【摘要】 根据pir AVp基因保守序列设计特异性引物和TaqMan–MGB探针,经优化反应体系及条件,建立检测致对虾急性肝胰腺坏死病副溶血弧菌(VpAHPND)的TaqMan–MGB探针荧光定量PCR方法;以重组质粒为标准品制作标准曲线,并进行特异性、敏感性、重复性及临床应用试验。结果表明:该方法定量范围宽,起始模板浓度范围为1×101～1×109拷贝/反应时,标准曲线具良好的线性关系;最低检测限约为10拷贝/反应,与对虾其他病原菌及病毒无交叉反应,重复试验变异系数为0.37%～0.47%,可在1 h内完成单个样品检测;对临床对虾样品及水样的检测结果表明,与世界动物卫生组织(OIE)推荐的套式PCR法相比,可提高VpAHPND的阳性检出率,且检测为阳性的样品包含全部套式PCR法检测为阳性的样品。可见,应用TaqMan–MGB探针建立的检测VpAHPND的荧光定量PCR方法具有灵敏度高、特异性强、重复性好、能快速定量等优点,可用于临床对虾样品及水样的VpAHPND检测。更多还原

【Abstract】 In this study,a pair of specific primers and fluorogenic-labeled TaqMan-Minor Groove Binder(TaqMan-MGB)probe were designed and synthesized based on the conserved region of pirAVp gene,then a real-time PCR method was developed to detect AHPND-causing Vibrio parahaemolyticus(VpAHPND).The results showed that the method had a wide quantitative range from 1×101 to 1×109 copies per reaction,with a good linear relationship in its standard curve.The real-time PCR method had a high sensitivity with the detection limit as low as to 10 copies per reaction for the purified recombinant plasmids of pTOPO-T-pir AVp,and the entire detection could be completed within 1 h for a single sample and it also had a high specificity in detecting DNA of VpAHPND.The variation coefficients among cycle thresholds(Cts) of the repeatability test were 0.37%-0.47%.Comparing with the nested PCR recommended by OIE,the TaqMan-MGB probe real-time PCR had better positive detection rate of VpAHPND.In conclusion,the TaqMan-MGB probe real-time PCR assay developed in the study is fast,sensitive,specific,quantitative and of high reproducibility,making it a ideal method for detecting VpAHPND in shrimp and water samples.更多还原