Detection of respiratory syncytial virus by RNA-polymerase chain reaction and differentiation of subgroups with oligonucleotide probes.

The polymerase chain reaction (RNA-PCR) was used for specific detection of respiratory syncytial virus (RSV) genomes in clinical specimens. A set of primers was selected from conserved regions of the 1B and N genes for detection of both subgroups. The primers were found to be RSV specific, all RSV strains generated a 218 bp product, and no RSV specific amplified product was obtained when nucleic acids from a variety of micro-organisms from the respiratory tract were subjected to the RNA-PCR. We took advantage of the sequence heterogeneity of the amplified products to discriminate between the A and B strains by hybridisation with subgroup specific oligonucleotide probes. This additional hybridisation assay increased the sensitivity of the RNA-PCR tenfold. The RNA-PCR was tested on clinical specimens from children with symptoms of an infection of the respiratory tract. The results were compared with isolation of RSV in cell culture and direct immunofluorescence. From 93 specimens tested, 31 were found positive by all three techniques. Six additional positive results were detected using RNA-PCR. From these 37 RSV positive specimens 33 (92%), including all 6 additional positives, were subgroup A and only 4 were subgroup B strains. Thus, the RNA-PCR is a specific and sensitive technique for the detection and subgroup classification of RSV genomes.

Prospective application of reverse transcriptase polymerase chain reaction for diagnosing influenza infections in respiratory samples from a children's hospital.

A prospective clinical evaluation of the reverse transcriptase polymerase chain reaction (RNA PCR) for detection of influenza viruses was carried out with specimens from 342 patients of a children's hospital in The Netherlands. The RNA PCR, carried out directly on the specimens without an organic extraction, showed a sensitivity and specificity which are superior to those of direct immunofluorescence and comparable to those of cell culture combined with immunofluorescence (culture/IF). Negative results can be obtained within 2 days by the RNA PCR but may take up to 14 days by culture/IF. Because culturing is the standard technique for the detection of respiratory viruses, at this moment there are no strong arguments to replace culture/IF with RNA PCR for the detection of influenza A virus.