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

J Med Virol. 1993 Jun;40(2):142-5. doi: 10.1002/jmv.1890400211.

Abstract

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.

MeSH terms

  • Aedes / microbiology
  • Animals
  • Dengue / microbiology
  • Dengue Virus / genetics
  • Dengue Virus / isolation & purification*
  • Female
  • Guanidines
  • Humans
  • Polymerase Chain Reaction / methods*
  • RNA, Viral / blood
  • RNA, Viral / genetics*
  • RNA, Viral / isolation & purification
  • RNA-Directed DNA Polymerase
  • Sensitivity and Specificity
  • Silicon Dioxide
  • Thiocyanates

Substances

  • Guanidines
  • RNA, Viral
  • Thiocyanates
  • guanidine thiocyanate
  • Silicon Dioxide
  • RNA-Directed DNA Polymerase