Department of Genetics, Stellenbosch University, Stellenbosch, South Africa.
Virol J. 2012 Sep 27;9:219. doi: 10.1186/1743-422X-9-219.
Grapevine leafroll-associated virus 3 (GLRaV-3) is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3.
In this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM) curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay.
A universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h) gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM.
The real-time RT-PCR HRM provides a sensitive, automated and rapid tool to detect and differentiate different variant groups in order to study the epidemiology of leafroll disease.
葡萄卷叶伴随病毒 3（GLRaV-3）是全球范围内引起卷叶病的主要致病因子。在南非发现了已知的六个 GLRaV-3 变体组中的四个，但它们对卷叶病的单独贡献尚不清楚。为了研究卷叶病的发病机制，需要一种敏感且准确的诊断检测方法，能够检测 GLRaV-3 的不同变体组。
在本研究中，开发了一种一步实时 RT-PCR，随后进行高分辨率熔解（HRM）曲线分析，用于同时检测和鉴定 GLRaV-3 变体组 I、II、III 和 VI。为每个变体组计算了熔点置信区间，以包含至少 90%观察到的所有熔点。为这四个变体组开发了多重 RT-PCR 方案，以评估实时 RT-PCR HRM 检测方法的功效。
设计了针对 GLRaV-3 热休克蛋白 70 同源物（Hsp70h）基因的通用引物组，能够检测 GLRaV-3 变体组 I、II、III 和 VI，并通过高分辨率熔解曲线分析对它们进行区分。使用 121 个 GLRaV-3 阳性样本对实时 RT-PCR HRM 和多重 RT-PCR 进行了优化。由于每个 GLRaV-3 组中观察到的熔解曲线存在相当大的变化，因此基于熔解点的范围和分布，为每个变体组计算了置信区间超过 90%。组 I 和 II 的区间无法区分，因此计算了组 I 和 II 变体同时检测的 95%联合置信区间。针对 GLRaV-3 ORF1a 开发了另外一对引物，可用于后续的实时 RT-PCR HRM 以区分组 I 和 II 的变体。此外，多重 RT-PCR 成功验证了实时 RT-PCR HRM 检测到的感染的 94.64%。
实时 RT-PCR HRM 提供了一种敏感、自动化和快速的工具，用于检测和区分不同的变体组，以研究卷叶病的流行病学。
