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

Abstract

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.

Comparison of a Multiplex Reverse Transcription-PCR-Enzyme Hybridization Assay with Conventional Viral Culture and Immunofluorescence Techniques for the Detection of Seven Viral Respiratory Pathogens

“…These findings are consistent with those of other studies previously reported, which have used monospecific or multiplex RT-PCR assays for the detection of viral infections (2,11,12,25). Although oligonucleotide hybridization confirms the specificity of the amplified PCR product, there is also evidence of improved sensitivity of PCR assays with oligonucleotide hybridization compared to the sensitivity of PCR assays with agarose gels for the detection of infection (10,28). RT-PCR-EHA incorporates both the RT-PCR technology and microplate hybridization for confirmation of results, with the combination of both techniques potentially augmenting the sensitivity of this assay.…”

PCR-Based Detection of an Emerging Avian Pneumovirus in US Turkey Flocks

“…8 Another approach tested for the detection of non-US APVs includes the use of digoxigenin-labeled (F gene based) primers for PCR and then immunochemiluminescent Southern blotting of the PCR product. 8,25 This method has the advantage of increased sensitivity and also reduced risk of false-positive reactions. More recently, an M gene-based PCR assay has been described for the detection of APV-US infection.…”

Clinical findings and unusual epidemiologic characteristics of human metapneumovirus infections in children in the region of Basel, Switzerland

“…The two hMPV RT-PCRs were integrated in the existing in-house RT-multiplex PCR as follows: primers for the hMPV N gene were combined with those for RSV, influenza viruses A and B (block 1), and primers for HMPV L gene were combined with those for adenoviruses and parainfluenza type 1 and type 3 viruses (block 2) [6,13,20,26,38]. RNA of RNA viruses was transcribed to cDNA before PCR was performed.…”