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.