Detection of HIV-1 by digoxigenin-labelled PCR and microtitre plate solution hybridisation assay and prevention of PCR carry-over by uracil-N-glycosylase

Julie A. King; Jonathan Ball

doi:10.1016/0166-0934(93)90008-f

Detection of HIV-1 by digoxigenin-labelled PCR and microtitre plate solution hybridisation assay and prevention of PCR carry-over by uracil-N-glycosylase

Julie A. King, Jonathan Ball

University Hospitals Birmingham NHS Foundation Trust

Research output: Contribution to journal › Article › peer-review

11 Citations (Scopus)

Abstract

An extremely sensitive and convenient microtitre plate solution hybridisation assay for the detection of HIV-1 PCR products was developed. The PCR product is labelled by direct incorporation of digoxigenin-dUTP and after denaturation is captured by a microtitre plate coated with a streptavidin-linked biotinylated probe. The PCR/probe hybrids are reacted with an alkaline phosphatase conjugated anti-digoxigenin antibody and detected using an alkaline phosphatase enzyme amplification system. The use of uracil-N-glycosylase and dUTP instead of dTTP in the PCR is used to effectively control carry-over from previous PCR products. The assay can detect single HIV-1 DNA molecules in a background DNA of 0.75μg.

Original language	English
Pages (from-to)	67-76
Number of pages	10
Journal	Journal of Virological Methods
Volume	44
Issue number	1
DOIs	https://doi.org/10.1016/0166-0934(93)90008-f
Publication status	Published - 1 Sept 1993
Externally published	Yes

Keywords

Digoxigenin
HIV-1
Hybridisation
PCR
UNG

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/0166-0934(93)90008-f

Cite this

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title = "Detection of HIV-1 by digoxigenin-labelled PCR and microtitre plate solution hybridisation assay and prevention of PCR carry-over by uracil-N-glycosylase",

abstract = "An extremely sensitive and convenient microtitre plate solution hybridisation assay for the detection of HIV-1 PCR products was developed. The PCR product is labelled by direct incorporation of digoxigenin-dUTP and after denaturation is captured by a microtitre plate coated with a streptavidin-linked biotinylated probe. The PCR/probe hybrids are reacted with an alkaline phosphatase conjugated anti-digoxigenin antibody and detected using an alkaline phosphatase enzyme amplification system. The use of uracil-N-glycosylase and dUTP instead of dTTP in the PCR is used to effectively control carry-over from previous PCR products. The assay can detect single HIV-1 DNA molecules in a background DNA of 0.75μg.",

keywords = "Digoxigenin, HIV-1, Hybridisation, PCR, UNG",

author = "King, \{Julie A.\} and Jonathan Ball",

year = "1993",

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doi = "10.1016/0166-0934(93)90008-f",

language = "English",

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journal = "Journal of Virological Methods",

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TY - JOUR

T1 - Detection of HIV-1 by digoxigenin-labelled PCR and microtitre plate solution hybridisation assay and prevention of PCR carry-over by uracil-N-glycosylase

AU - King, Julie A.

AU - Ball, Jonathan

PY - 1993/9/1

Y1 - 1993/9/1

N2 - An extremely sensitive and convenient microtitre plate solution hybridisation assay for the detection of HIV-1 PCR products was developed. The PCR product is labelled by direct incorporation of digoxigenin-dUTP and after denaturation is captured by a microtitre plate coated with a streptavidin-linked biotinylated probe. The PCR/probe hybrids are reacted with an alkaline phosphatase conjugated anti-digoxigenin antibody and detected using an alkaline phosphatase enzyme amplification system. The use of uracil-N-glycosylase and dUTP instead of dTTP in the PCR is used to effectively control carry-over from previous PCR products. The assay can detect single HIV-1 DNA molecules in a background DNA of 0.75μg.

AB - An extremely sensitive and convenient microtitre plate solution hybridisation assay for the detection of HIV-1 PCR products was developed. The PCR product is labelled by direct incorporation of digoxigenin-dUTP and after denaturation is captured by a microtitre plate coated with a streptavidin-linked biotinylated probe. The PCR/probe hybrids are reacted with an alkaline phosphatase conjugated anti-digoxigenin antibody and detected using an alkaline phosphatase enzyme amplification system. The use of uracil-N-glycosylase and dUTP instead of dTTP in the PCR is used to effectively control carry-over from previous PCR products. The assay can detect single HIV-1 DNA molecules in a background DNA of 0.75μg.

KW - Digoxigenin

KW - HIV-1

KW - Hybridisation

KW - PCR

KW - UNG

U2 - 10.1016/0166-0934(93)90008-f

DO - 10.1016/0166-0934(93)90008-f

M3 - Article

SN - 0166-0934

VL - 44

SP - 67

EP - 76

JO - Journal of Virological Methods

JF - Journal of Virological Methods

IS - 1

ER -

Detection of HIV-1 by digoxigenin-labelled PCR and microtitre plate solution hybridisation assay and prevention of PCR carry-over by uracil-N-glycosylase

Abstract

Keywords

UN SDGs

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