Establishment of a detection method for Campylobacter jejuni based on RAA-CRISPR/Cas13a

Luo Ruxing; Liang Zili; He Qifu; Zhang Yan; Zhang Zhidong; Guo Zijing

doi:10.20154/j.cnki.issn1672-9692.2025.08.003

Abstract：The study aims to combine recombinase aided amplification(RAA)technology with the Cas13a protein technology associated with regularly spaced clustered short palindromic repeat sequences(CRISPR-Cas13a)to establish a detection method for Campylobacter jejuni based on RAA-CRISPR/cas13a.Specific CRISPR RNA sequences and RAA primers were designed targeting a conserved gene of Campylobacter jejuni.Following RAA amplification,fluorescence detection was performed using CRISPR-Cas13a.Key components of the detection system were optimized,and the specificity,sensitivity,and repeatability of the optimized method were evaluated.Clinical samples were further analyzed and compared with existing quantitative real-time PCR(qPCR).Results demonstrated that the optimized method exhibited excellent specificity with no cross-reactivity against multiple foodborne pathogens.The limit of detection reached 9.3 copies/μL,indicating high sensitivity.Intra-and inter-assay coefficients of variation were below 4.15％and 3.51％,respectively,confirming strong repeatability.When testing 40 pig diarrheal fecal samples,the detection rate of the RAA-CRISPR/Cas13a method was 30％(12/40),outperforming qPCR(20％,8/40).All 12 positive samples were further confirmed as Campylobacter jejuni by sequencing,validating the accuracy of the method.This study successfully developed a highly sensitive and specific RAA-CRISPR/Cas13a method for detecting Campylobacter jejuni,providing a novel technical approach for its surveillance.The method demonstrates promising potential for applications in foodborne disease monitoring and animal disease prevention and control.