An Innovative and Efficient Fluorescent Detection Technique for <i>Salmonella</i> in Animal-Derived Foods Using the CRISPR/Cas12a-HCR System Combined with PCR/RAA

Wang Y., Du P., Shao Y., Wang W., Liu Y., Ma Y., ...More

JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol.72, no.15, pp.8831-8839, 2024 (SCI-Expanded, Scopus) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 72 Issue: 15
  • Publication Date: 2024
  • Doi Number: 10.1021/acs.jafc.3c08829
  • Journal Name: JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Analytical Abstracts, Applied Science & Technology Source, Aquatic Science & Fisheries Abstracts (ASFA), BIOSIS, Biotechnology Research Abstracts, CAB Abstracts, Chemical Abstracts Core, Chimica, Compendex, Environment Index, Food Science & Technology Abstracts, Pollution Abstracts, Veterinary Science Database, DIALNET
  • Page Numbers: pp.8831-8839
  • Keywords: animal-derived foods, CRISPR/Cas12a-HCR, fluorescence rapid detection, PCR/RAA, Salmonella, specific target
  • Ataturk University Affiliated: Yes

Abstract

Here, we present a method for Salmonella detection using clustered regularly interspaced short palindromic repeats associated with the CRISPR-associated protein 12a-hybridization chain reaction (CRISPR/Cas12a-HCR) system combined with polymerase chain reaction/recombinase-assisted amplification (PCR/RAA) technology. The approach relies on the Salmonella invA gene as a biorecognition element and its amplification through PCR and RAA. In the presence of the target gene, Cas12a, guided by crRNA, recognizes and cleaves the amplification product, initiating the HCR. Fluorescently labeled single-stranded DNA (ssDNA) H1 and H2 were introduced, and the Salmonella concentration was determined based on the fluorescence intensity from the triggered HCR. Both assays demonstrate high specificity, sensitivity, simplicity, and rapidity. The detection range was 2 x 10(1)-2 x 10(9) CFU/mL, with an LOD of 20 CFU/mL, and the entire process enabled specific and rapid Salmonella detection within 85-105 min. Field-incurred spiked recovery tests were conducted in mutton and beef samples using both assays, demonstrating satisfactory recovery and accuracy in animal-derived foods. By combining CRISPR/Cas12a with hybridization chain reaction technology, this study presents a rapid and sensitive Salmonella detection method that is crucial for identifying pathogenic bacteria and monitoring food safety.