Detection of hepatitis A virus by hybridization with single-stranded RNA probes

Abstract

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.

Detection of Norwalk virus and hepatitis A virus in shellfish tissues with the PCR

“…After linearization of the plasmid DNA with XbaI (Promega), RNA was synthesized from 1 g of plasmid with 45 U of SP6 polymerase (Bethesda Research Laboratories, Gaithersburg, Md.) in a total volume of 20 l, as previously described (11). The plasmid DNA was removed by digestion with RNase-free DNase (Promega), and the RNA was precipitated twice in ammonium acetate and ethanol to remove the remaining nucleoside triphosphates.…”

“…The HAV primer (HAVp5), which consisted of the sequence of the upstream primer followed by a 25-base deletion plus an additional 20 bases of virus-specific sequence, was 5Ј-ATGTTTTGCTCCTCTTTATCATGCTAT GTCTGGTGGTTTTTCAACAAC-3Ј. For the HAV internal standard, primer HAVp5 was used in a PCR with HAVp3 and pGHAV1307A plasmid DNA (11). After linearization of the plasmid DNA with SpeI (Promega), RNA was synthesized from 1 g of plasmid with 10 U of T7 polymerase (Promega) in a total volume of 20 l (11).…”

Specific detection of enteroviruses in clinical samples by molecular hybridization using poliovirus subgenomic riboprobes

“…All specimens except those of CSF were suspended in 5 ml of transport medium (Eagle minimal essential medium supplemented with 5 mg of bovine serum albumin per ml, 4.76 mg of HEPES [N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid], 1,500 U of penicillin per ml, and 1 mg of streptomycin per ml) and mixed. One part was used for infection of permissive MRC5, buffalo green monkey, or rhabdosarcoma cells to constitute the cell lysates.…”

“…Detection of various viral RNAs by molecular hybridization with radiolabeled cDNA or cRNA probes has been reported recently (3)(4)(5)(6)(7)(8)(11)(12)(13). We have developed a sensitive and specific test for the detection of enteroviruses by using subgenomic cRNA transcripts of poliovirus type 1 synthesized in vitro (riboprobes) (2).…”

Immunoaffinity concentration and purification of waterborne enteric viruses for detection by reverse transcriptase PCR

“…Nucleic acid methods, especially in vitro enzymatic amplification of specific viral genomic nucleic acid sequences by PCR followed by detection of the amplified nucleic acid by hybridization, have emerged recently as sensitive and specific approaches for the detection of many viruses (1,3,6,13,14,22,26). A major obstacle to such nucleic acid detection is interference with the nucleic acid polymerase enzymes by inhibitory agents in the sample.…”