大口黑鲈虹彩病毒TaqMan荧光定量PCR检测方法的建立与应用

Establishment and application of TaqMan qPCR method for largemouth bass ranavirus

为建立大口黑鲈虹彩病毒(LMBV)荧光定量PCR检测方法,本研究基于LMBV的主要衣壳蛋白(MCP)基因设计特异性引物及Taq Man探针,通过反应条件的优化,建立了LMBV的TaqMan荧光定量PCR检测方法。结果显示,该方法以pMD-LMBV-MCP重组质粒为标准品,建立的标准曲线在4.6×10^(2)拷贝/μL~4.6×10^(8)拷贝/μL浓度范围内与Ct值具有良好的线性关系,相关系数(R2)为1,扩增效率(E)为99.2%。利用本实验建立的方法对传染性脾肾坏死病毒、大口黑鲈弹状病毒、神经坏死病毒、鲤春病毒血症病毒、草鱼呼肠弧病毒、传染性造血器官坏死病毒及LMBV进行检测,结果显示,该方法仅对LMBV产生特异性扩增,其他病原均为阴性,特异性强;该方法对重组质粒标准品的检测下限为46拷贝/μL,是常规PCR检测方法(4.6×10^(3)拷贝/μL)的100倍,敏感性高;组内和组间重复性试验的变异系数均小于2.5%,重复性好。利用该方法对30份临床样品进行检测,结果显示LMBV阳性率为40%,显著高于常规PCR的13.3%;另外利用该方法对人工感染LMBV的大口黑鲈各组织进行病毒载量测定,结果显示其胃、肝、肠、脾和心脏的病毒载量高于其他组织。本研究建立的Taq Man荧光定量PCR检测方法特异性强、灵敏度高、重复性好,可用于临床LMBV的检测和流行病学调查,为LMBV感染的早期诊断提供可靠的检测方法。

To establish a qPCR detection method for the detection of largemouth bass ranavirus(LMBV). In this study,specific primers and Taq Man probes were designed based on the main capsid protein(MCP) gene of LMBV. Through the optimization of reaction conditions, a Taq Man qPCR detection method for LMBV was established. In this study, the recombinant plasmid of p MD-LMBV-MCP was used as standard, and the obtaining standard curve has a good linear relationship in the concentration range of 4.6×10^(2) copies/μL-4.6×10^(8) copies/μL, and the correlation coefficient(R^(2)) is 1. The amplification efficiency(E) of Taq Man q PCR is 99.2%;this method only produces specific amplification for LMBV, but not for ISKNV, MSRV, NNV,SVCV, GCRV and IHNV;the detection limit of this method is 46 copies/μL of p MD-LMBV-MCP plasmid, which is 100 times more sensitive than that of a conventional PCR(4.6×10^(3) copies/μL) suggesting a high sensitivity of Taq Man q PCR. The coefficients of variation for intra-assay and inter-assay reproducibilities were less than 2.5%;Thirty clinical samples were tested by this method,and the results showed that the positive detection rate of LMBV was 40%, which was significantly higher than that of conventional PCR(13.3%);This method was used to determine the viral load of each tissue obtained from largemouth bass artificially infected with LMBV. The results showed that the viral loads of the stomach, liver, intestine, spleen and heart were higher than those of other tissues. The Taq Man q PCR detection method established in this study has strong specificity, high sensitivity, and good reproducibility. It can be used for clinical LMBV detection and epidemiological investigation, and provides a reliable detection method for the early diagnosis of LMBV.