Detection of respiratory syncytial virus antigen and nucleic acid in clinical specimens using synthetic oligonucleotides

Cubie, Heather; Inglis, J.M.; McGowan, A.

doi:10.1016/0166-0934(91)90118-j

Cited by 10 publications

(3 citation statements)

References 15 publications

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“…One of the reasons may be the lack of a confirmatory assay by oligonucleotide hybridisation, which also increases the sensitivity of a PCR assay [Claas et al, 19891. Two RSV antigenic subgroups, A and B, have been recognised by their reaction with panels of monoclonal antibodies [Anderson et al, 1985;Mufson et al, 19851. Based on differences in nucleic acid sequences between the two subgroups, nucleic acid hybridisation assays have been developed to differentiate between subgroup A and B RSV [Cubie et al, 1991;Sullender et al, 1990;Sullender and Wertz, 19911. However, only a few sequences of RSV subgroups have been published.…”

Section: Introductionmentioning

confidence: 99%

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

Milaan

Sprenger

Rothbarth

et al. 1994

Journal of Medical Virology

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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.

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Section: Introductionmentioning

confidence: 99%

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

Milaan

Sprenger

Rothbarth

et al. 1994

Journal of Medical Virology

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“…The PCR assay provides a new approach to BRSV detection (5). Combined reverse transcription-PCR methods have been published for the detection of HRSV and BRSV (8,(19)(20)(21)24). The HRSV PCR assay has been evaluated both on viruses grown in cell culture and on clinical specimens, but the BRSV PCR assay has not yet been applied to clinical material.…”

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confidence: 99%

Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples

et al. 1994

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Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme Scal, which specifically cleaved products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (890%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and sensitive means for the detection of BRSV infection in cattle. Considering its higher specificity, the PCR-F assay can be recommended as the method of choice in the analysis of clinical specimens of BRSV.

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“…The use of 32Plabeled cDNA probes has allowed the detection of RSV genomic RNA or mRNA by either dot blot or Northernblot assays. The use of cDNA probes allows the distinc-tion of subgroups and serotypes in RSV [Cubie et al, 1991;Sullender et al, 19901. Although those probes showed a high degree of discrimination, the use of radioactive isotopes limits their use as a tool in clinical laboratories [Sullender and Wertz, 19911.…”

Section: Introductionmentioning

confidence: 99%

Respiratory syncytial virus detection by dot blot hybridization with a nonradioactive synthetic oligo deoxynucleotide probe

Hernández

Fernández

Valenzuela

et al. 1992

Journal of Medical Virology

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A synthetic oligodeoxynucleotide corresponding to a region of the nucleocapside gene (N) of respiratory syncytial virus (RSV), was used as a DNA probe to develop a nonradioactive hybridization assay for the detection of RSV. The probe was labeled by incorporation of biotin-7-dATP to the 3' end by a reaction catalyzed by terminal deoxynucleotydil transferase. The dot blot hybridization assay was found to be specific for RSV when tested against RSV isolates (subgroups A and B) obtained from cell cultures and isolates of adenovirus, reovirus, rotavirus, and pararotavirus. The assay detected both RSV subgroups (A and B) without significant differences. The dot blot hybridization assay using the nonradioactive probe led to similar results to indirect immunofluorescence (IFI) when tested against a panel of 64 clinical samples from nasopharyngeal secretions of infants with clinical symptoms of respiratory disease. This assay may provide the basis for a rapid, simple, and inexpensive method for routine RSV diagnosis.

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Detection of respiratory syncytial virus antigen and nucleic acid in clinical specimens using synthetic oligonucleotides

Cited by 10 publications

References 15 publications

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

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

Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples

Respiratory syncytial virus detection by dot blot hybridization with a nonradioactive synthetic oligo deoxynucleotide probe

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