虾虹彩病毒TaqMan-MGB探针荧光定量PCR检测方法的建立

Development of TaqMan-MGB probe fluorescence quantitative PCR for detection of shrimp iridescent virus

【目的】建立检测虾虹彩病毒(SIV)的TaqMan-MGB探针荧光定量PCR,为实现虾虹彩病毒病(SIVD)快速诊断及疫情监测提供一种新的技术手段。【方法】根据SIV的MCP基因保守序列设计特异性引物和TaqMan-MGB探针,将目的片段克隆至pMD18-T载体制备重组质粒pMD18-T-MCPSIV;优化反应体系及扩增条件后建立检测SIV的TaqMan-MGB探针荧光定量PCR,以pMD18-T-MCPSIV为标准品绘制标准曲线,并通过特异性、敏感性、重复性试验及临床应用验证其实用性。【结果】制备的重组质粒(pMD18-T-MCPSIV)DNA稳定性好,满足作为标准品的要求;标准品起始模板范围为2.0×101~2.0×109拷贝/反应时,建立的标准曲线具有良好线性关系。优化后的TaqMan-MGB探针荧光定量PCR灵敏度高,对重组质粒的检测灵敏度可达20拷贝/反应,对虾组织SIV的检测灵敏度约10拷贝/mg,其临床检测的敏感性与套式PCR相当;与虾类其他常见病原无交叉反应,组内和组间变异系数分别为0.27%~0.54%和0.61%~0.95%,且扩增与产物分析同步完成,整个检测过程仅需1 h左右。采用建立的TaqMan-MGB探针荧光定量PCR与目前农业农村部推荐的套式PCR同时对276份临床虾样品进行SIV检测,结果显示,两种方法的大部分检测结果一致,符合率为98.6%,但检测低拷贝数样品时前者具有更高的灵敏度。2018和2019年广西虾样品的SIV阳性检出率分别为19.7%和7.5%,且养殖凡纳滨对虾、罗氏沼虾和红螯螯虾均检测到SIV阳性。【结论】基于SIV MCP基因保守序列建立的TaqMan-MGB探针荧光定量PCR检测方法具有灵敏度高、特异性强、重复性好、定量范围宽及简单快速等优点,适用于虾类样品SIV检测,可为SIVD快速诊断及疫情监测提供一种新的技术手段。

【Objective】The TaqMan-MGB probe fluorescence quantitative PCR detection method for shrimp iridescent virus(SIV)was developed in order to provide a new detection technology for clinical diagnosis and epidemic monitoring of shrimp iridescent virus disease(SIVD).【Method】A pair of specific primers and a fluorogenic-labeled TaqMan-Minor Groove Binder(TaqMan-MGB)probe were designed based on the conserved sequence of MCP gene in SIV,and the target fragments were cloned into the pMD18-T vector to construct pMD18-T-MCPSIV recombinant plasmids. Then the TaqMan-MGB probe fluorescence quantitative PCR detection method was developed after optimizing reaction system and amplification,the standard curve was established for quantitative analysis with pMD18-T-MCPSIV as standards and the specificity,sensitivity,reproducibility tests and detection of clinical samples were carried out to evaluate the applicability of this method.【Result】The prepared recombinant plasmid DNA of pMD18-T-MCPSIV had good stability and could meet the requirements of the standards,and the standard curve showed a good linear relationship with quantitative range from 2.0×101 to 2.0×109 copies/reaction. The optimized TaqMan-MGB probe fluorescence quantitative PCR method had a high sensitivity with the detection limit as low as to 20 copies/reaction for the pMD18-T-MCPSIV and approximately 10 copies/mg for the shrimp tissue samples,and its clinical detection sensitivity was comparable to nested PCR. It had no cross reaction with common shrimp pathogens,the variation coefficients of intra-group and inter-group were 0.27%-0.54% and 0.61%-0.95%,amplification could be completed together with products analysis and the entire detection could be completed within 1 h for a single sample. The TaqMan-MGB probe fluorescence quantitative PCR and nested PCR method recommended by Ministry of Agriculture and Rural Affairs were adopted to detect 276 clinical samples,the results showed that most of the results were the same with coincidence rate of 98.6%. But TaqMan-MGB probe fluorescence quantitative PCR had better sensitivity in detecting samples with low copies SIV. The SIV positive rates of Guangxi were 19.7% in 2018 and 7.5% in 2019,respectively. SIV positive samples were detected in aquaculture Litopenaeus vannamei,Macrobrachium rosenbergii and Cherax quadricarinatus. 【Conclusion】The TaqMan-MGB probe fluorescence quantitative PCR assay based on MCP gene conservative sequence in SIV developed in the study is fast,sensitive,specific,reproducible and of wide quantitative range,making it an ideal method for detecting SIV in shrimp clinical samples and a new technique for the diagnosis and monitoring of SIVD.