title: Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma.
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.
authors.affiliation: Oxford University Clinical Research Unit, Hospital for Tropical Diseases, 190 Ben Ham Tu, Quan 5, Ho Chi Minh City, Viet Nam. kiendth@oucru.org
authors.forename: Kien Duong Thi
authors.initials: KD
authors.lastname: Hue
authors.forename: Trung Vu
authors.initials: TV
authors.lastname: Tuan
authors.forename: Hanh Tien Nguyen
authors.initials: HT
authors.lastname: Thi
authors.forename: Chau Tran Nguyen
authors.initials: CT
authors.lastname: Bich
authors.forename: Huy Huynh Le
authors.initials: HH
authors.lastname: Anh
authors.forename: Bridget A
authors.initials: BA
authors.lastname: Wills
authors.forename: Cameron P
authors.initials: CP
authors.lastname: Simmons
mesh_terms: D003715:Dengue / Q000175:diagnosis* / Q000821:virology; D003716:Dengue Virus / Q000235:genetics*; D007240:Equartevirus / Q000235:genetics; D006801:Humans; D057230:Limit of Detection; D008828:Microbiological Techniques; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000097:blood*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017361:Viral Nonstructural Proteins / Q000235:genetics
chemical_list: D012367:RNA, Viral; D017361:Viral Nonstructural Proteins