摘要
针对对虾急性肝胰腺坏死病(acute hepatopancreatic necrosis disease,AHPND)病原菌检测,建立了一种重组酶介导等温扩增(recombinase acid amplification,RAA)检测方法。根据pVA1质粒保守序列设计特异性探针和引物,分别以不同稀释浓度梯度的含pirB靶序列的重组质粒pMD18-T-pirB为模板进行荧光RAA法检测,评价其灵敏度;对溶藻弧菌、河流弧菌、创伤弧菌等病原菌进行荧光RAA法检测,评价其特异性;取8.2×10^(3 )copies/μL浓度的pMD18-T-pirB重组质粒进行荧光RAA试验,评价其重复性;对人工感染样品用RAA方法进行应用试验,以水产行业标准(SC/T 7233—2020)推荐的荧光探针PCR法作为平行对照。结果显示:该方法可对pirB靶序列基因片段进行有效扩增,最低检出限为8.2×101 copies/μL,与其他病原菌无交叉反应,重复试验变异系数为0.41%,与荧光探针PCR方法符合率为100%。结果表明,该方法特异性强、灵敏度高、重复性好、操作步骤简单,可用于对虾样品的AHPND现场检测。本研究为AHPND快速检测提供了技术支撑。
A recombinase acid amplification(RAA)assay was established for detecting the pathogen of acute hepatopancreatic necrosis disease(AHPND).Specific probes and primers were designed according to the conserved sequence of pVA1 plasmid for detection by the established method,taking the recombinant plasmid pMD18-T-pirB containing pirB target sequence at various dilution concentration gradient as a template,followed by evaluation on its sensitivity;the method was used to detect such pathogenic bacteria as Vibrio alginolyticus,Vibrio fluvialis and Vibrio vulnificus,followed by evaluation on its specificity;and then to detect pMD18-T-pirB at the concentration of 8.2×10^(3) copies/μL,followed by evaluation on its repeatability;application test was conducted for artificially infected samples by the established method,taking the fluorescent probe PCR assay recommended by the aquatic industry standard(SC/T 7233—2020)as a parallel control.The results showed that pirB target sequence gene fragment could be effectively amplified by the established method,with a minimum detection limit of 8.2×101 copies/μL,no cross reaction with other pathogenic bacteria was observed,the variable coefficient of repeatability test was 0.41%,and the coincidence rate with fluorescent probe PCR was 100%.In conclusion,the established method could be used for field detection of AHPND in shrimp samples with its advantages of strong specificity,high sensitivity,good repeatability and simple operation,by which,future rapid detection of AHPND was technically supported.
作者
陈平亚
吴山楠
何翠华
张亮亮
吴少荣
王绥家
吴毓浪
Chen Pingya;Wu Shannan;He Cuihua;Zhang Liangliang;Wu Shaorong;Wang Suijia;Wu Yulang(Technical Center of Haikou Customs,Haikou,Hainan 570311,China)
出处
《中国动物检疫》
CAS
2022年第9期128-133,共6页
China Animal Health Inspection
基金
海南省重点研发计划项目(ZDYF2020088)。
