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对虾传染性皮下及造血组织坏死病毒(IHHNV) TaqMan MGB检测方法的建立
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Abstract:
本研究建立了一种对虾传染性皮下及造血组织坏死病毒(Infectious Hypodermal and Hematopoietic Necrosis Virus, IHHNV) TaqMan MGB探针荧光定量PCR检测方法。根据GenBank中已登录的IHHNV基因序列(GenBank登录号：AF218226)设计引物和探针建立的检测方法，采用构建的pMD-18-IHHNV质粒建立的标准曲线为y = ？0.3084X + 12.631，R2 = 0.9995。另外该定量检测方法的最低检出限为5 copies/μl，与对虾其它常见疫病均无交叉反应，且不同浓度标准品组内的变异系数(CV)在0.08%~0.26%之间，与普通PCR检测结果比较显示对虾样品的IHHNV阳性检出率提高，说明该方法具有良好的灵敏度、特异性和重复性，能够为对虾传染性皮下及造血组织坏死病的早期快速诊断提供技术支撑。
This study established a contagious subcutaneous and hematopoietic tissue necrosis virus TaqMan in shrimp MGB probe fluorescence quantitative PCR detection method. The primers and probes for this method were designed based on the IHHNV gene sequence already registered in GenBank (GenBank accession number: AF218226). The standard curve was established using the constructed pMD-18-IHHNV plasmid, with the equation y = ？0.3084X + 12.631 and R2 = 0.9995. In addition, the minimum detection limit of this quantitative detection method is 5 copies/μl. This method has no cross reactivity with other common diseases of shrimp. The coefficient of variation (CV) within different concentration standard groups of this method ranges from 0.08% to 0.26%. Compared with ordinary PCR detection results, the IHHNV positive detection rate of shrimp samples has increased. This method demonstrates good sensitivity, specificity, and reproducibility. This method can provide technical support for the early and rapid diagnosis of infectious subcutaneous and hematopoietic tissue necrosis disease in shrimp.
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