Comparison of colorimetric, fluorescent, and enzymatic amplification substrate systems in an enzyme immunoassay for detection of DNA-RNA hybrids

Coutlée, François; Viscidi, Raphael P.; Yolken, Robert H.

doi:10.1128/jcm.27.5.1002-1007.1989

Cited by 39 publications

(12 citation statements)

References 34 publications

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“…Genital tract biopsy specimens from female patients (57 specimens from 30 patients) and a laryngeal papilloma biopsy specimen from a child, collected from 1986 to 1988 and stored at -70°C, were processed for RNA detection. On the basis of a previous virological investigation of portions of some of these biopsy specimens by Southern hybridization NOVEL ASSAY FOR DETECTION OF HPV 16 AND HPV 18 mRNAs and/or ViraPap (a dot blot screening test from Life Technologies for HPV 6,11,16,18,31,33,and 35), the specimens were characterized as containing HPV 16 or HPV 18 (diagnosis by Southern hybridization, 5 specimens), containing other HPV types (positive by ViraPap but negative for HPV 16 and HPV 18 by Southern hybridization, 13 specimens), not containing HPV (15 specimens), and not tested for HPV (25 specimens).…”

Section: Methodsmentioning

confidence: 99%

Detection of transcripts of human papillomaviruses 16 and 18 in cancer-derived cell lines and cervical biopsies by enzyme immunoassay for DNA-RNA hybrids following solution hybridization

et al. 1991

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mRNAs of human papillomaviruses (HPV) 16 and 18 were detected in cancer-derived cell lines and genital tract biopsy specimens by a novel hybridization assay. Biotinylated whole genomic HPV DNA probes were hybridized in solution to extracted total nucleic acids. Hybrids between the labeled probes and RNA transcripts were captured on a microplate coated with an antibiotin antibody. Bound hybrids were incubated with a j8-galactosidase-labeled monoclonal antibody to DNA-RNA hybrids and measured by the addition of a fluorogenic substrate. HPV 18 and HPV 16 mRNAs were detected in nucleic acids from 2.3 x i03 HeLa cells and 104 SiHa cells, respectively. The specificity of the assay for mRNA was demonstrated by the low reactivity of nucleic acids from SiHa cells after treatment with T1 RNase and by the selective reactivity of cellular nucleic acids which bound to an oligo(dT) column. With HPV 16 subgenomic probes, E6-E7 transcripts but not L1-L2 transcripts were detected in SiHa cells. Tests of 58 biopsy specimens from 31 patients showed that the detection of HIPV 16 and HPV 18 transcripts in tissue specimens was feasible. Analysis of biopsy specimens with subgenomic probes revealed HPV 16 E6-E7 transcripts in all specimens that reacted with the whole genomic probe, while L1-L2 transcripts were found infrequently.

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Section: Methodsmentioning

confidence: 99%

Detection of transcripts of human papillomaviruses 16 and 18 in cancer-derived cell lines and cervical biopsies by enzyme immunoassay for DNA-RNA hybrids following solution hybridization

et al. 1991

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“…The specificity of PCR-EIA was initially evaluated on defined virus types. One nanogram of each plasmid containing inserts of DNA of HPV types 6,11,16,18,31,33,35, and 45 was tested in the PCR-EIA with each type-specific nested RNA probe ( Table 2). The reactivities of the RNA probes with buffer alone were subtracted from each value.…”

Section: Design Of the Nonisotopic Assay For Detection Of Amplifiedmentioning

confidence: 99%

“…Detection and typing of HPV DNA in clinical specimens. In order to investigate if detection of HPV types 6,11,16,18,31,33,35, and 45 can be accomplished in clinical specimens by PCR-EIA combined with the L1 consensus PCR assay, 66 cervicovaginal lavage samples from 65 women were processed and analyzed by PCR-EIA and a standard dot blot assay. The generic probe used in this dot blot test is not the most sensitive (2,20), but it detects commonly encountered genital types (18) (Fig.…”

Section: Design Of the Nonisotopic Assay For Detection Of Amplifiedmentioning

confidence: 99%

Detection of human papillomavirus DNA in cervical lavage specimens by a nonisotopic consensus PCR assay

