Identification of enteroviruses in clinical specimens by competitive PCR followed by genetic typing using sequence analysis
Identification of enteroviruses in clinical specimens by competitive PCR followed by genetic typing using sequence analysis
复制标题
DOI：
10.1128/jcm.34.2.313-318.1996
复制
发表时间：
1996-02-01
影响因子：
9.4
通讯作者：
Hyypia, T
中科院分区：
文献类型：
--
作者：
Arola, A;Santti, J;Hyypia, T
建立了一种基于竞争性PCR的肠道病毒RNA检测和定量方法，特别强调了对污染和潜在抑制因子的控制。选择来自5'非翻译区和VP2衣壳蛋白编码区的保守部分的寡核苷酸引物，以根据cDNA扩增子的长度区分肠道病毒和鼻病毒。从埃可病毒11基因组的截短cDNA拷贝转录的RNA用作逆转录反应加PCR的内部对照。这使得通过琼脂糖凝胶电泳和临床标本中病毒RNA的非放射性定量可以简单地区分对照和病毒PCR产物。通过直接对聚合酶链反应产物进行测序，然后对数据进行计算机分析，可以监测潜在的实验室污染，并且可以将临床样品中的肠道病毒分为四个不同的簇，从而可以对病毒进行基因分型。所述方法可应用于肠道病毒感染的诊断和流行病学。
A sensitive method based on competitive PCR was developed to detect and quantitate enteroviral RNA in clinical specimens, with special emphasis on controlling contamination and the presence of potential inhibitory factors in the specimens. Oligonucleotide primers from the conserved parts of the 5' untranslated and VP2 capsid protein-coding regions were selected to differentiate between enteroviruses and rhinoviruses on the basis of the length of the cDNA amplicons. RNA transcribed from a truncated cDNA copy of the echovirus 11 genome was used as an internal control for the reverse transcription reaction add PCR. This allowed simple differentiation of the control and viral PCR products from each other by agarose gel electrophoresis and nonradioactive quantitation bf the viral RNA in the clinical specimens. By direct sequencing of the PCR products and subsequent computer analysis of the data, potential laboratory contaminations could be monitored and enteroviruses from clinical samples could be grouped into four distinct clusters, thus enabling genetic typing of the viruses. The described method can be applied to the diagnosis and epidemiology of enteroviral infections.