Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses.
BACKGROUND
Dengue is emerging as a major public health concern in many parts of the world. The development of a one-step, single tube, rapid, and multiplex reverse transcription polymerase chain reaction (M-RT-PCR) for simultaneous detection and typing of dengue virus using serotype specific primers during acute phase of illness is reported.


RESULTS
An optimal assay condition with zero background was established having no cross-reaction with closely related members of flavivirus (Japanese encephalitis, West Nile, Yellow fever) and alphavirus (Chikungunya). The feasibility of M-RT-PCR assay for clinical diagnosis was validated with 620 acute phase dengue patient sera samples of recent epidemics in India. The comparative evaluation vis a vis conventional virus isolation revealed higher sensitivity. None of the forty healthy serum samples screened in the present study revealed any amplification, thereby establishing specificity of the reported assay for dengue virus only.


CONCLUSIONS
These findings clearly suggested that M-RT-PCR assay reported in the present study is the rapid and cost-effective method for simultaneous detection as well as typing of the dengue virus in acute phase patient serum samples. Thus, the M-RT-PCR assay developed in this study will serve as a very useful tool for rapid diagnosis and typing of dengue infections in endemic areas.
Saxena
Parag
P
Division of Virology, Defence Research & Development Establishment, Jhansi Road, Gwalior 474 002, MP, India. paragsaxena_viro@rediffmail.com
Dash
Paban Kumar
PK
Santhosh
S R
SR
Shrivastava
Ambuj
A
Parida
Manmohan
M
Rao
Pv Lakshmana
PL
D000818:Animals; D002460:Cell Line; D017931:DNA Primers; D003715:Dengue / Q000175:diagnosis*; D003716:Dengue Virus / Q000145:classification / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping; D019562:Viral Load
D017931:DNA Primers; D012367:RNA, Viral