实时荧光重组酶聚合酶扩增技术快速检测创伤弧菌方法的建立  

Establishment of a rapid detection method for Vibrio vulnificus based on real⁃time fluorescent recombinase polymerase amplification

作　　者：禹乐[1] 朱启淦 温路生[1] 许家伦[1] 郭健莲[2] 梁声强 孟加榕[1] YU Le;ZHU Qigan;WEN Lusheng;XU Jialun;GUO Jianlian;LIANG Shengqiang;MENG Jiarong(Department of Pathology,909th Hospital,Dongnan Hospital Affiliated to Xiamen University,Zhangzhou 363000,China;Department of Clinical Laboratory,909th Hospital,Dongnan Hospital Affiliated to Xiamen University)

机构地区：[1]第九○九医院,厦门大学附属东南医院病理科,漳州363000 [2]第九○九医院,厦门大学附属东南医院检验科

出　　处：《山西医科大学学报》2025年第2期213-218,共6页Journal of Shanxi Medical University

基　　金：福建省自然科学基金项目(2023J011843,2023J011841);福建省漳州市科技拥军项目(ZZ2024KD04);福建省漳州市科技项目(ZZ2021J09);联勤保障部队第九○九医院自主项目(22MS001)。

摘　　要：目的 建立一种基于实时荧光重组酶聚合酶扩增(recombinant polymerase amplification,RPA)技术快速检测创伤弧菌的方法。方法 根据编码创伤弧菌溶血素vvhA基因的保守序列设计RPA引物,通过琼脂糖凝胶电泳筛选出最佳引物;根据筛选出的引物设计实时荧光探针,并建立实时荧光RPA检测体系。通过检测金黄色葡萄球菌、粪肠球菌、铜绿假单胞菌、河流弧菌、溶藻弧菌等10种其他菌种评价检测体系的特异性;通过检测梯度稀释的创伤弧菌基因组DNA评价检测体系的灵敏度。用qPCR试剂盒平行检测10例模拟临床样本,验证检测体系的性能。结果 基于创伤弧菌vvhA基因保守序列建立起实时荧光RPA检测体系,该体系对金黄色葡萄球菌等10种其他菌种基因组DNA均无交叉反应。检测创伤弧菌的灵敏度为1×10^(2)CFU/mL;检测模拟临床样本结果与qPCR方法的结果一致,准确率和检出率均为100%。结论 本研究建立的实时荧光RPA检测创伤弧菌方法灵敏度高、特异性强,操作简单快速,为创伤弧菌的现场快速检测提供一种新途径。Objective To establish a method for rapid detection of Vibrio vulnificus based on real-time recombinant polymerase ampli-fication(RPA).Methods RPA primers were designed according to the conserved sequence of vvhA gene encoding Vibrio vulnificus hemolysin,and the optimal primers were screened by agarose gel electrophoresis.Real-time fluorescent probes were designed according to the screened primers,and a real-time fluorescent RPA detection system was established.The specificity of the detection system was evaluated by detecting ten other strains such as Staphylococcus aureus,Enterococcus faecalis,Pseudomonas aeruginosa,Vibrio fluvialis,and Vibrio alginolyticus.The sensitivity of the detection system was evaluated by detecting gradient-diluted Vibrio vulnificus genomic DNA.A qPCR kit was used to parallelly detect ten simulated clinical samples to verify the performance of the detection system.Results A real-time fluorescent RPA detection system based on the conserved sequence of the vvhA gene of Vibrio vulnificus was suc-cessfully established.The system had no cross-reaction with genomic DNA of ten other strains such as Staphylococcus aureus.The sen-sitivity for detecting Vibrio vulnificus by this system was 1×10^(2) CFU/mL.The results were in full accordance with those obtained by qPCR,with both the accuracy and the detection rate reaching 100%.Conclusion The established real-time fluorescent RPA method for detecting Vibrio vulnificus has high sensitivity,strong specificity,and simple and rapid operation,which provides a new approach for on-site rapid detection of Vibrio vulnificus.

关 键 词：创伤弧菌 vvhA基因 重组酶聚合酶扩增 核酸扩增 分子检测 快速检测 

分 类 号：R446[医药卫生—诊断学]