Background: The TRUGENE HIV-1 genotyping assay and OpenGene DNA Sequencing System are designed to sequence the PR- and RT-coding regions of HIV-1 pol. Studies were undertaken to determine the performance characteristics of this assay, including the effect of RNA extraction method, anticoagulant, specimen handling, presence of interfering substances, and HIV-1 RNA copy number. Overall accuracy of the assay was also evaluated using a panel of plasma samples spiked with molecular infectious clones (MIC) of HIV-1 with a variety of mutations in PR and RT.

Methods: Plasma samples containing wild-type and mutant virus were spiked with MIC of HIV-1 or obtained from HIV-infected subjects.  Virus titer was determined by RT-PCR. Extraction methods tested included standard and ultrasensitive AMPLICOR HIV-1 MONITOR, QIAGEN Viral RNA Extraction Mini Kit, QIAGEN Ultra HIV Extraction, and NASBA Manual HIV-1 Quantitative NucliSens.   Sequence data from test sites were compared to a consensus sequence to determine the percentage of agreement.

Results:  Agreement between test sequences and the consensus sequence at the nucleotide level was excellent overall (97.5-100%).  Similar results were obtained regardless of extraction method, use of EDTA or ACD as anticoagulant, and despite presence of triglycerides, bilirubin, hemoglobin, antiretroviral drugs, HIV-2, HCV, HBV, CMV, HTLV-I, or HTLV-II.  Samples with HIV-1 RNA titers >1000 copies/mL gave consistent results. To determine assay precision a panel of 10 plasma samples with 10 MIC carrying a total of 72 PR and RT mutations was tested 10 times. 31/31 (100%) PR mutations and 40/41 (97.6%) RT mutants were detected consistently, excluding 2 samples contaminated at the test lab with another HIV-1 sequence.

Conclusions: The TRUGENE HIV-1 Genotyping  Kit and OpenGene DNA Sequencing System consistently generate highly accurate sequence data when tested with HIV MIC and patient samples.