Detection of hepatitis A virus by hybridization with single-stranded RNA probes.
An improved method of dot-blot hybridization to detect hepatitis A virus (HAV) was developed with single-stranded RNA (ssRNA) probes. Radioactive and nonradioactive ssRNA probes were generated by in vitro transcription of HAV templates inserted into the plasmid pGEM-1. 32P-labeled ssRNA probes were at least eightfold more sensitive than the 32P-labeled double-stranded cDNA counterparts, whereas biotin-labeled ssRNA probes showed a sensitivity comparable with that of the 32P-labeled double-stranded cDNA counterparts. Hybridization of HAV with the ssRNA probes at high stringency revealed specific reactions with a high signal-to-noise ratio. The differential hybridization reactions seen with probes of positive and negative sense (compared with HAV genomic RNA) were used to detect HAV in clinical and field samples. A positive/negative ratio was introduced as an indicator that permitted a semiquantitative expression of a positive HAV reaction. Good agreement of this indicator was observed with normal stool samples and with HAV-seeded samples. By using this system, HAV was detected in estuarine and freshwater samples collected from a sewage-polluted bayou in Houston and a saltwater tributary of Galveston Bay.
D002648:Child; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D005189:False Positive Reactions; D005243:Feces / Q000382:microbiology*; D006507:Hepatovirus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D011237:Predictive Value of Tests; D012313:RNA / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D014158:Transcription, Genetic; D014871:Water Microbiology
D004279:DNA, Viral; D012367:RNA, Viral; D012313:RNA