Elia Gabriella, Cavalli Alessandra, Desario Costantina, Lorusso Eleonora, Lucente Maria Stella, Decaro Nicola, Martella Vito, Buonavoglia Canio
Department of Animal Health and Well-being, Faculty of Veterinary Medicine of Bari, S.p. per Casamassima km 3, 70010 Valenzano, Bari, Italy.
J Virol Methods. 2007 Dec;146(1-2):202-8. doi: 10.1016/j.jviromet.2007.06.017. Epub 2007 Aug 10.
A TaqMan real-time RT-PCR assay was developed for detection of RNA transcripts produced by replicating CPV-2. A pair of primers and a TaqMan probe targeting the spliced NS2 mRNA were designed. A synthetic DNA fragment was constructed to mimic the spliced NS2 mRNA by PCR-based gene assembly and was used for generation of standard RNAs. The detection limit of the assay was 1x10(2) RNA copies and standard curve displayed a linear range from 1x10(2) to 1x10(9) copies and a good reproducibility. The assay was then applied to determine the mRNA loads in the tissues of dogs naturally infected by CPV-2. mRNA was detected in a variety of tissues, including the central nervous system.
开发了一种TaqMan实时逆转录聚合酶链反应（RT-PCR）检测方法，用于检测复制型犬细小病毒2型（CPV-2）产生的RNA转录本。设计了一对靶向剪接后NS2 mRNA的引物和一个TaqMan探针。通过基于聚合酶链反应（PCR）的基因组装构建了一个合成DNA片段，以模拟剪接后的NS2 mRNA，并用于生成标准RNA。该检测方法的检测限为1×10²个RNA拷贝，标准曲线显示线性范围为1×10²至1×10⁹个拷贝，且具有良好的重复性。然后将该检测方法应用于测定自然感染CPV-2的犬组织中的mRNA载量。在包括中枢神经系统在内的多种组织中检测到了mRNA。
