ULTRA-RAPID, SIMPLE, SENSITIVE, AND ECONOMICAL SILICA METHOD FOR EXTRACTION OF DENGUE VIRAL-RNA FROM CLINICAL SPECIMENS AND MOSQUITOS 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 mul) 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-I virus was also detected in field-caught Aedes aegypti mosquitoes by silica RT-PCR.