São Paulo State University, Laboratório de Virologia e Patologia Animal, Curso de Medicina Veterinária, Campus de Araçatuba, São Paulo, Brazil.
J Virol Methods. 2010 Feb;163(2):509-12. doi: 10.1016/j.jviromet.2009.11.013. Epub 2009 Nov 14.
An in situ polymerase chain reaction (IS-PCR) hybridisation assay was carried out on the brains of 20 cattle infected naturally with bovine herpesvirus type 5 (BoHV-5). Sections from the olfactory bulb and the frontal cortex of each sample were analysed using IS-PCR followed by hybridisation targeting the BoHV-5 US9 gene using a biotinylated primer. Each of the IS-PCR and hybridisation steps was optimised, and three different methods for detecting the virus were used. No false positive signals were observed in any negative control sample (n=20), resulting in a specificity of 100%. The results of IS-PCR hybridisation analysis of the olfactory bulb and the frontal cortex be compared directly with the results obtained using virus isolation, and the specificity and sensitivity were calculated. The most suitable method of visualisation was the peroxidase/3'-3-diaminobenzidine (DAB) detection system coupled with the use of the fluorescent dye Cy3. Using either of these methods, 80% of the positive samples (16 out of 20 samples) were identified using olfactory bulb sections. This is the first report using IS-PCR hybridisation for the direct detection of BoHV-5 DNA in clinical samples, and it provides an additional method for veterinary virology.
对 20 头自然感染牛疱疹病毒 5 型（BoHV-5）的牛的脑组织进行了原位聚合酶链反应（IS-PCR）杂交检测。使用 IS-PCR 对每个样本的嗅球和额皮质进行分析，然后使用生物素标记的引物针对 BoHV-5 US9 基因进行杂交。优化了 IS-PCR 和杂交的每一步，并使用三种不同的方法检测病毒。在任何阴性对照样本（n=20）中均未观察到假阳性信号，特异性为 100%。直接比较嗅球和额皮质的 IS-PCR 杂交分析结果与使用病毒分离获得的结果，并计算特异性和敏感性。最适合的可视化方法是过氧化物酶/3'-3-二氨基联苯胺（DAB）检测系统与荧光染料 Cy3 的联合使用。使用这两种方法中的任何一种，都可以使用嗅球切片鉴定 80%的阳性样本（20 个样本中的 16 个）。这是首次使用 IS-PCR 杂交直接检测临床样本中的 BoHV-5 DNA，为兽医病毒学提供了一种额外的方法。
