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Genotypic resistance testing in HIV by arrayed primer extension.

Michaela Schanné1, Jochen Bodem, Aslihan Gerhold-Ay

  • 1Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany. m.schanne@dkfz.de

Analytical and Bioanalytical Chemistry
|January 19, 2008
PubMed
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Arrayed Primer Extension (APEX) offers a rapid, cost-effective method for human immunodeficiency virus (HIV) genotypic resistance testing. This assay detects mutations linked to antiretroviral therapy failure, aiding in patient treatment monitoring.

Area of Science:

  • Molecular Biology
  • Virology
  • Genetics

Background:

  • Monitoring antiretroviral therapy (ART) for human immunodeficiency virus (HIV) requires analysis of drug resistance mutations.
  • Standard sequencing methods for genotypic resistance testing can be time-consuming and expensive.

Purpose of the Study:

  • To establish and validate Arrayed Primer Extension (APEX) as a rapid and economical alternative for HIV genotypic resistance testing.
  • To develop a microarray-based assay for detecting key drug resistance mutations in HIV protease and reverse transcriptase.

Main Methods:

  • Arrayed Primer Extension (APEX) assay utilizing immobilized oligonucleotide primers and fluorescently labeled dideoxynucleotides.
  • Primer extension reaction on hybridized template DNA, where sequence-specific color patterns indicate mutations.

Related Experiment Videos

  • Design of a microarray targeting 26 codons in HIV protease and 33 in reverse transcriptase, using degenerate oligonucleotides to reduce primer numbers.

    • Main Results:

    • Successful establishment and application of APEX for HIV genotypic resistance testing.
    • Demonstrated ability to detect mutations associated with resistance to HIV protease and reverse transcriptase inhibitors.
    • Validation performed on DNA from 94 HIV protease-resistant and 48 HIV reverse transcriptase-resistant patients, including diverse HIV subtypes.

    Conclusions:

    • APEX is a viable, rapid, and economical method for genotypic resistance testing in HIV patients.
    • The APEX assay effectively identifies drug resistance mutations, supporting personalized antiretroviral therapy.
    • The method's adaptability to various HIV subtypes enhances its utility in global HIV management.