鱼类产品中嗜水气单胞菌重组酶聚合酶扩增结合CRISPR/Cas12a快速检测及耐药性分析

Rapid detection and drug resistance analysis of Aeromonas hydrophila recombinase polymerase amplification combined with CRISPR/Cas12a in fish products

中文关键词:  嗜水气单胞菌  RPA-CRISPR/Cas12a  LAMP  SYBG实时荧光PCR  耐药表型  耐药基因

英文关键词:Aeromonas hydrophila  recombinase polymerase amplification  clustered regularly interspaced short palindromic repeats-associated protei  loop-mediated isothermal amplification  real-time fluorescence quantitative polymerase chain reaction  drug-resistant phenotype  drug-resistant gene

中文摘要:

      目的 建立重组酶聚合酶扩增(recombinase polymerase amplification, RPA)结合簇状规则间隔短链重复序列及其相关蛋白(clustered regularly interspaced short palindromic repeats-associated protein, CRISPR-Cas)新方法, 检测鱼类产品中嗜水气单胞菌, 并分析其耐药性。方法 采用基于最新的CRISPR基因编辑技术建立RPA结合CRISPR/Cas12a新方法, 快速检测嗜水气单胞菌, 同时采用环介导等温扩增(loop-mediated isothermal amplification, LAMP)方法比对两种方法的检出率。筛查嗜水气单胞菌分离株的耐药性, 采用纸片扩散法测定耐药表型, 采用荧光染料(SYBR green, SYBG)实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction, qPCR)测定耐药基因, 分析耐药表型与耐药基因结果的一致性。结果 在74例鱼类样品中, 基于RPA-CRISPR/Cas12a和LAMP同时检测出6例嗜水气单胞菌。分析耐药性测试结果发现, 分离株对5大类10种抗生素的耐药情况差异较大, 耐药表型与耐药基因结果一致性较高。结论 所建立的RPA-CRISPR/Cas12a方法适用于嗜水气单胞菌快速检测。而细菌耐药性的产生机制比较复杂, 耐药表型的产生在一定程度上受耐药基因调控。研究结果为水产品中嗜水气单胞菌快速检测和耐药性筛查提供新思路和方法。

英文摘要:

      Objective To establish a new method using recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats-associated protein (CRISPR-Cas) to detect Aeromonas hydrophila in fish products and analyze its drug resistance. Methods A new method of RPA combined with CRISPR/Cas12a was established based on the latest CRISPR gene editing technology to rapidly detect Aeromonas hydrophila, and the loop-mediated isothermal amplification (LAMP) method was used to compare the results of the two methods. To screen the drug resistance of Aeromonas hydrophila isolates, the disk diffusion method was used to determine the drug resistance phenotype, SYBR Green (SYBG) real-time fluorescence quantitative polymerase chain reaction (qPCR) was used to determine the drug resistance genes, and the consistency of the drug resistance phenotype and drug resistance gene results was analyzed. Results Among 74 fish samples, 6 cases of Aeromonas hydrophila were simultaneously detected based on RPA-CRISPR/Cas12a and LAMP. Analysis of the drug resistance test results found that the isolates had great differences in resistance to 10 antibiotics in 5 major categories, and the results of drug-resistant phenotypes and drug-resistant genes were highly consistent. Conclusion The established RPA-CRISPR/Cas12a method is suitable for rapid detection of Aeromonas hydrophila. The mechanism of bacterial drug resistance is relatively complex, and the generation of drug-resistant phenotypes is regulated by drug-resistant genes to a certain extent. The research results provide new ideas and methods for rapid detection and drug resistance screening of Aeromonas hydrophila in aquatic products.