Liu Jing-bo, Zhang Zheng
Department of Clinical Laboratory, Peking University People's Hospital, 11 Xizhimen South Road, Xicheng District, Beijing 100044, China.
J Virol Methods. 2008 Apr;149(1):129-35. doi: 10.1016/j.jviromet.2007.12.011. Epub 2008 Feb 15.
Ganciclovir (GCV) is an antiviral drug that is used to treat cytomegalovirus (CMV) infection. However, long-term monotherapy does not commonly result in complete suppression of viral replication and is associated with the emergence of resistant mutants. In this study, a method for detecting CMV resistance mutations was carried out by real-time amplification refractory mutation system PCR (real-time ARMS PCR) using SYBR Green I fluorescent dye. Three recombinant plasmids were constructed by overlapping extension PCR to be used as standard mutation or wild-type models. Four pairs of primers were used to amplify the approximately 150 bp of the UL97 gene spanning codon 460, where mutations associated with resistance to GCV invariably occur. As little as 20% mutants DNA in 10(7)copies/ml mixture DNA were detected. Though this approach was not more sensitive than PCR-restriction fragment length polymorphism (RFLP) for the detection of the presence of mixtures, it was a high-throughput and automation method, and the specific mutation type can be deduced by the real-time ARMS PCR data. Overall, this study has demonstrated an approach that could be a sensitive and rapid method for the detection of GCV resistance-associated mutation in CMV.
更昔洛韦（GCV）是一种用于治疗巨细胞病毒（CMV）感染的抗病毒药物。然而，长期单一疗法通常不会完全抑制病毒复制，且与耐药突变体的出现有关。在本研究中，采用实时扩增阻滞突变系统PCR（实时ARMS PCR）并使用SYBR Green I荧光染料，开展了一种检测CMV耐药突变的方法。通过重叠延伸PCR构建了三个重组质粒，用作标准突变或野生型模型。使用四对引物扩增跨越密码子460的约150 bp的UL97基因，与GCV耐药相关的突变总是发生在此处。在10⁷拷贝/ml的混合DNA中，低至20%的突变体DNA也能被检测到。虽然这种方法在检测混合物存在时不如PCR-限制性片段长度多态性（RFLP）敏感，但它是一种高通量且自动化的方法，并且可以通过实时ARMS PCR数据推断出特定的突变类型。总体而言，本研究展示了一种可能成为检测CMV中GCV耐药相关突变的灵敏且快速的方法。
