Virology Laboratory, Centre for Animal Disease Research and Diagnosis (CADRAD), Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, U.P. 243122, India.
J Virol Methods. 2010 Oct;169(1):198-201. doi: 10.1016/j.jviromet.2010.06.007. Epub 2010 Jun 25.
The present study describes the development of SYBR Green based real-time polymerase chain reaction (real-time PCR) for detection and quantitation of canine parvovirus type 2 (CPV 2) in faecal samples of dogs. In this assay, the primers were designed and custom-synthesized based on nucleotide sequence of VP2 gene of CPV 2. A standard curve was plotted using 10-fold serial dilution of standard plasmid DNA and Ct value. The standard curve was found to be linear over a 10(-7) dilution. The real-time PCR results were expressed as the number of DNA copies of CPV 2 per mg of faecal samples and showed range of 1.0 x 10(3) to 7.0 x 10(9) copies of viral DNA per mg of stool samples. The analytical sensitivity of the SYBR Green based real-time PCR was shown to be equivalent to 10 copies. Faecal samples (47) from dogs suspected of CPV 2 infection were analyzed by real-time PCR, haemagglutination (HA) assay and by a conventional PCR and 24, 20 and 22 samples were found positive for CPV 2, respectively. Comparison between the results of three different assays revealed that real-time PCR is more sensitive than HA and conventional PCR and allow the detection of low titers of CPV 2 in infected dogs.
本研究描述了基于 SYBR Green 的实时聚合酶链反应（real-time PCR）检测和定量犬细小病毒 2 型（CPV 2）在犬粪便样本中的方法。在该检测方法中，引物是根据 CPV 2 的 VP2 基因序列设计并定制合成的。通过对标准质粒 DNA 进行 10 倍系列稀释和 Ct 值绘制标准曲线。发现标准曲线在 10(-7)稀释范围内呈线性。实时 PCR 结果表示粪便样本中 CPV 2 的 DNA 拷贝数，其范围为 1.0 x 10(3)至 7.0 x 10(9)拷贝/毫克粪便样本。结果表明，基于 SYBR Green 的实时 PCR 的分析灵敏度相当于 10 拷贝。对疑似 CPV 2 感染的犬的粪便样本（47 个）进行实时 PCR、血凝（HA）检测和常规 PCR 分析，结果分别有 24、20 和 22 个样本为 CPV 2 阳性。三种不同检测方法的结果比较表明，实时 PCR 比 HA 和常规 PCR 更敏感，并能检测到感染犬中低滴度的 CPV 2。
