RAPID DETECTION AND TYPING OF DENGUE VIRUSES FROM CLINICAL-SAMPLES BY USING REVERSE TRANSCRIPTASE-POLYMERASE CHAIN-REACTION
RAPID DETECTION AND TYPING OF DENGUE VIRUSES FROM CLINICAL-SAMPLES BY USING REVERSE TRANSCRIPTASE-POLYMERASE CHAIN-REACTION
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DOI：
10.1128/jcm.30.3.545-551.1992
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发表时间：
1992-03-01
影响因子：
9.4
通讯作者：
VORNDAM, AV
中科院分区：
文献类型：
--
作者：
LANCIOTTI, RS;CALISHER, CH;VORNDAM, AV
我们报告了用于检测和分型登革热病毒的快速检测方法的开发和应用。 寡核苷酸共有引物设计用于与四种登革热病毒类型中的任何一种退火，并在逆转录酶聚合酶链式反应 (PCR) 中扩增 511 bp 产物。 首先，我们在引物 D2 存在的情况下在逆转录酶反应中产生了部分病毒基因组的 cDNA 副本，然后在添加引物 D1 的情况下进行标准 PCR（35 个循环的热变性、退火和引物延伸）。 RT-PCR 产生的双链 DNA 产物通过两种方法进行分型：511 bp 扩增产物与登革热病毒类型特异性探针的点印迹杂交，或使用类型特异性引物进行第二轮 PCR 扩增（巢式 PCR），产生独特大小的 DNA 产物，可诊断每种登革热病毒血清型。 积累的数据表明，可以从病毒血症人血清样本中准确检测登革热病毒并进行分型。
We report on the development and application of a rapid assay for detecting and typing dengue viruses. Oligonucleotide consensus primers were designed to anneal to any of the four dengue virus types and amplify a 511-bp product in a reverse transcriptase-polymerase chain reaction (PCR). First, we produced a cDNA copy of a portion of the viral genome in a reverse transcriptase reaction in the presence of primer D2 and then carried out a standard PCR (35 cycles of heat denaturation, annealing, and primer extension) with the addition of primer D1. The resulting double-stranded DNA product of the RT-PCR was typed by two methods: dot blot hybridization of the 511-bp amplified product to dengue virus type-specific probes or a second round of PCR amplification (nested PCR) with type-specific primers, yielding DNA products the unique sizes of which were diagnostic for each dengue virus serotype. The accumulated data demonstrated that dengue viruses can be accurately detected and typed from viremic human serum samples.