Elimination of background signals in a modified polymerase chain reaction-based reverse transcriptase assay

Maudru, Tom; Peden, Keith

doi:10.1016/s0166-0934(97)00067-0

Cited by 28 publications

(17 citation statements)

References 22 publications

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“…This signal is 100-to 1000-fold lower than with HIV-1 MAL at its lowest point and is most likely due to released DNA-dependent DNA polymerases from lysed cells in the culture. All the PBRT assays detect activity in lysed cells (9,10,15). Also, the signal in the HIV-1 MAL cultures is not due to residual input virus, since the inoculum of a virus that does not replicate cannot be detected after five days (unpublished results).…”

Section: Adaptation Of the Fluorogenic 5 ′ ′ -Nuclease Chemistry To Amentioning

confidence: 89%

“…Because some thermostable DNA polymerases have RNA-dependent DNA polymerase activity, background signals can be generated (10,14). Additionally, the assay is not selective for retroviruses, because lysates of all cells tested have PBRT activity (9,10,15).…”

Section: Adaptation Of the Fluorogenic 5 ′ ′ -Nuclease Chemistry To Amentioning

confidence: 99%

“…The legend for Figure 1 gives the sequences of the TaqMan primers and the probe designed for use. To assess the sensitivity of these new primers in comparison with the primers used previously (10,14,15), we carried out our version of the PBRT assay (10). Using (i) avian myeloblastoma virus (AMV) RT to generate the cDNA and primer extension and (ii) polyacrylamide gel electrophoresis to analyze the PCR product (10), we found that the sensitivity of the new primers with the PBRT assay was equivalent to the sensitivity of the original primers (14), which were able to detect between 100 and 1000 pU of AMV RT (data not shown).…”

Section: Adaptation Of the Fluorogenic 5 ′ ′ -Nuclease Chemistry To Amentioning

confidence: 99%

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Adaptation of the Fluorogenic 5′-Nuclease Chemistry to a PCR-Based Reverse Transcriptase Assay

Maudru

Peden

1998

BioTechniques

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Section: Adaptation Of the Fluorogenic 5 ′ ′ -Nuclease Chemistry To Amentioning

confidence: 89%

Section: Adaptation Of the Fluorogenic 5 ′ ′ -Nuclease Chemistry To Amentioning

confidence: 99%

Section: Adaptation Of the Fluorogenic 5 ′ ′ -Nuclease Chemistry To Amentioning

confidence: 99%

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Adaptation of the Fluorogenic 5′-Nuclease Chemistry to a PCR-Based Reverse Transcriptase Assay

Maudru

Peden

1998

BioTechniques

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“…This cDNA is then amplified by PCR, and the PCR product can be detected and quantified. These assays have the potential to detect a single retroviral virion (Heneine et al, 1995 ;Maudru & Peden, 1997 ;Pyra et al, 1994 ;Silver et al, 1993) and thus should be Author for correspondence : Keith Peden.…”

mentioning

confidence: 99%

“…Virus production was measured by a conventional RT (Peden & Martin, 1995) or by a PBRT assay (Maudru & Peden, 1997) except that the read-out was modified. Amplified products of the PCR were subjected to electrophoresis in 2 % (w\v) agarose gels.…”

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confidence: 99%

Detection of human immunodeficiency virus type 1 after infection of unstimulated peripheral blood mononuclear cells.

Shapiro

Maudru

Peden

1999

Journal of General Virology

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Application of a highly sensitive PCR-based reverse transcriptase (RT) assay to the analysis of the infection of CD4 M cell lines with human immunodeficiency virus type 1 (HIV-1) demonstrated that virus production can be detected as early as 24 h after infection. Most of the signal at 24 h was due to virus production, as it could be substantially reduced by prior treatment with the RT inhibitor zidovudine. Virus production at 24 and 48 h was unaffected by the protease inhibitor indinavir. Infection of unstimulated peripheral blood mononuclear cells (PBMC) with a macrophage-tropic HIV-1 isolate yielded increasing virus production for 2-3 weeks, while infection with a T-cell line-tropic isolate yielded only low and sporadic virus production. Productive infection of unstimulated PBMC by the macrophage-tropic virus required functional Gag matrix and Vpr proteins ; therefore, the monocyte-derived macrophage is probably the virus-producing cell in these cultures.Studies on human immunodeficiency virus type 1 (HIV-1) replication in vitro most commonly use a p24 antigen ELISA or reverse transcriptase (RT) assay to quantify virus released into the culture supernatant from infected cells. The p24 assay is capable of detecting HIV-1 between the pg and ng range, while conventional RT assays are about 10 to 100 times less sensitive. Several highly sensitive RT assays have been described that are approximately 10'-fold more sensitive than conventional RT assays (Heneine et al., 1995 ;Pyra et al., 1994 ;Silver et al., 1993). These assays rely on the ability of a retroviral RT to copy an exogenous RNA template of known sequence into a complementary DNA (cDNA). This cDNA is then amplified by PCR, and the PCR product can be detected and quantified. These assays have the potential to detect a single retroviral virion (Heneine et al

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Enhanced Detection of Airborne Microbial Contaminants

Stetzenbach

2003

Encyclopedia of Environmental Microbiology

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Elimination of background signals in a modified polymerase chain reaction-based reverse transcriptase assay

Cited by 28 publications

References 22 publications

Adaptation of the Fluorogenic 5′-Nuclease Chemistry to a PCR-Based Reverse Transcriptase Assay

Adaptation of the Fluorogenic 5′-Nuclease Chemistry to a PCR-Based Reverse Transcriptase Assay

Detection of human immunodeficiency virus type 1 after infection of unstimulated peripheral blood mononuclear cells.

Enhanced Detection of Airborne Microbial Contaminants

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