Development of a fluorogenic RT-PCR system for quantitative identification of dengue virus serotypes 1-4 using conserved and serotype-specific 3′ noncoding sequences
Development of a fluorogenic RT-PCR system for quantitative identification of dengue virus serotypes 1-4 using conserved and serotype-specific 3′ noncoding sequences
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DOI：
10.1016/s0166-0934(01)00280-4
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发表时间：
2001-06-01
影响因子：
3.1
通讯作者：
Kanesa-thasan, N
中科院分区：
文献类型：
--
作者：
Houng, HSH;Chen, RCM;Kanesa-thasan, N
建立了一种快速诊断登革热病毒血症的荧光逆转录聚合酶链式反应(RT-PCR)系统。利用登革病毒3‘端非编码序列建立了4种不同的登革病毒血清型特异性RT-PCR方法，用于定量鉴定4种不同的登革病毒血清型。用含有两个登革热特异性反义引物(DV-L1和DV-L2)的通用RT引物可将4种登革热病毒的RNA转录成互补DNA(CDNA)。用4个血清型特异的正义引物进行5‘-3’外切酶分析，可在血清型水平上定量鉴定登革热病毒的DNA。针对登革热类型的荧光RT-PCR可以以类似的低检测下限检测到四种登革热类型中的每一种，即每毫升血清中有20-50个空斑形成单位。用4个登革热参考血清型和134份临床标本组成的两个小组，以细胞培养中的病毒分离为标准，验证了登革热血清型RT-PCR方法的敏感性和特异性。通过对1999年至2000年收集的波多黎各血清样本的分析，该方法显示出高水平的检测灵敏度和特异度，分别为92.8%和92.4%。分别针对所有四种登革热病毒血清型。(C)2001 Elsevier Science B.V.保留所有权利。
A fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) system was developed for use as a rapid diagnostic lest for determining dengue viremia. The dengue virus 3 ' -noncoding sequence was utilized to formulate serotype-specific RT-PCR assays for quantitative identification of the four different dengue virus serotypes. A generic RT primer set containing two dengue specific anti-sense primers (DV-LI and DV-L2) could be used to transcribe extracted viral RNA of all four dengue virus types to complimentary DNA (cDNA). The resultant dengue viral cDNA could be quantitatively identified at the serotype level by the 5 ' -3 ' exonuclease assay using four serotype-specific sense primers. The fluorogenic dengue type-specific RT-PCR can detect each of the four dengue types at similar low detection limits, i.e. 20-50 plaque forming units per milliliter of serum. Two panels with four dengue reference serotypes and 134 clinical samples were used to validate detection sensitivity and specificity of the dengue serotype RT-PCR assay, using virus isolation in cell culture as the criterion standard. By analyzing sera samples from Puerto Rico that were collected from 1999 through 2000, the assay demonstrated high level detection sensitivity and specificity of 92.8 and 92.4%. respectively, for all four dengue virus serotypes. (C) 2001 Elsevier Science B.V. All rights reserved.