Ultra-rapid, simple, sensitive, and economical silica method for extraction of dengue viral RNA from clinical specimens and mosquitoes by reverse transcriptase-polymerase chain reaction.
A rapid, simple and efficient single-tube procedure is described for the isolation of dengue virus RNA from small amount of serum (10 microliters) followed by a reverse transcriptase-polymerase chain reaction (RT-PCR). Recovery of RNA is based on the lysing and nuclease-inactivating properties of guanidinium thiocyanate in the presence of silica. The silica RT-PCR can be completed within 5 hours starting from RNA extraction to agarose gel electrophoresis. All of the 63 dengue-3 culture-positive sera were RT-PCR-positive (virus titres: < 10(2) to 11(10.69.). Of 33 culture-negative acute sera from serologically confirmed dengue fever patients collected during dengue-3 epidemic, 4 were RT-PCR-positive. RT-PCR was also positive in 29 of 30 dengue-1 culture-positive sera (virus titres range: < 10(2) to 10(8.69). Dengue-1 virus was also detected in field-caught Aedes aegypti mosquitoes by silica RT-PCR.
Chungue
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E
Institut Territorial de Recherches Médicales Louis Malardé, Papeete, Tahiti.
Roche
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C
Lefevre
M F
MF
Barbazan
P
P
Chanteau
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S
D000330:Aedes / Q000382:microbiology; D000818:Animals; D003715:Dengue / Q000382:microbiology; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D006146:Guanidines; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide; D013861:Thiocyanates
D006146:Guanidines; D012367:RNA, Viral; D013861:Thiocyanates; C054436:guanidine thiocyanate; D012822:Silicon Dioxide; D012194:RNA-Directed DNA Polymerase