End-point limiting-dilution real-time PCR assay for evaluation of hepatitis C virus quasispecies in serum: Performance under optimal and suboptimal conditions
End-point limiting-dilution real-time PCR assay for evaluation of hepatitis C virus quasispecies in serum: Performance under optimal and suboptimal conditions
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DOI：
10.1016/j.jviromet.2008.05.005
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发表时间：
2008-08-01
影响因子：
3.1
通讯作者：
Khudyakov, Yury
中科院分区：
文献类型：
--
作者：
Ramachandran, Sumathi;Xia, Guo-liang;Khudyakov, Yury
建立了终点限制稀释实时聚合酶链式反应(EPLD-PCR)检测丙型肝炎病毒准种的方法。它包括使用限制稀释法从血清样本中分离出丙型肝炎病毒基因组高变区1(Hvr1)的单个共存序列变体。将EPLD-PCR应用于丙型肝炎暴发研究，提供了对事件病例和慢性病例之间的流行病学关系的洞察。当应用于来自感染患者的纵向研究的样本时，来自每个采样时间点的hvr1序列被观察为不同的系统发育簇。对这些患者产生的EPLD-PCR产物进行熔融峰分析，可以在不进行克隆测序的情况下用于评估HVR1序列的异质性。此外，为了更好地理解单分子PCR的机制，我们在最佳和次佳的退火温度下进行了实验。在所有测试的温度条件下，来自主要准种系统发育簇的HVR1变体都可以被扩增，这表明成功的HVR1准种分析并不取决于开始制备的cDNA被稀释到单分子状态。结果发现，在次优温度下进行的EPLD-PCR得到的唯一序列变异的分布与在最适温度下进行的PCR所得到的分布略有不同。因此，可以操纵EPLD-PCR条件来获得不同亚群的丙型肝炎病毒HVR1准种，从而提高准种的检测范围。尽管在不同条件下进行的EPLD-PCR检测到的准种种群略有不同，但本研究所得到的准种样本完全适用于不同临床和流行病学环境下的分子流行病学调查。爱思唯尔出版公司(Elsevier B.V.)
An approach for determination of hepatitis C virus (HCV) quasispecies by end-point limiting-dilution real-time PCR (EPLD-PCR) is described. It involves isolation of individual coexisting sequence variants of the hypervariable region 1 (HVR1) of the HCV genome from serum specimens using a limiting-dilution protocol. EPLD-PCR applied to an HCV outbreak study provided insights into the epidemiological relationships between incident and chronic cases. When applied to samples from a longitudinal study of infected patients, HVR1 sequences from each sampling time-point were observed to group as distinct phylogenetic clusters. Melting peak analysis conducted on EPLD-PCR products generated from these patients could be used for evaluation of HVR1 sequence heterogeneity without recourse to clonal sequencing, Further, to better understand the mechanism of single-molecule PCR, experiments were conducted under optimal and suboptimal annealing temperatures. Under all temperature conditions tested, HVR1 variants from the major phylogenetic clusters Of quasispecies could be amplified, revealing that Successful HVR1 quasispecies analysis is not contingent to dilution of starting cDNA preparations to a single-molecule state. It Was found that EPLD-PCR conducted at Suboptimal annealing temperatures generated distributions Of unique-sequence variants slightly different from the distribution obtained by PCR conducted at the optimal temperature. Hence, EPLD-PCR conditions can be manipulated to access different subpopulations of HCV HVR1 quasispecies, thus, improving the range of the quasispecies detection. Although EPLD-PCR conducted at different conditions detect slightly different quasispecies populations, as was shown it) this study, the resulted samples of quasispecies are completely suitable for molecular epidemiological investigation in different clinical and epidemiological settings. Published by Elsevier B.V.