建立两步 SYBR Green I 实时 RT-PCR 检测方法用于检测和定量临床样品中的小反刍兽疫病毒。
Development of a two-step SYBR Green I based real time RT-PCR assay for detecting and quantifying peste des petits ruminants virus in clinical samples.
机构信息
Department of Veterinary Microbiology, Madras Veterinary College, Chennai 600007, India.
Department of Veterinary Microbiology, Madras Veterinary College, Chennai 600007, India.
出版信息
J Virol Methods. 2014 Dec;209:25-9. doi: 10.1016/j.jviromet.2014.08.017. Epub 2014 Sep 4.
A two-step SYBR Green I based real time RT-PCR targeting the matrix (M) gene of Peste des petits ruminants virus (PPRV) was developed. The specificity of the assay was assessed against viral nucleic acid extracted from a range of animal viruses of clinical and structural similarities to PPRV including canine distemper virus, measles virus, bluetongue virus and Newcastle disease virus. But none of the viruses and no template control showed an amplification signal. Sensitivity of the same assay was assessed based on plasmid DNA copy number and with respect to infectivity titre. The lower detection limit achieved was 2.88 plasmid DNA copies/μl with corresponding Ct value of 35.93. Based on tissue culture infectivity titre the lower detection limits were 0.0001TCID50/ml and 1TCID50/ml for the SYBR green I based real time RT-PCR and conventional RT-PCR, respectively. The calculated coefficient of variations values for intra- and inter-assay variability were low, ranging from 0.21% to 1.83% and 0.44% to 1.97%, respectively. The performance of newly developed assay was evaluated on a total of 36 clinical samples suspected of PPR and compared with conventional RT-PCR. The SYBR Green I based real time RT-PCR assay detected PPRV in 32 (88.8%) of clinical samples compared to 19 (52.7%) by conventional RT-PCR. Thus, the two-step SYBR Green I based real time RT-PCR assay targeting the M gene of PPRV reported in this study was highly sensitive, specific and reproducible for detection and quantitation of PPRV nucleic acids.
建立了一种基于两步 SYBR Green I 的实时 RT-PCR 方法，用于检测小反刍兽疫病毒（PPRV）的基质（M）基因。该方法的特异性通过针对与 PPRV 具有临床和结构相似性的一系列动物病毒的病毒核酸进行评估，包括犬瘟热病毒、麻疹病毒、蓝舌病病毒和新城疫病毒。但没有一种病毒或无模板对照显示扩增信号。该检测方法的灵敏度基于质粒 DNA 拷贝数和感染性滴度进行评估。实现的最低检测限为 2.88 个质粒 DNA 拷贝/μl，相应的 Ct 值为 35.93。基于组织培养感染性滴度，基于 SYBR 绿色 I 的实时 RT-PCR 和常规 RT-PCR 的最低检测限分别为 0.0001TCID50/ml 和 1TCID50/ml。内和间分析变异性的计算变异系数值较低，范围分别为 0.21%至 1.83%和 0.44%至 1.97%。总共对 36 份疑似 PPR 的临床样本进行了新开发检测方法的性能评估，并与常规 RT-PCR 进行了比较。与常规 RT-PCR 相比，基于两步 SYBR Green I 的实时 RT-PCR 方法在 32 份（88.8%）临床样本中检测到 PPRV，而在 19 份（52.7%）临床样本中检测到 PPRV。因此，本研究报道的针对 PPRV M 基因的两步 SYBR Green I 基于实时 RT-PCR 检测方法对 PPRV 核酸的检测和定量具有高度的敏感性、特异性和可重复性。
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