嗜水气单胞菌RPA-LFD快速检测方法的建立

ESTABLISHMENT OF A METHOD FOR RAPID DETECTION OF AEROMONAS HYDROPHILA BY RECOMBINASE POLYMERASE AMPLIFICATION(RPA)COMBINED WITH LATERAL FLOW STRIPS(LFD)

文章旨在建立一种针对嗜水气单胞菌的敏感性高、特异性强的快速临床诊断方法。研究根据嗜水气单胞菌促旋酶B亚单位(gyrase subunit B,gyrB)基因的保守序列,设计特异性重组酶聚合酶扩增技术(RPA)扩增引物,通过结合琼脂糖凝胶电泳(AGE)和侧流层析试纸条(LFD)的方法进行反应条件的优化、特异性及灵敏度检测。实验结果表明,所建立的嗜水气单胞菌RPA-LFD快速检测方法可在38℃最适温度下30min内无干扰地检测到嗜水气单胞菌,对嗜水气单胞菌纯培养物和基因组DNA的最小检出限为10^(3)cfu/mL和100 pg/μL。利用建立的RPA-LFD与普通PCR同时检测杂交鲟鱼处理临床样品的结果表明,两种方法的结果一致。综上所述,通过对RPA反应条件的探索和优化,成功建立了嗜水气单胞菌快速检测方法。该方法操作简便,反应迅速,与普通PCR相比,不需要昂贵的仪器,为未来嗜水气单胞菌细菌性疾病的早期诊断提供了更有效的技术支持。

In order to establish a rapid detection method for Aeromonas hydrophila utilizing recombinase polymerase amplification(RPA)technology,we designed specific RPA primers based on the conserved sequence of the gyrB gene of Aeromonas hydrophila.The research included the optimization of reaction conditions and an assessment of specificity and sensitivity by both agarose gel electrophoresis(AGE)and lateral flow dipstick(LFD)chromatography.The experimental results showed that the established rapid detection for Aeromonas hydrophila could detect Aeromonas hydrophila within 30min at the optimal temperature of 38℃.The minimum limit of detection(LOD)for Aeromonas hydrophila pure culture and genomic DNA using the RPA-LFD method was determined to be 10^(3) cfu/mL and 100 pg/μL,respectively.Furthermore,the simultaneous detection of clinical samples from hybridized sturgeon,treated using our established RPA-LFD method,exhibited consistent results when compared to conventional PCR.In conclusion,the recombinase polymerase amplification combined with lateral flow test strip(RPA-LFD)method of Aeromonas hydrophila was successfully established by exploring and optimizing RPA reaction conditions.The method was characterized by its simplicity,rapid reaction time,and lack of reliance on expensive instrumenttion compared with conventional PCR.Importantly,this method provides effective technical support for the early diagnosis of sturgeon suffering from bacterial diseases caused by Aeromonas hydrophila in the future.