A simple and sensitive DNA hybridization assay used for the routine diagnosis of human parvovirus B19 infection

Hicks, Karen E.; Beard, Stuart; Cohen, B. J.; Clewley, Jonathan P.

doi:10.1128/jcm.33.9.2473-2475.1995

Cited by 25 publications

(5 citation statements)

References 17 publications

(16 reference statements)

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“…The use of PCR amplimers as probes for in situ hybridization will avoid the necessity for the laborious procedures used in the preparation of plasmid-derived probes and should also avoid the disadvantages of possible cross-contamination between residual bacterial plasmid and/or host chromosomal DNA and bacterial DNA present in a contaminated specimen (vector homology) (11).…”

Section: Discussionmentioning

confidence: 99%

“…Purchase of labelled probes for in situ hybridization is expensive and the preparation of plasmid-derived probes is laborious and time consuming. There is also the possibility of cross-hybridization between residual bacterial plasmid and/or host chromosomal DNA due to vector homology (11). With the advent of PCR technology, amplifying and labelling DNA to produce specific and sensitive probes can be done relatively easily.…”

mentioning

confidence: 99%

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A simple and rapid technique for the detection of Epstein‐Barr virus DNA in HIV‐associated oral hairy leukoplakia biopsies

Mabruk¹,

Antonio²,

Flint³

et al. 2000

J Oral Pathology Medicine

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A method of generating nucleic acid probes by polymerase chain reaction (PCR) for the detection of Epstein-Barr virus (EBV)-DNA by in situ hybridization in oral hairy leukoplakia (OHL) lesions is described. This method has the advantage over older methods of being cheaper, quicker and retaining sensitivity and specificity. Purified PCR products of Epstein-Barr virus DNA of 110 bp and 328 bp were labelled with biotin by nick translation or random primer labelling and were compared in in situ hybridization experiments with probes prepared by incorporation of biotin-labelled nucleotides in the PCR reaction mixture, with EBV viral DNA as a template. These probes were applied to 18 OHL tongue biopsies known to be positive for EBV-DNA, using a commercially available biotin-labelled BamHI "V" fragment EBV-DNA probe. To determine the specificity of the probes, we applied them to 20 normal tongue tissue samples and to 12 biopsies taken from keratotic tongue lesions from patients without risk factors for HIV infection and known to be negative for EBV-DNA. Clear positive signals for EBV-DNA were detected in all 18 cases of OHL biopsies using the amplimer of 328 bp labelled by PCR and random primer labelling. However, nick translation labelling was less efficient and sensitive. All control specimens were negative for EBV-DNA.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

A simple and rapid technique for the detection of Epstein‐Barr virus DNA in HIV‐associated oral hairy leukoplakia biopsies

Mabruk¹,

Antonio²,

Flint³

et al. 2000

J Oral Pathology Medicine

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“…Several techniques have been used to detect parvovirus B19 viraemia, including an assay for circulating viral antigen [2], nucleic acid hybridisation assays [3][4][5], PCR assays [6][7][8][9] and, more recently, haemagglutination assays [10,11]. The maximum sensitivity of detection of 208 Vox Sang 1997;73:207-211 Saldanha/Minor/B19 Collaborative Study Group B19 DNA by DNA hybridisation or haemagglutination assays is 10 6 -10 7 genome equivalents/ml [10,12]. Thus, these assays are sufficiently sensitive for detecting viraemia in single donations where the level of circulating virus may be as high as 10 12 genomes/ml.…”

Section: Introductionmentioning

confidence: 99%

Collaborative Study to Assess the Suitability of a Proposed Working Reagent for Human Parvovirus B19 DNA Detection in Plasma Pools by Gene Amplification Techniques

Saldanha¹,

Minor²

1997

Vox Sanguinis

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Background and objectives: A collaborative study was done to examine the sensitivity and specificity of assays for the detection of human parvovirus B19 DNA in plasma pools by PCR techniques and to establish a working reagent for B19 DNA testing of plasma pools. Materials and methods: Duplicate samples consisting of a tenfold dilution series of a positive cryosupematant diluted in B19 DN A-negative cryosupematant were sent to 17 laboratories. Results: The sensitivity of the assays varied: 2 laboratories were able to detect the 10^-7 dilution while 1 laboratory failed to detect B19 DNA in any samples. In addition, 5 laboratories obtained false-positive results. Conclusions: In general, laboratories using assays optimised for rapid detection of B19 DNA in serum samples did not perform well, indicating that such rapid methods are not adequate for examination of plasma pools. The 10^-6 dilution was detected by approximately half the laboratories and could be used as the working reagent.

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“…Currently, the methods used to detect B19-positive sera mainly consist of hybridization assays with labeled probes (2,11,21) or PCRs (5,6,15). The nucleic acid hybridization assay can detect the medium to high levels of viremia (above 10 4 genome copies of B19 DNA) which occur in the acute phase of B19 infection, and the PCR assay can detect even very low viral titers (between 1 and 100 B19 genome copies).…”

mentioning

confidence: 99%

Dot immunoperoxidase assay for detection of parvovirus B19 antigens in serum samples

et al. 1997

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We describe a simple and rapid dot immunoperoxidase assay for the direct detection of parvovirus B19 capsid antigens in human sera. The assay was performed with serum specimens dotted onto nylon membranes. VP1 and VP2 B19 antigens, which represent 4 and 96% of the capsid, respectively, were detected with a pool of monoclonal antibodies directed against the two proteins, and the complex was visualized by immunoperoxidase staining. The assay could be performed in about 4 h, and positive results were revealed at the end of the reaction as dark blue spots on the nylon membrane at the site of positive specimens. A total of 541 serum samples from different subjects and with different laboratory evaluations with regard to B19 infection were analyzed. The results obtained by the dot immunoperoxidase assay were compared with the results obtained for the presence of B19 DNA by dot blot hybridization and nested PCR. With optimized working conditions, the dot immunoperoxidase assay was able to detect the presence of B19 with a sensitivity comparable or slightly higher than that achieved by dot blot hybridization but less than that achieved by nested PCR. Since the level of sensitivity of the dot immunoperoxidase assay proved to be appropriate for the detection of acute B19 infection, and since the cost, time to a result, and versatility of the assay are important issues, from our evaluation, the dot immunoperoxidase assay described may be particularly suitable for large-scale screening of samples and a good alternative to DNA detection methods in the routine laboratory evaluation of B19 infection.

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A simple and sensitive DNA hybridization assay used for the routine diagnosis of human parvovirus B19 infection

Cited by 25 publications

References 17 publications

A simple and rapid technique for the detection of Epstein‐Barr virus DNA in HIV‐associated oral hairy leukoplakia biopsies

A simple and rapid technique for the detection of Epstein‐Barr virus DNA in HIV‐associated oral hairy leukoplakia biopsies

Collaborative Study to Assess the Suitability of a Proposed Working Reagent for Human Parvovirus B19 DNA Detection in Plasma Pools by Gene Amplification Techniques

Dot immunoperoxidase assay for detection of parvovirus B19 antigens in serum samples

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