College of Agronomy and Biotechnology, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing 100193, People's Republic of China.
Virol J. 2013 Aug 10;10:255. doi: 10.1186/1743-422X-10-255.
Co-infections of Apple chlorotic leaf spot virus (ACLSV) and Cherry green ring mottle virus (CGRMV) in peach is common in China and have resulted in significant yield reductions. A reliable, sensitive and quantitive method is needed to detect and distinguish between ACLSV and CGRMV in peach.
We developed a sensitive and specific SYBR Green-I based RT-qPCR for the quantification of ACLSV and CGRMV in different peach tissues, and a duplex RT-qPCR system to detect ACLSV and CGRMV simultaneously. The RT-qPCR method was optimized using standard samples transcribed by the T7 Large Scale RNA Production System in vitro. The peach genes, RNA Polymerase subunit II (RPII) and Ubiquitin 10 (UBQ10), which were used as the internal controls for the quantification assay also showed good expression stability in this system. Single RT-qPCR assays showed that CGRMV in peach accumulates to a higher level than ACLSV. The detection limits of the duplex RT-qPCR assay were 10² and 10⁴ copies for ACLSV and CGRMV, respectively. The sensitivity of the duplex RT-qPCR was as high as RT-qPCR and higher than RT-PCR.
The SYBR Green-I RT-qPCR assay provided a sensitive, specific and reliable method for the detection and quantification of ACLSV and CGRMV in different peach tissues. The duplex RT-qPCR system provided a sensitive and specific method to detect and differentiate between ACLSV and CGRMV in a single sample. This RT-qPCR assay could be a useful tool for the routine diagnosis of these two viruses and for disease epidemiology studies in peach orchards.
在中国，桃果实感染苹果褪绿叶斑病毒（ACLSV）和樱桃绿环斑驳病毒（CGRMV）的现象较为常见，这导致了显著的产量下降。因此，需要一种可靠、敏感和定量的方法来检测和区分桃果实中的 ACLSV 和 CGRMV。
我们开发了一种基于 SYBR Green-I 的敏感和特异的 RT-qPCR 方法，用于检测和定量不同桃组织中的 ACLSV 和 CGRMV，以及一种用于同时检测 ACLSV 和 CGRMV 的双重 RT-qPCR 系统。该 RT-qPCR 方法使用 T7 大规模 RNA 生产系统体外转录的标准样品进行优化。桃基因 RNA 聚合酶亚基 II（RPII）和泛素 10（UBQ10）被用作定量测定的内参基因，在该系统中也表现出良好的表达稳定性。单重 RT-qPCR 检测结果表明，桃果实中 CGRMV 的积累水平高于 ACLSV。双重 RT-qPCR 检测的灵敏度分别为 ACLSV 和 CGRMV 的 10²和 10⁴拷贝。该双重 RT-qPCR 系统的灵敏度与 RT-qPCR 相当，高于 RT-PCR。
SYBR Green-I RT-qPCR 检测方法为检测和定量不同桃组织中的 ACLSV 和 CGRMV 提供了一种敏感、特异和可靠的方法。双重 RT-qPCR 系统为在单个样本中同时检测和区分 ACLSV 和 CGRMV 提供了一种敏感和特异的方法。该 RT-qPCR 检测方法可用于这两种病毒的常规诊断和桃果园疾病流行学研究。
