title: Comparison of four RNA extraction methods for the detection of small round structured viruses in faecal specimens.
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.
authors.affiliation: Enteric and Respiratory Virus Laboratory, Central Public Health Laboratory, London, UK.
authors.forename: A D
authors.initials: AD
authors.lastname: Hale
authors.forename: J
authors.initials: J
authors.lastname: Green
authors.forename: D W
authors.initials: DW
authors.lastname: Brown
mesh_terms: D001483:Base Sequence; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D002614:Chelating Agents; D002850:Chromatography, Gel; D004279:DNA, Viral; D003902:Detergents; D003911:Dextrans; D005243:Feces / Q000821:virology*; D006146:Guanidines; D006801:Humans; D008969:Molecular Sequence Data; D009663:Norwalk virus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D012117:Resins, Synthetic; D012680:Sensitivity and Specificity; D013861:Thiocyanates
chemical_list: D002593:Cetrimonium Compounds; D002614:Chelating Agents; D004279:DNA, Viral; D003902:Detergents; D003911:Dextrans; D006146:Guanidines; D012367:RNA, Viral; D012117:Resins, Synthetic; D013861:Thiocyanates; C024997:Chelex 100; C054436:guanidine thiocyanate; C025614:sephadex; D000077286:Cetrimonium