Rapid and Sensitive Detection of Salmonella spp. Using CRISPR-Cas13a Combined With Recombinase Polymerase Amplification

• Published in: Frontiers in microbiology Vol. 12; p. 732426
• Main Authors: An, Bailin, Zhang, Hongbin, Su, Xuan, Guo, Yue, Wu, Tao, Ge, Yiyue, Zhu, Fengcai, Cui, Lunbiao
• Format: Journal Article
• Language: English
• Published: Frontiers Media S.A 18.10.2021
• Subjects: CRISPR-Cas13a; foodborne disease; Microbiology; molecular detection; RPA; Salmonella
• ISSN: 1664-302X; 1664-302X
• DOI: 10.3389/fmicb.2021.732426

Salmonella spp. is one of the most common foodborne disease-causing pathogens that can cause severe diseases in very low infectious doses. Rapid and sensitive detecting Salmonella spp. is advantageous to the control of its spread. In this study, a conserved short fragment of the Salmonella invA gene was selected and used to design primers and specific crRNA (CRISPR RNA) for establishing a one-tube and two-step reaction system for Salmonella spp. detection, by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats associated protein 13a) cleavage. The established one-tube RPA-Cas13a method can complete the detection within 20 min and the two-step RPA-Cas13a method detection time within 45 min. The designed primers were highly specific to Salmonella spp. and had no cross-reaction with the other nine diarrheal bacteria. The one-tube RPA-Cas13a could detect the Salmonella genome with the limit of 10 2 copies, which was the same as real-time polymerase chain reaction (PCR), but less sensitive than two-step RPA-Cas13a (10 0 copies). The detection results of one-tube or two-step RPA-Cas13a and real-time PCR were highly consistent in clinical samples. One-tube RPA-Cas13a developed in this study provides a simple, rapid, and specific detection method for Salmonella spp. While two-step assay was more sensitive and suitable for samples at low abundance.