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

• Published in: Journal of virological methods 2011-11, Vol.177 (2), p.168-173
• Main Authors: Hue, Kien Duong Thi, Tuan, Trung Vu, Thi, Hanh Tien Nguyen, Bich, Chau Tran Nguyen, Anh, Huy Huynh Le, Wills, Bridget A., Simmons, Cameron P.
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; dengue; Dengue - diagnosis; Dengue - virology; Dengue virus; Dengue Virus - genetics; Equartevirus - genetics; Fundamental and applied biological sciences. Psychology; genes; hospitals; Humans; Limit of Detection; Microbiological Techniques; Microbiology; Multiplex Polymerase Chain Reaction - methods; One step real time multiplex RT-PCR; patients; Plasmids - genetics; Plasmids - metabolism; quality control; Reproducibility of Results; reverse transcriptase polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA; RNA, Viral - blood; Sensitivity and Specificity; Techniques used in virology; Validation; Viral Nonstructural Proteins - genetics; Virology; viruses
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2011.08.002

► Validation of internally controlled one step real time multiplex DENV RT-PCR assay. ► The sensitivity, specificity, linearity, reproducibility and precision are tested. ► The assay can detect from 0.5 to 1 infectious particle per mL. ► The assay is rapid and has been operationally characterized in dengue patients. 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.