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Related Experiment Videos

Quantitative, competitive PCR assay for HIV-1 using a microplate-based detection system

P C Guenthner1, C E Hart

  • 1Centers for Disease Control and Prevention, Atlanta, GA, USA.

Biotechniques
|May 20, 1998
PubMed
Summary

A new quantitative competitive PCR (QC-PCR) assay quantifies human immunodeficiency virus Type 1 (HIV-1) DNA and RNA. This microplate-based method offers a cost-effective way to measure HIV-1 viral loads in various biological samples.

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Area of Science:

  • Molecular Biology
  • Virology
  • Biotechnology

Background:

  • Accurate quantification of human immunodeficiency virus Type 1 (HIV-1) DNA and RNA is crucial for monitoring disease progression and treatment efficacy.
  • Existing methods may require optimization for broad applicability across diverse biological matrices.

Purpose of the Study:

  • To develop and validate a quantitative competitive PCR (QC-PCR) assay in a microplate format for precise HIV-1 DNA/RNA quantification.
  • To adapt a previously described QC-PCR technique for enhanced detection and quantification in a high-throughput format.

Main Methods:

  • Modification of a competitive PCR assay using an internal standard with an 80-bp deletion in the HIV-1 gag target sequence.
  • Introduction of a non-HIV, 31-bp insert into the internal standard for specific probe hybridization.

Related Experiment Videos

  • Utilized biotinylated primers for streptavidin-coated microplate binding, followed by denaturation and probing with digoxigenin (Dig)-labeled probes.
  • Detection of hybridized probes via anti-Dig antibody conjugates for luminometry or optical densitometry.

    • Main Results:

    • The developed QC-PCR assay accurately quantifies HIV-1 gag sequences over a range of 100-10,000 copies.
    • The assay demonstrated efficacy in quantifying HIV-1 DNA/RNA in various source materials, including plasma, vaginal lavage, and cultured cells.
    • The microplate format facilitates convenient and cost-effective quantification of viral loads.

    Conclusions:

    • The microplate-based QC-PCR assay provides a reliable and efficient method for quantifying HIV-1 proviral DNA and viral RNA.
    • This assay is suitable for diverse clinical and research applications involving various body fluids, cells, and tissues.
    • The enhanced detection strategy improves the precision and applicability of QC-PCR for HIV-1 load determination.