Improved method for purification of viral RNA from fecal specimens for rotavirus detection.
An improved procedure to recover and purify double-stranded RNA (dsRNA) from fecal specimens is described. Guanidine isothiocyanate, hydroxyapatite, and cetyltrimethylammonium bromide were used to extract and purify rotavirus dsRNA from fecal specimens. The method is very efficient and easy to perform, and precludes the use of toxic substances such as phenol, chloroform, and Freon. It yields RNA free of enzymatic inhibitors, permitting its detection by reverse transcription-polymerase chain reaction assays. In addition, it was demonstrated that during initial clarification of the fecal suspension, the pellet must be washed at least twice to avoid massive losses of virus, viral protein, or viral nucleic acid retained in the solid debris.
Santos
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Division of Molecular Biological Research and Evaluation, Food and Drug Administration, Washington, DC 20204.
Gouvea
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D001483:Base Sequence; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D011232:Chemical Precipitation; D003967:Diarrhea / Q000175:diagnosis* / Q000382:microbiology; D017886:Durapatite; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000737:chemistry / Q000382:microbiology*; D006146:Guanidines; D006801:Humans; D017879:Isothiocyanates; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012330:RNA, Double-Stranded / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification*; D012401:Rotavirus / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis* / Q000382:microbiology; D013048:Specimen Handling
D002593:Cetrimonium Compounds; D006146:Guanidines; D017879:Isothiocyanates; D012330:RNA, Double-Stranded; D012367:RNA, Viral; C054435:guanidine isothiocyanate; D017886:Durapatite; D000077286:Cetrimonium