Universal primers that amplify RNA from all three flavivirus subgroups.
Universal primers that amplify RNA from all three flavivirus subgroups.
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DOI：
10.1186/1743-422x-5-16
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发表时间：
2008-01-24
期刊：
影响因子：
4.8
通讯作者：
Gibbs MJ
中科院分区：
文献类型：
--
作者：
Maher-Sturgess SL;Forrester NL;Wayper PJ;Gould EA;Hall RA;Barnard RT;Gibbs MJ
黄病毒属内的物种在世界各地造成公共卫生问题。亚洲登革热和日本脑炎病毒病例的增加，非洲和南美洲黄热病病毒的频繁爆发，以及西尼罗河病毒在整个美洲的持续传播，表明黄病毒病的地理负担。黄病毒感染通常与其他发热性疾病混淆。在这里，我们回顾了已发表的引物的特异性，并描述了一种新的通用引物对，可以检测范围广泛的黄病毒，包括从每个公认的亚组的病毒。对257个已发表的全长黄病毒基因组的生物信息学分析揭示了先前未被引物靶向的保守区域。从这些区域设计了两个简并引物Flav 100 F和Flav 200 R，并用于产生800个碱基对的cDNA产物。扩增的区域编码部分甲基转移酶和大部分RNA依赖性RNA聚合酶（NS 5）编码序列。一步RT-PCR测试是成功的，使用标准条件与RNA超过60种不同的黄病毒株代表约50个物种。对来自每个病毒分离株的cDNA进行测序，然后用于系统发育分析和数据库搜索以确认模板RNA的身份。全面的测试揭示了这些引物的广泛特异性。我们简要地讨论了这些通用引物的优点和用途。
Species within the Flavivirus genus pose public health problems around the world. Increasing cases of Dengue and Japanese encephalitis virus in Asia, frequent outbreaks of Yellow fever virus in Africa and South America, and the ongoing spread of West Nile virus throughout the Americas, show the geographical burden of flavivirus diseases. Flavivirus infections are often indistinct from and confused with other febrile illnesses. Here we review the specificity of published primers, and describe a new universal primer pair that can detect a wide range of flaviviruses, including viruses from each of the recognised subgroups. Bioinformatic analysis of 257 published full-length Flavivirus genomes revealed conserved regions not previously targeted by primers. Two degenerate primers, Flav100F and Flav200R were designed from these regions and used to generate an 800 base pair cDNA product. The region amplified encoded part of the methyltransferase and most of the RNA-dependent-RNA-polymerase (NS5) coding sequence. One-step RT-PCR testing was successful using standard conditions with RNA from over 60 different flavivirus strains representing about 50 species. The cDNA from each virus isolate was sequenced then used in phylogenetic analyses and database searches to confirm the identity of the template RNA. Comprehensive testing has revealed the broad specificity of these primers. We briefly discuss the advantages and uses of these universal primers.