Seah C L, Chow V T, Chan Y C
Department of Microbiology, Faculty of Medicine, National University of Singapore, Kent Ridge, 0511, Singapore.
Clin Diagn Virol. 1995 Aug;4(2):113-20. doi: 10.1016/0928-0197(94)00063-z.
More rapid, specific and sensitive tests for the laboratory diagnosis of dengue virus infections are needed.
To develop a semi-nested polymerase chain reaction (PCR) assay based on primers within the NS3 gene for the simultaneous detection and typing of dengue viruses in human sera.
A first round of single-step reverse transcription-polymerase chain reaction (RT-PCR) was carried out with a pair of consensus primers, followed by a second round of semi-nested amplification using the upstream consensus primer and four type-specific down-stream primers. The sensitivity and specificity of the semi-nested PCR assay were determined using plaque- or TCID(50)-titrated virus-infected tissue culture fluid, and total RNA extracted from C6/36 cells infected with dengue and other flaviviruses, respectively. A retrospective study was performed on acute sera from thirteen patients with dengue (confirmed by virus isolation) employing semi-nested PCR in parallel with virus re-isolation and a single-step RT-PCR method for the typing of dengue viruses in human sera.
The semi-nested PCR assay could detect up to 1 pfu of dengue virus, but not other flaviviruses. The semi-nested PCR and single-step RT-PCR assays correctly typed dengue viruses in twelve and five sera, respectively, whereas none of the sera was positive by virus re-isolation.
This semi-nested PCR assay is a sensitive and specific tool for the detection and typing of dengue viruses from viremic human sera.
需要更快速、特异且灵敏的检测方法用于登革病毒感染的实验室诊断。
基于NS3基因内的引物开发一种半巢式聚合酶链反应（PCR）检测方法，用于同时检测和分型人血清中的登革病毒。
第一轮采用一对共有引物进行单步逆转录聚合酶链反应（RT-PCR），随后第二轮使用上游共有引物和四种型特异性下游引物进行半巢式扩增。使用蚀斑或半数组织培养感染剂量（TCID₅₀）滴定的病毒感染组织培养液，以及分别从感染登革病毒和其他黄病毒的C6/36细胞中提取的总RNA，测定半巢式PCR检测方法的敏感性和特异性。对13例登革热患者（经病毒分离确诊）的急性血清进行回顾性研究，同时采用半巢式PCR、病毒再分离以及单步RT-PCR方法对人血清中的登革病毒进行分型。
半巢式PCR检测方法可检测低至1个蚀斑形成单位（pfu）的登革病毒，但不能检测其他黄病毒。半巢式PCR和单步RT-PCR检测方法分别在12份和5份血清中正确分型登革病毒，而病毒再分离检测所有血清均为阴性。
这种半巢式PCR检测方法是一种灵敏且特异的工具，可用于从病毒血症患者血清中检测和分型登革病毒。
