Immunodetection of DNA with biotinylated RNA probes: A study of reactivity of a monoclonal antibody to DNA-RNA hybrids

Coutlée, François; Bobo, Linda; Mayur, Kumudini; Yolken, Robert H.; Viscidi, Raphael P.

doi:10.1016/0003-2697(89)90399-0

Cited by 50 publications

(26 citation statements)

References 27 publications

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“…Improved sensitivity and precision have obvious analytical advantages, but additionally the specificity improvement increases the attractiveness of physical methods for PCR product detection and quantitation, such as electrophoresis and HPLC (37). HPLC has much better precision and dynamic range than the isotopic or enzyme-linked DNA probe approaches currently favored for quantitative PCR (38)(39)(40)(41). The conventional arguments against physical (as opposed to probed) identification of amplified DNA lose force when single-copy sensitivity protects against false negatives and increased specificity reduces the chance of false positives.…”

Section: Resultsmentioning

confidence: 99%

Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications

Chou¹,

Russell²,

Birch³

et al. 1992

Nucl Acids Res

677

315

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A Hot Start Polymerase Chain Reaction (PCR) entails the withholding of at least one reagent from the reaction mixture until the reaction tube temperature has reached 60-80 degrees C. Hot Start amplification with an AmpliWax vapor barrier uses a layer of solid wax to separate the retained reagent(s) and the test sample from the bulk of the reagents until the first heating step of automated thermal cycling melts the wax and convectively mixes the two aqueous layers. Wax-mediated Hot Start PCR greatly increases the specificity, yield, and precision of amplifying low copy numbers of three HIV targets. In the presence of 1 microgram of human placental DNA (1.6 x 10(5) diploid genomes) the specificity improvement entails considerable to complete reduction in the amplification of mis-primed sequences and putative primer oligomers. When mis-priming is negligible, the procedural improvement still suppresses putative primer oligomerization. Hot Start PCR with an AmpliWax vapor barrier permits routine amplification of a single target molecule with detection by ethidium stained gel electrophoresis; nonisotopically visualized probing suffices for confirmation. The improved amplification performance is evident for target copy numbers below approximately 10(3).

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Section: Resultsmentioning

confidence: 99%

Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications

Chou¹,

Russell²,

Birch³

et al. 1992

Nucl Acids Res

677

315

View full text Add to dashboard Cite

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“…The base pair numbers are from Wain-Hobson et al (37). Primer pairs SK38/SK39 (28) and 01/02 (8) amplified DNA segments of the gag gene of HIV-1 of 115 bp (bp 1090 to 1204) and 525 bp (bp 753 to 1277), respectively. The DNA fragment generated by amplification of HIV-1 with N1/N2 (bp 811 to 1203) was nested within the segment amplified with 01/02, while sequences generated by amplification with N3/N4 (bp 1118 to 1176) were nested within the DNA segment generated with SK38/SK39.…”

Section: Methodsmentioning

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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“…In the late 1980s, there was a sudden boom of interest in the study of immunodetection of DNA. Various methods of immunodetection were published and amongst them is a study by Coutlée et al [1] where they studied the immunodetection of DNA using biotinylated RNA probes. From then on, numerous studies on immunodetection of DNA using enzyme linked immunosorbent assay techniques were published, which subsequently lead to the introduction of polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA).…”

Section: Introductionmentioning

confidence: 99%

Application of PCR-ELISA in Molecular Diagnosis

Sue

Yeap

Omar

et al. 2014

BioMed Research International

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Polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) is an immunodetection method that can quantify PCR product directly after immobilization of biotinylated DNA on a microplate. This method, which detects nucleic acid instead of protein, is a much more sensitive method compared to conventional PCR method, with shorter analytical time and lower detection limit. Its high specificity and sensitivity, together with its semiquantitative ability, give it a huge potential to serve as a powerful detection tool in various industries such as medical, veterinary, and agricultural industries. With the recent advances in PCR-ELISA, it is envisaged that the assay is more widely recognized for its fast and sensitive detection limit which could improve overall diagnostic time and quality.

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Immunodetection of DNA with biotinylated RNA probes: A study of reactivity of a monoclonal antibody to DNA-RNA hybrids

Cited by 50 publications

References 27 publications

Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications

Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications

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

Application of PCR-ELISA in Molecular Diagnosis

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