Development And Preliminary Application Of CRISPR-Cas12b-based Campylobacter Jejuni Detection System

The bio-safety problems caused by pathogenic microorganisms have been widely concerned by the society.As an important zoonotic pathogen that can cause many diseases including human bacterial enteritis and Guillain-Barre syndrome(GBS),Campylobacter jejuni threatens the food safety and public health seriously.Therefore,detection of C.jejuni in samples associated with public health and food safety,as well as animal husbandry and veterinary,and entry-exit inspection and quarantine,is mandantory and routinely performed.However,the traditional cultivation-based C jejuni detection methods are time-consuming due to the complex sample processing steps,and thus unsuitable for on-site rapid detection.Therefore,it is urgent to develop new detection methods to meet the needs of on-site detection of C.jejuni.The CRISPR-Cas-based nucleic acid detection systems have shown great potential for rapid and accurate detection of pathogens’ however,identification of pathogen-specific and-conserved sequence signatures of the given pathogen,which is the critical step for the CRISPR-Cas-based pathogen detection system assembly,is not easy to perform.Here,we developed a bioinformatics analysis pipeline to identify pathogen-specific and-conserved sequence signatures using C.jejuni as a model,and developed a CRISPR-Cas12b-based C.jejuni detection system to detect C.jejuni contatmination rapidly and accurately.All the 2495 Campylobacter genome sequences accessible in the NCBI refseq database were downloaded,and the too fragemented(scaffolds>100)genomes and genomes showing genome-wise average nucleotide identity(ANI)values lower than the species demarcation threshold of 95%when compared with the C.jejuni type strain NCTC11351T,were discarded from further analyses.Consequently,1037 high-quality C.jejuni and genomes were obtained.600 high-quality C.coli genomes were also extracted based on the same procedure.Through comparative analysis between the representative C.jejuni and C.coli genomes using Neptune software,a total of 15 C.jeuni-specific and-conserved sequence signatures(score>0.95)were obtained.Finally,a 20 bp protospacer sequence which fitted the requirements for CRISPR-Cas12b-based detection system assembly was identified from a representative sequence signature,and the results demonstrated that this 20 bp protospacer sequence was very conserved among the C.jejuni population(identical in 1024 of the 1037 analyzed C.jejuni genomes),while no identical sequence was identified in non-C.jejuni genomes.The full-length sequence of the Cas12b-encoding gene was amplified,cloned into the pET-28a vector,and transformed into E.coli strain BL21(DE3).Cas12b protein was then expressed and purified.The Cas12b-compatible sgRNA was designed based on the obtained 20 bp protospacer sequence,and the CRISPR-Casl2b-based C jejuni detection system was assembled.To evaluate the specificity of the CRISPR-Cas12b system for C.jejuni detection,the DNA samples of C jejuni and other foodborne pathogenic bacteria were employed as targets,and the DNA samples were added to the detection system individually or mixed.The results demonstrated that the CRISPR-Cas12b-based detection system was activated only when C.jejuni DNA was present in the system,indicative of high specificity of the detection system.The detection threshold of the CRISPR-Cas12b-based detection system was 204 ag/μL,and the sensitivity reached sub-micromolar level.The reliability of the CRISPR-Cas12b-based detection system was further evaluated based on the simulated C.jejuni contamination experiment under laboratory conditions.The results demonstrated that the detection threshold of this detection system could reach 10 CFU/g chicken sample,which was～10 times more sensitive than the traditional cultivation-based C.jejuni detection methods.We then applied the CRISPR-Cas12b-based C.jejuni detection system to detect C.jejuni contamination in retailed chicken samples and the associated environmental samples,and the results demonstrated that the sensitivity of this detection system(82.2%)was higher than the cultivation-based C.jejuni detection methods(68.6%).The sample-to-answer time of this detection system was-40 minutes,indicating that the detection system has a high application potential for rapid and accurate on-site detection of C.jejuniThis study provides an example of bioinformatics-guided pathogen-specific target sequence identification for rapid and accurate onsite pathogen detection,and assembled a rapid and accurate CRISPR-Cas12b-based detection system for C.jejuni detection for the first time,and this detection system shows a high application potential in food safety and public health.