Rapid detection, serotyping and quantitation of dengue viruses by TaqMan real-time one-step RT-PCR.
The use of the polymerase chain reaction (PCR) in molecular diagnosis is now accepted worldwide and has become an essential tool in the research laboratory. In the laboratory, a rapid detection, serotyping and quantitation, one-step real-time RT-PCR assay was developed for dengue virus using TaqMan probes. In this assay, a set of forward and reverse primers were designed targeting the serotype conserved region at the NS5 gene, at the same time flanking a variable region for all four serotypes which were used to design the serotype-specific TaqMan probes. This multiplex one-step RT-PCR assay was evaluated using 376 samples collected during the year 2003. These groups included RNA from prototype dengue virus (1-4), RNA from acute serum from which dengue virus was isolated, RNA from tissue culture supernatants of dengue virus isolated, RNA from seronegative acute samples (which were culture and IgM negative) and RNA from samples of dengue IgM positive sera. The specificity of this assay was also evaluated using a panel of sera which were positive for other common tropical disease agents including herpes simplex virus, cytomegalovirus, measles virus, varicella-zoster virus, rubella virus, mumps virus, WWF, West Nile virus, Japanese encephalitis virus, S. typhi, Legionella, Leptospira, Chlamydia, and Mycoplasma. The sensitivity, specificity and real-time PCR efficiency of this assay were 89.54%, 100% and 91.5%, respectively.
Kong
Yong Yean
YY
Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Thay
Chong Heng
CH
Tin
Tan Chong
TC
Devi
Shamala
S
D001483:Base Sequence; D003715:Dengue / Q000175:diagnosis / Q000821:virology*; D003716:Dengue Virus / Q000145:classification* / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping / Q000379:methods; D017361:Viral Nonstructural Proteins / Q000235:genetics
C096041:NS5 protein, flavivirus; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins