使用五条NS3基因引物对登革病毒进行快速单步逆转录聚合酶链反应分型

Rapid, single-step RT-PCR typing of dengue viruses using five NS3 gene primers.

作者信息

Seah C L, Chow V T, Tan H C, Can Y C

机构信息

Department of Microbiology, Faculty of Medicine, National University of Singapore.

出版信息

J Virol Methods. 1995 Feb;51(2-3):193-200. doi: 10.1016/0166-0934(94)00104-o.

DOI:10.1016/0166-0934(94)00104-o

PMID:7738139

Abstract

In order to detect and type dengue viruses in serum specimens, four type-specific downstream primers were designed for use with a consensus upstream primer in a reverse transcription and polymerase chain reaction (RT-PCR) assay. RT-PCR using these five primers amplified NS3 gene fragments of diagnostic sizes of 169, 362, 265 and 426 base pairs for dengue virus types 1, 2, 3 and 4, respectively, but not for Japanese encephalitis, Kunjin and yellow fever viruses. The conventional two-step RT-PCR procedure was simplified by combining RT and PCR in a single-step format with a "hot start". This RT-PCR protocol was applied successfully to dengue virus-spiked serum and dengue patient serum samples, and could detect as few as one PFU of dengue virus. This assay offers a rapid, specific and sensitive molecular technique for the simultaneous detection and typing of dengue viruses.

摘要

为了检测血清标本中的登革病毒并进行分型，设计了四种型特异性下游引物，与一条共有上游引物一起用于逆转录聚合酶链反应（RT-PCR）检测。使用这五种引物进行的RT-PCR分别扩增出了登革病毒1型、2型、3型和4型诊断大小为169、362、265和426个碱基对的NS3基因片段，但对日本脑炎病毒、库京病毒和黄热病病毒无扩增。通过将逆转录（RT）和聚合酶链反应（PCR）以单步“热启动”形式结合，简化了传统的两步RT-PCR程序。该RT-PCR方案成功应用于登革病毒加标血清和登革热患者血清样本，并且能够检测低至一个噬斑形成单位（PFU）的登革病毒。该检测方法为登革病毒的同时检测和分型提供了一种快速、特异且灵敏的分子技术。