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 Huea b✉, Trung Vu Tuanb, Hanh Tien Nguyen Thib, Chau Tran Nguyen Bichb, Huy Huynh Le Anhb, Bridget A. Willsb c, Cameron Paul Simmonsb d
- aOxford University Clinical Research Unit, Hospital for Tropical Diseases, 190 Ben Ham Tu, Quan 5, Ho Chi Minh City, Viet Nam. kiendth@oucru.org
- bOxford University Clinical Research Unit, Hospital for Tropical Diseases, 190 Ben Ham Tu, Quan 5, Ho Chi Minh City, Viet Nam
- cCentre for Tropical Medicine, CCVTM, University of Oxford, Churchill Hospital, Oxford, UK
- dCenter for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Center for Clinical Vaccinology and Tropical Medicine, Oxford, UK
- ✉Corresponding author
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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.
TL;DRAn 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 and was validated for sensitivity, specificity, linearity, reproducibility and precision. (Semantic Scholar)