Improved detection of rotavirus RNA in dot-blot hybridization assay by chromatographic extraction and acid denaturation of double-stranded RNA.
One of the commonest methods of denaturing nucleic acids, denaturation by heat, was found to be ineffective for double-stranded (ds)RNA when RNA preparations contain 200 mM or higher concentrations of NaCl. We report acid denaturation of dsRNA (incubation for 10 min in the presence of 80 mM HCl) was particularly useful for such preparations. When genomic dsRNAs of rotavirus were extracted by ion-exchange chromatography columns (Extractor) and then denatured by acid, the detection of rotavirus RNA in stool specimens by the previously reported dot-blot hybridization assay (Yamakawa, K. et al. (1989). Molecular and Cellular Probes 3, 397-401.) was significantly improved.
Yamakawa
K
K
Research Center, Toyobo Co., Ltd, Shiga, Japan.
Nakagomi
O
O
D002852:Chromatography, Ion Exchange; D005243:Feces / Q000737:chemistry / Q000382:microbiology; D005759:Gastroenteritis / Q000175:diagnosis* / Q000382:microbiology; D006801:Humans; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D012330:RNA, Double-Stranded / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis* / Q000382:microbiology
D012330:RNA, Double-Stranded; D012367:RNA, Viral