山西医科大学学报2025，Vol.56Issue(2)：213-218,6.DOI:10.13753/j.issn.1007-6611.2025.02.015
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 am-plification
摘要
Abstract
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 1×102 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Key words
Vibrio vulnificus/vvhA gene/RPA/nucleic acid amplification/molecular detection/rapid detection分类
医药卫生引用本文复制引用
禹乐,朱启淦,温路生,许家伦,郭健莲,梁声强,孟加榕..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[J].山西医科大学学报,2025,56(2):213-218,6.基金项目
福建省自然科学基金项目(2023J011843,2023J011841) （2023J011843,2023J011841）
福建省漳州市科技拥军项目(ZZ2024KD04) （ZZ2024KD04）
福建省漳州市科技项目(ZZ2021J09) （ZZ2021J09）
联勤保障部队第九○九医院自主项目(22MS001) （22MS001）
