猪细环病毒k2型SYBR GreenⅠ实时荧光定量PCR方法的建立

摘要

In order to rapidly and quantitatively detect Porcine Torque teno sus virus k2 (PTTSuVk2) , a SYBR GreenⅠ-based quantitative real time PCR assay was developed using specifi cally paired primers targeting to the ORF2 gene of PTTSuVk2. The PCR assay was linear in the range of 1.92×102~1.92×107 copies per microliter. A series of experiments were carried out to assess the sensitivity, specifi city and reproducibility of the method, followed by the intra-assay and inter-assay CVs for CT values obtained with the standard plasmids. The melting curve analysis using SYBR GreenⅠ dye showed one specifi c peak with a melting temperature (Tm) at (83.83±0.08)℃ without primer-dimers peak. The intra-assay CVs were equal or less than 1.69 % and the inter-assay CVs were equal or less than 2.19 %. No cross-reaction occurred with nucleic acids extracted from Porcine circovirus (type 1 and type 2), Porcine parvovirus,Porcine bovavirus type 5 and Porcine torque teno virus 1a and 1b. The results demonstrated that the assay was specifi c and reproducible, suggesting the quantitative PCR might be used for rapid laboratory diagnosis and epidemiological investigation for PTTSuVk2.%为建立检测猪细环病毒k2型（Porcine Torque teno sus virus k2，PTTSuVk2）SYBR GreenⅠ实时荧光定量PCR方法，本研究根据PTTSuVk2 ORF2基因序列特征设计引物，建立基于SYBR GreenⅠ检测PTTSuVk2的实时荧光定量PCR方法，该方法在PTTSuVk2 ORF2基因含量为1.92×102~1.92×107 copies/μL反应范围内有很好的线性关系。扩增产物溶解曲线分析仅出现单一特异峰，无引物二聚体，Tm值为（83.83±0.08）℃，对猪圆环病毒（Porcine circovirus type 1 and type 2，PCV1和PCV2）、猪细小病毒（Porcine parvovirus，PPV）、猪博卡病毒5型（Porcine bovavirus type 5，PboV5）、猪细环病毒1a型（PTTSuV1a）和1b型（PTTSuV 1b）核酸均无阳性信号扩增，可重复性好，组内变异系数为0.39%～1.69%，组间变异系数0.69%～2.19%。本方法的建立可用于定量分析PTTSuVk2感染噬性组织器官和感染程度，为PTTSuVk2的流行病学研究及临床诊断等提供了一种可靠的方法。