SEMI-NESTED PCR USING NS3 PRIMERS FOR THE DETECTION AND TYPING OF DENGUE VIRUSES IN CLINICAL SERUM SPECIMENS

被引：36

作者：

SEAH, CLK ^{[1
]}

CHOW, VTK ^{[1
]}

CHAN, YC ^{[1
]}

机构：

[1] NATL UNIV SINGAPORE,FAC MED,DEPT MICROBIOL,SINGAPORE 0511,SINGAPORE

来源：

CLINICAL AND DIAGNOSTIC VIROLOGY | 1995年 / 4卷 / 02期

关键词：

DENGUE VIRUSES; TYPING; SEMI-NESTED PCR; NS3 GENE PRIMERS;

D O I：

10.1016/0928-0197(94)00063-Z

中图分类号：

Q93 [微生物学];

学科分类号：

071005 ; 100705 ;

摘要：

Background: More rapid, specific and sensitive tests for the laboratory diagnosis of dengue virus infections are needed. Objective: 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. Study design: 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 downstream primers. The sensitivity and specificity of the semi-nested PCR assay were determined using plaque- or TCID50-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. Results: 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. Conclusions: This semi-nested PCR assay is a sensitive and specific tool for the detection and typing of dengue viruses from viremic human sera.

引用

页码：113 / 120

页数：8

共 16 条

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