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为建立一种灵敏、特异、快速地检测虾肝肠胞虫的方法,以虾肝肠胞虫的保守序列为靶序列,利用Primer Premier 5.0和Primer Express 2.0软件分别设计引物和TaqMan探针,开展了虾肝肠胞虫的TaqMan荧光定量PCR检测技术研究。将虾肝肠胞虫目标片段克隆到p MD 19-T载体上构建重组质粒,然后以10倍梯度稀释的重组质粒作为标准品。根据标准品拷贝数与Ct值的关系绘制标准曲线,其斜率为-3.239,相关系数r~2=0.996。重复性试验结果表明,该方法 Ct值的变异系数为0.94%~1.13%,具有良好的稳定性。特异性检测结果显示,该方法对白斑综合征病毒、传染性皮下及造血组织坏死病毒、高致病性副溶血弧菌、微孢子虫感染的对虾肌肉组织等的检测结果均为阴性,表明无交叉反应,具有良好特异性。应用建立的方法对宁波和嘉兴地区15批凡纳滨对虾样品进行检测,发现有6批感染虾肝肠胞虫。利用标准曲线对感染虾肝肠胞虫的虾不同组织进行了定量分析。本研究建立的虾肝肠胞虫TaqMan荧光定量PCR方法灵敏度高、特异性强,对虾肝肠胞虫病的快速诊断与病原定量检测有重要意义,可广泛应用于实验室和现场快速检测。
In order to establish a sensitive, specific and rapid method for the detection of E.coli, we designed the primers and TaqMan probes by using the conserved sequences of E.coli and E.coli as the target sequences. Study on TaqMan Fluorescence Quantitative PCR Detection of. The target fragment was cloned into p MD 19-T vector to construct a recombinant plasmid, and then a 10-fold gradient of the recombinant plasmid was used as a standard. According to the standard copy number and Ct value of the relationship between the standard curve, the slope of -3.239, the correlation coefficient r ~ 2 = 0.996. Repeatability test results show that the Ct value of the coefficient of variation coefficient of 0.94% to 1.13%, with good stability. Specificity test results showed that this method was negative for white spot syndrome virus, infectious subcutaneous and hematopoietic tissue necrosis virus, highly pathogenic Vibrio parahaemolyticus, microsporidian infected shrimp muscle tissue and the like, indicating that none Cross-reaction, with good specificity. Fifteen batches of Litopenaeus vannamei samples from Ningbo and Jiaxing were detected by using the established method. Six batches of shrimp infected with shrimp and intestine were found. The standard curve was used to quantitatively analyze different tissues of shrimp infected with shrimp and hepatocytes. The TaqMan fluorescent quantitative PCR method established in this study has high sensitivity and specificity and is of great significance in the rapid diagnosis and pathogen quantitative determination of hepatobiliary and cystosis in shrimp, and can be widely used in laboratories and on-the-spot rapid detection.