Comparison of four RNA extraction methods for the detection of small round structured viruses in faecal specimens.
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.
Hale
A D
AD
Enteric and Respiratory Virus Laboratory, Central Public Health Laboratory, London, UK.
Green
J
J
Brown
D W
DW
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
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