Comparison of four RNA extraction methods for the detection of small round structured viruses in faecal specimens

J Virol Methods. 1996 Apr 5;57(2):195-201. doi: 10.1016/0166-0934(95)01966-9.

Abstract

Four methods for extraction of the RNA genome of small round structured viruses (SRSVs) from faecal specimens by reverse transcription polymerase chain reaction (RT-PCR) were evaluated. The efficiency of recovery of viral RNA and removal of amplification inhibitors were compared. RNA extraction using the metal chelating agent Chelex-100 and Sephadex G200 column chromatography were the most sensitive, detecting a 10(-4) dilution of a known SRSV positive specimen. Guanidinium thiocyanate (GTC) with adsorption of viral RNA onto silica was 10-fold less sensitive. The fourth method, based on PEG precipitation followed by phenol/chloroform extraction with the addition of the detergent cetyltrimethylammonium bromide (CTAB), only detected a 1 in 10 dilution of the positive specimen. The CTAB method was 2- to 50-fold less sensitive than the GTC/silica method when dilution series of three further SRSV positive specimens were tested. Thirty-six SRSV negative faecal specimens were spiked with virus and RT-PCR performed following RNA extraction by each of the four techniques in order to assess the ability of these methods to remove amplification inhibitors. The GTC/silica method successfully removed inhibitors from all samples whereas partial or complete inhibition was observed in seven (19%) specimens following extraction by the CTAB method, 17 (47%) by the Sephadex method, and 20 (56%) by the Chelex method. We conclude that, of these four methods, the GTC/silica method is the most appropriate for the extraction of viral RNA from faecal samples prior to RT-PCR for detecting SRSVs.

Publication types

  • Comparative Study

MeSH terms

  • Base Sequence
  • Cetrimonium
  • Cetrimonium Compounds
  • Chelating Agents
  • Chromatography, Gel
  • DNA, Viral
  • Detergents
  • Dextrans
  • Feces / virology*
  • Guanidines
  • Humans
  • Molecular Sequence Data
  • Norwalk virus / genetics
  • Norwalk virus / isolation & purification*
  • Polymerase Chain Reaction / methods*
  • RNA, Viral / analysis*
  • Resins, Synthetic
  • Sensitivity and Specificity
  • Thiocyanates

Substances

  • Cetrimonium Compounds
  • Chelating Agents
  • DNA, Viral
  • Detergents
  • Dextrans
  • Guanidines
  • RNA, Viral
  • Resins, Synthetic
  • Thiocyanates
  • Chelex 100
  • guanidine thiocyanate
  • sephadex
  • Cetrimonium