Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma

Kien Duong Thi Hue, Trung Vu Tuan, Hanh Tien Nguyen Thi, Chau Tran Nguyen Bich, Huy Huynh Le Anh, Bridget A. Wills, Cameron P. Simmons

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78 Citations (Scopus)

Abstract

Dengue is mosquito-borne virus infection that annually causes ~50 million clinically apparent cases worldwide. An internally controlled one-step real-time multiplex RT-PCR assay was developed for detection and quantitation of DENV RNA in plasma sample by using specific primers and fluorogenic TaqMan probes. All primers and probes targeted sequences near the 3' end of the NS5 gene. The method comprised two multiplex assays and was validated for sensitivity, specificity, linearity, reproducibility and precision. An internal control template was spiked into each clinical specimen to provide quality assurance for each experimental step. The assay allowed for detection of between 0.5 and 3 infectious particles per mL, is rapid and has been operationally characterized in 287 Vietnamese dengue patients from two therapeutic intervention trials at the Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam.

Original languageEnglish
Pages (from-to)168-173
Number of pages6
JournalJournal of Virological Methods
Volume177
Issue number2
DOIs
Publication statusPublished - Nov 2011
Externally publishedYes

Keywords

  • Dengue virus
  • One step real time multiplex RT-PCR
  • Validation

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