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87 PCR-Based Qualitative DNA Assay with a Broad Subtype Detection Range
C. Christopherson* and K. K. Y. Young
Roche Molecular Systems, Alameda, CA, USA
Background: A nucleic acid-based test for the detection of HIV-1 can be an important tool for vaccine trials to differentiate true infections from vaccinations.
Methods: We have developed a prototype PCR-based HIV-1 DNA qualitative test. The assay incorporates the primers SK145-SKCC1B, which are contained in the AMPLICOR HIV-1 MONITOR v1.5 RNA quantitative assay and have been shown to amplify the different subtypes of HIV-1 Group M with equal efficiency. In addition, the assay incorporates an internal control (IC) that is coamplified with the test sample. The IC is introduced into each sample at 30 copies to maximize sensitivity to inhibitors and provides assurance that a negative result is not due to compromised amplification.
Results: The test uses a microwell-based detection format with a sensitivity of 10 copies. Results from a panel representing 5 subtype A, 10 subtype B, 3 subtype D, 10 subtype E, 5 subtype F, 2 subtype G, and 1 subtype H showed that the assay has broad subtype detection range.
Conclusions: This assay will prove to be particularly useful in evaluating samples in which antibody test results are not informative. Furthermore, the broad subtype detection range will be critical to trials outside of the US in which non-subtype B isolates of HIV-1 are common.
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