Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions.
A method was developed for the purification of rotavirus RNA from fecal extracts in order to permit the sensitive identification of group A rotavirus in fecal specimens by the polymerase chain reaction. Sequential reactions with reverse transcriptase and Taq polymerase with directed primers from rotavirus gene 6 yielded characteristic 259-base-pair fragments that were then visualized by silver stain on a polyacrylamide gel. As few as 500 genomic copies of purified rotavirus RNA could be detected in this manner. However, when the method was applied to fecal samples with added rotavirus virions, inhibition was noted in many of the fecal extracts which were tested. The inhibition could be reversed by dilution of the fecal extract, but sensitivity was also reduced by a corresponding dilutional factor. The inhibition was quantitatively removed by an added step in the extraction process that utilized chromatographic cellulose fiber powder (CF11 powder) to purify the rotavirus RNA during a series of rapid washing and elution steps. After CF11 purification, rotavirus RNA could be detected in experimental fecal samples at dilutions 1,000- to 10,000-fold beyond the detection limits of standard techniques such as enzyme immunoassay and the direct visualization of RNA following polyacrylamide gel electrophoresis. Furthermore, following purification by CF11, rotavirus RNA could be detected in all of seven enzyme-linked immunosorbent assay-positive fecal samples obtained from a child with rotavirus gastroenteritis; when CF11 purification was not performed, rotavirus RNA could be detected in only four of these samples, even after the removal of inhibitors by dilution of the extracts. Large-scale identification of rotavirus in fecal specimens may be possible by use of CF11 purification of viral RNA prior to sequential reactions with reverse transcriptase and Taq polymerase in a modified polymerase chain reaction.
Wilde
J
J
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Eiden
J
J
Yolken
R
R
D005243:Feces / Q000382:microbiology*; D005759:Gastroenteritis / Q000175:diagnosis / Q000453:epidemiology; D006801:Humans; D007223:Infant; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000096:biosynthesis*; D012194:RNA-Directed DNA Polymerase; D012401:Rotavirus / Q000235:genetics* / Q000302:isolation & purification; D012400:Rotavirus Infections / Q000175:diagnosis* / Q000453:epidemiology; D012680:Sensitivity and Specificity; D014481:United States / Q000453:epidemiology
D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase