| In recent years,antibiotic-resistant bacterial infections have been causing hundreds of thousands of deaths worldwide every year,which pose serious threats to human health.Polymyxin and tigecycline are considered as the last clinical means for the treatment of infective diseases caused by antibiotic-resistant gram-negative bacteria;however,more and more strains isolated from the human-and animal-originating samples are found to be resistant to polymyxin and tigecycline recently,which seriously threaten the public health.Mcr-1 and tet(X4)are the most prevalent genes that endow bacteria resistance to polymyxin and tigecycline,respectively.Under such circumstances,it is urgent to develop rapid detection systems for mcr-1 and tet(X4)detection to meet the on-site detection requirments.In this study,we developed a RPA/CRISPR-Cas12b-based detection system to determine mcr-1 or tet(X4)contamination rapidly,and the detection results can also provide guidelines for the isolation and identification of mcr-1 or tet(X4)positive strains.The conserved and specific protpspacer sequences that are critical for the cleavage activity activation of Cas12b were identified in the mcr-1 and tet(X4)genes by multiple sequence alignment and online BLAST analyses.Then,sgRNAs were designed and prepared based on the protospacers,and the efficiency of the sgRNAs was confirmed by the successful cis-and trans-cleavage activity activation of CRISPR-Cas12b.Based on the designed sgRNAs,the CRISRP-Cas12b-based mcr-1 and tet(X4)detection systems were developed.The specificity of the developed mcr-1 and tet(X4)detection systems were determined,and the results showed that the detection system was activatied and bright fluorescence showed up only when mcr-1-positive or tet(X4)-positive bacterial genomic DNA was present in the system,while the DNA samples from other mcr-1-negative or tet(X4)-negative strains could not activate the CRISPR-Cas12b-based detection system,demonstrating that the specificity of the developed CRISRP-Cas12b-based mcr-1 and tet(X4)detection systems was very high.Three primer pairs were then designed from the conserved regions of mcr-1 and tet(X4)sequences containing the protospacers,respectively,and the RPA amplification methods were developed accrodingly.Then,RPA amplication was performed in the same PCR tube with the Cas12b detection step to improve the sensitivity and reduce the probability of contamination of the detection systems,and the optimal reaction temperature and time of the RPA/CRISPR-Cas 12b-based detection systems were explored.The optimal reaction temperature and time of the RPA-CRISPR-Cas 12bbased mcr-1 detection system were 43.3℃ and 45 min,respectively,while the values for the RPA/CRISPR-Cas12b-based tet(X4)detection system were 42℃ and 30 min,respectively.The specificity of the developed mcr-1 and tet(X4)detection systems were determined,and the results showed that the DNA samples from other mcr-1-negative or tet(X4)-negative strains could not activate the RPA/CRISPR-Cas12b-based detection systems.The RPA/CRISPR-Cas12b-based detection systems could be activated by 6.25 copies/μL of mcr-1 gene or 9 copies/μL of tet(X4)gene,indicative of high sensitivity of the developed detection systems.Finally,the reliability of the developed detection systems were determined based on simulated contaiminated pork samples under laboratory conditions.The results demonstrated that the DNA samples extracted from the pork samples without catamination of mcr-1-or tet(X4)-positive strains could not activate the detection systems,while the samples contaminated with as low as 7 CFU/g of mcr-1-or tet(X4)-positive strains activated the systems and strong fluorescence signals were observed.These results further demonstrated the RPA/CRISPR-Cas12b-based detection systems exhibited high sensitivity and specificity for mcr-1 and tet(X4)detection.The detection systems were then applied to determine whether the pork and environmental samples collected from the local market were contaiminated with mcr-1 or tet(X4),and 5 and 7 samples among the total 40 samples were identified to be mcr-1 and tet(X4)positive,respectively.Based on these results,4 mcr1-positive and 12 tet(X4)-positive strains were obtained by bacterial cultivation method.A representative mcr-1-positive and a tet(X4)-positive strains were selected for whole genome sequencing using Nanopore third-generation sequencing technology.The genetic background of the mcr-1 and tet(X4)were identified,which provides novel insights for continuous monitoring of the prevalence and spread of mcr-1 and tet(X4)resistance genes.Overall,the RPA/CRISPR-Cas12b-based mcr-1 and tet(X4)detection systems were developed in this study,and the detection systems exhibited high specificity and sensitivity,with a sample-to-result time being～1 h.The developed detection systems can identify mcr1 and tet(X4)contaminated samples rapidly,the results can also provide guidelines for isolation and identification of drug-resistant bacteria,and thus show promising potential in clinic and food safety applications. |
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