摘要：为了准确、特异、高效地检测猪繁殖与呼吸综合征病毒2型(PRRSV2),本研究根据GenBank上登录的PRRSV2不同谱系流行毒株的ORF6基因序列,选取保守区域设计合成了1对特异性检测引物和1条mgb探针,经优化退火温度、引物和探针比例等反应条件及敏感性、特异性、重复性试验,建立了检测PRRSV2的taqman mgb荧光定量PCR方法。结果:以猪繁殖与呼吸综合征病毒1型(PRRSV1)、伪狂犬病病毒(PRV)、猪瘟病毒(CSFV)、猪圆环病毒2型(PCV2)、猪圆环病毒3型(PCV3)、猪流行性腹泻病毒(PEDV)及猪细小病毒(PPV)等阳性核酸为模板,均无扩增;对标准质粒的最低检测下限为1×10^(1)copies/μL,建立的标准曲线呈现良好的线性关系,相关系数R2为0.994;稳定性和重复性好,批内和批间变异系数均小于1.78%。应用该方法对保留的30份临床阳性样品进行检测,结果阳性100%,Ct值在22~36间。综上,本研究建立的taqman mgb荧光定量PCR方法可用于临床和实验室中PRRSV2的快速检测,为猪繁殖与呼吸综合征的防控提供了技术支持。

摘要：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.