嗜水气单胞菌纳米PCR检测方法的建立  

Establishment of a Nano-PCR Detection Method for Aeromonas hydrophila

作　　者：韩雅静 赵奇 石有权 安瑞 赵云萍 史秋梅 吴同垒 高桂生 HAN Yajing;ZHAO Qi;SHI Youquan;AN Rui;ZHAO Yunping;SHI Qiumei;WU Tonglei;GAO Guisheng(Hebei Key Laboratory of Preventive Veterinary Medicine,Qinhuangdao 066004,China;Luanzhou Agriculture and Rural Bureau,Tangshan 063700,China;Agriculture and Rural Bureau of Qianxi County,Tangshan 064300,China)

机构地区：[1]河北省预防兽医学重点实验室,河北秦皇岛066004 [2]滦州市农业农村局,河北唐山063700 [3]迁西县农业农村局,河北唐山064300

出　　处：《中国兽医杂志》2025年第2期47-51,共5页Chinese Journal of Veterinary Medicine

基　　金：河北省科技厅循环养殖鱼类技术的应用与示范(202867012);河北省第三期现代农业产业技术体系技术河北省创新团队建设项目(HBCT2023220207)。

摘　　要：为了快速检测水生动物的嗜水气单胞菌(A.hydrophila)感染,本试验根据嗜水气单胞菌溶血素hlyA基因序列设计引物,通过对退火温度和引物体积的优化,建立了嗜水气单胞菌的纳米聚合酶链式反应(Nano-PCR)检测方法,分析其敏感性、特异性和临床样本检测结果,并与普通聚合酶链式反应(PCR)方法进行比较。结果显示,优化建立的纳米PCR方法成功扩增出约280 bp的嗜水气单胞菌特异性条带;最小检测浓度为1.61 pg,敏感性是普通PCR(最小检测浓度为16.1 pg)的10倍;纳米PCR和普通PCR方法对创伤弧菌(V.vulnificus)、迟缓爱德华菌(E.tarda)和维氏气单胞菌(A.veronii)均无扩增;对18份病料的阳性检出率为72%,与普通PCR结果一致。结果表明,本试验建立的检测嗜水气单胞菌的纳米PCR方法较普通PCR方法具有更高的敏感性和特异性,为水生动物嗜水气单胞菌感染的鉴别诊断和防治提供了技术支持。To rapidly detect Aeromonas hydrophila(A.hydrophila)infections in aquatic animals,this study designed primers based on the hlyA hemolysin gene sequence of A.hydrophila.A nano-polymerase chain reaction(nano-PCR)detection method was established by optimizing annealing temperature and primer volume.The sensitivity,specificity,and clinical sample detection results of the nano-PCR method were analyzed and compared with those of conventional PCR.The results showed that the optimized nano-PCR successfully amplified a specific A.hydrophila band of approximately 280 bp.The minimum detection concentration was 1.61 pg,and the sensitivity is 10 times higher than that of conventional PCR(with a minimum detection concentration of 16.1 pg).Both nano-PCR and conventional PCR showed no amplification for Vibrio vulnificus,Edwardsiella tarda,and Aeromonas veronii.The positive detection rate for 18 clinical samples was 72%,consistent with the results of conventional PCR.These findings indicate that the nano-PCR method developed in this study has higher sensitivity and specificity than conventional PCR,providing technical support for the differential diagnosis and control of A.hydrophila infections in aquatic animals.

关 键 词：嗜水气单胞菌 纳米PCR hlyA 临床检测 

分 类 号：S855.1[农业科学—临床兽医学]