nicking endonuclease owing to its recognition sequence toward the hairpin probe. DNA polymerase can extend the 3'-end of the nicked site, which simultaneously displaces the newly synthesized strand. Thus, a large number of single-stranded DNA (ssDNA) were produced in the circle of nicking, polymerization, and strand displacement to achieve exponential amplification. The resultant amplified ssDNA products are subsequently recognized by CRISPR/Cas12a, resulting in the emission of a fluorescence signal. The detection system demonstrates a limit of detection of 10 fM for synthetic DNA and exhibits a strong linear relationship between 10 fM and 100 nM. Furthermore, the EXPAR-CRISPR/Cas12a detection system successfully identifies extracted genomic DNA samples containing Salmonella strains less than one hour, achieving a detection threshold of 1 pg/μL. This assay not only offers rapid results, requiring less than one hour for sample-to-answer outcomes, but is also cost-effective, minimizes aerosol risks, and provides exceptional specificity and sensitivity for the detection of Salmonella.