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

Abstract

A rapid, simple and efficient single‐tube procedure is described for the isolation of dengue virus RNA from small amount of serum (10 μl) 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: 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: 2 to 10^8.69). Dengue‐1 virus was also detected in fieldcaught Aedes aegypti mosquitoes by silica RT‐PCR.