Assessment of the quantitative real-time polymerase chain reaction using a cDNA standard for human group A rotavirus
Assessment of the quantitative real-time polymerase chain reaction using a cDNA standard for human group A rotavirus
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DOI：
10.1016/j.jviromet.2006.06.028
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发表时间：
2006-11-01
影响因子：
3.1
通讯作者：
Ahn, Byung-Yoon
中科院分区：
文献类型：
--
作者：
Min, Bok-Soon;Noh, Yoon-Ju;Ahn, Byung-Yoon
核酸扩增技术经常用于病毒性疾病的快速诊断。在这项研究中，使用病毒VP 4基因特异性引物和商业SYBR绿色试剂的实时聚合酶链反应方案进行了评价，用于定量测量人粪便标本中的人轮状病毒（HRV）RNA。SYBR绿色I检测包括分析PCR产物的解链温度和在最佳温度下测量荧光。该测定导致对范围从低（< 10（2）轮状病毒cDNA拷贝/反应）到高数量（> 10（6）轮状病毒cDNA拷贝/反应）的靶的灵敏且可再现的检测。与柯萨奇病毒、埃可病毒、脊髓灰质炎病毒、甲型肝炎病毒和腺病毒的粗细胞培养液无交叉反应。用HRV cDNA标准品进行的分析显示出高重复性，变异系数（CV）为0.2- 0.9%。三个不同实验室的日常性能显示CV不大于8%，表明变异处于中等水平。结果表明，该方法用于临床标本中轮状病毒的定量分析是可行的。(c)2006 Elsevier B. V.保留所有权利。
Nucleic acid amplification techniques are used frequently for rapid diagnosis of viral diseases. In this study, a real-time polymerase chain reaction protocol that uses primers specific for the viral VP4 gene and the commercial SYBR Green reagent were evaluated for the quantitative measurement of human rotavirus (HRV) RNA in human stool specimens. SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. The assay resulted in a sensitive and reproducible detection of targets ranging from low (< 10(2) rotavirus cDNA copies/reaction) to high numbers (> 10(6) rotavirus cDNA copies/reaction). No cross-reaction was found with crude cell culture stocks of coxsackievirus, echovirus, poliovirus, hepatitis A virus and adenovirus. Analysis with the HRV cDNA standard demonstrated high reproducibility with a coefficient of variation (CV) of 0.2-0.9%. Daily performance among three different laboratories showed a CV no greater than 8%, indicating an intermediate level of variation. These results demonstrate the feasibility of this method for quantitative analysis of human rotavirus in clinical samples. (c) 2006 Elsevier B.V. All rights reserved.