多重 PCR 结合限制性长度多态性分析对牛疱疹病毒 5 分离株的亚型分型。
Multiplex PCR followed by restriction length polymorphism analysis for the subtyping of bovine herpesvirus 5 isolates.
机构信息
Instituto de Virología, Centro de Investigaciones en Ciencias Veterinarias y, Agronómicas (CICVyA), Instituto de tecnología Agropecuaria (INTA), N. Repetto, y Los Reseros S/N, CC25, (B1712WAA), Castelar, Buenos Aires, Argentina.
出版信息
BMC Vet Res. 2013 Jun 4;9:111. doi: 10.1186/1746-6148-9-111.
BACKGROUND
Several types and subtypes of bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) have been associated to different clinical conditions of cattle, making type/subtype differentiation essential to understand the pathogenesis and epidemiology of BoHV infections. BoHV-5 subtyping is currently carried out by BstEII restriction enzyme analysis (REA) of the complete virus genome. This method allowed the description of three subtypes, one of which is the most widespread while the remaining two have so far only been found in South America. The present work describes a multiplex PCR followed by REA for BoHV-5 subtyping.
RESULTS
The method consists in the simultaneous amplification of glycoprotein B and UL54 gene fragments of 534 and 669 base pairs (bp), respectively, BstEII digestion of amplicons, separation of products in 1% agarose gels, and analysis of fragment length polymorphims. The multiplex PCR detected up to 227 BoHV-5 genome copies and 9.2 × 105 BoHV-5 genome copies when DNA was extracted from purified virus or infected tissue homogenates, respectively. The applicability of multiplex PCR-REA was demonstrated on 3 BoHV-5 reference strains. In addition, subtyping of two new isolates and seventeen previously reported ones (17 BHV-5a and 2 BHV-5b) by this method gave coincident results with those obtained with the classic BstEII REA assay.
CONCLUSIONS
Multiplex PCR-REA provides a new tool for the fast and simple diagnosis and subtyping of BoHV-5.
背景
几种类型和亚型的牛疱疹病毒 1 和 5（BoHV-1 和 BoHV-5）与牛的不同临床情况有关，因此对其进行分型/亚型区分对于了解 BoHV 感染的发病机制和流行病学至关重要。BoHV-5 的亚分型目前是通过 BstEII 限制性内切酶分析（REA）对完整病毒基因组进行的。这种方法描述了三种亚型，其中一种最为广泛，而其余两种迄今为止仅在南美洲发现过。本研究描述了一种用于 BoHV-5 亚分型的多重 PCR 后 REA 方法。
结果
该方法包括同时扩增糖蛋白 B 和 UL54 基因片段，分别为 534 和 669 个碱基对（bp），BstEII 消化扩增产物，在 1%琼脂糖凝胶中分离产物，并分析片段长度多态性。该多重 PCR 可检测到多达 227 个 BoHV-5 基因组拷贝，当从纯化病毒或感染组织匀浆中提取 DNA 时，可检测到 9.2×105 个 BoHV-5 基因组拷贝。该多重 PCR-REA 方法在 3 种 BoHV-5 参考株上的适用性得到了验证。此外，通过该方法对 2 种新分离株和之前报道的 17 种（17 种 BoHV-5a 和 2 种 BoHV-5b）进行了亚分型，其结果与经典的 BstEII REA 检测方法一致。
结论
多重 PCR-REA 为 BoHV-5 的快速、简便诊断和亚分型提供了一种新工具。
Zotero 插件