1995

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A gene amplification method that combines PCR with an enzyme immunoassay (PCR-EIA) for quantitation of amplified DNA was developed for the detection of human papillomavirus (HPV). Samples were amplified with consensus primers MY09 and MY11. Amplified DNA products were reacted in solution with type-specific nested RNA probes labelled with digoxigenin-11-UTP. Hybrids were captured on a microtiter plate coated with an antidigoxigenin antibody. Bound DNA-RNA hybrids were quantitated by the addition of an alkaline phosphatase-labelled monoclonal antibody directed against DNA-RNA hybrids and a fluorogenic substrate. The detection limit of PCR-EIA was six copies of HPV type 18 DNA in the original specimen. The assay was used to assess HPV infection of the uterine cervixes of 65 women referred to a colposcopy clinic. In 66 cervicovaginal lavage specimens, all 23 HPV strains detected by a standard isotopic PCR assay were also detected by the PCR-EIA (sensitivity, 100%; 95% confidence interval, 85.2 to 100%). Forty-two of the 43 samples that did not contain HPV types 6/11, 16, 18, 31, 33, 35, and 45 were also negative by PCR-EIA, for a specificity of 97.7%. Low-level cross-reactivity was encountered between HPV types 18 and 45 as well as between types 33 and 58. PCR-EIA provides a convenient means of objectively measuring PCR-amplified HPV DNA from common genital HPV types.

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“…degradation is required to reach comparable levels of sensitivity (10). The method is versatile and can be applied to any PCR assay by selecting a nested set of primers to prepare an RNA probe.…”

Section: A-pcr For B-globinmentioning

confidence: 99%

Evaluation of infection with human immunodeficiency virus type 1 by using nonisotopic solution hybridization for detection of polymerase chain reaction-amplified proviral DNA

et al. 1991

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A convenient assay combining solution hybridization and enzyme immunoassay for DNA-RNA hybrids (polymerase chain reaction-enzyme immunoassay [PCR-EIAJ) was developed to detect human immunodeficiency virus type 1 (HIV-1) provirus amplified by the PCR and was compared with oligomer hybridization with 32P-labeled SK19. In PCR-EIA, a fragment of the HIV-1 gag gene from peripheral blood mononuclear cells was first amplified with primer pair SK38/SK39 or 01/02. PCR-amplified material was reacted in solution with a biotinylated RNA probe. Biotinylated hybrids were measured in a microtiter-plate EIA with antibiotin antibody and a p-D-galactosidase-conjugated monoclonal antibody to DNA-RNA hybrids. Ten copies of HIV-1 DNA could be detected by PCR-EIA by using two different sets of primers. HIV-1 DNA was detected in 104 of 108 peripheral blood mononuclear cell samples by using SK38/39 and oligomer hybridization, in 104 of 108 samples by using SK38/SK39 and PCR-EIA, and in 104 of 108 samples by using 01/02 and PCR-EIA. HIV-1 provirus was detected in 107 of 108 samples by using a combination of two sets of primers. One sample from a seropositive patient was negative in all three PCR assays, and six samples gave discordant results between primer pairs. Six of the latter samples scored negative in a PCR for I-globin but became positive when the sample was diluted before amplification. When applied to clinical samples, PCR-EIA generated results similar to those of an isotopic assay for detection of ampliffied DNA.

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Comparison of colorimetric, fluorescent, and enzymatic amplification substrate systems in an enzyme immunoassay for detection of DNA-RNA hybrids

Cited by 39 publications

References 34 publications

Detection of transcripts of human papillomaviruses 16 and 18 in cancer-derived cell lines and cervical biopsies by enzyme immunoassay for DNA-RNA hybrids following solution hybridization

Detection of transcripts of human papillomaviruses 16 and 18 in cancer-derived cell lines and cervical biopsies by enzyme immunoassay for DNA-RNA hybrids following solution hybridization

Detection of human papillomavirus DNA in cervical lavage specimens by a nonisotopic consensus PCR assay

Evaluation of infection with human immunodeficiency virus type 1 by using nonisotopic solution hybridization for detection of polymerase chain reaction-amplified proviral DNA

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