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Improved conditions for extraction and amplification of human immunodeficiency virus type 1 RNA from plasma samples
M L Villahermosa1, M Thomson, E Vázquez de Parga
1Instituto de Salud Carlos III, Centro Nacional de Biología Fundamental, Area de Patogenia Viral, Madrid, Spain.
Objectives:
We attempted to define optimal conditions for amplification of low copy number HIV-1 RNA sequences in plasma samples, applying improved conditions for nucleic acid extraction and amplification.
Methods:
Several methodologic parameters were evaluated, including methods of RNA extraction, volumes of plasma samples, proportion of extracted RNA used as a template for amplification, and reverse transcriptase-DNA polymerase enzyme combination employed in cDNA synthesis and polymerase chain reaction amplification.
Results:
With this improved assay, we were able to obtain sufficient amounts of amplified material for direct sequencing in 97% of all plasma samples in our study, including 88% of samples with viral loads <80 copies/mL, 78% of samples with viral loads <50 copies/mL, and even 2 (67%) of 3 samples with <20 copies/mL.
Conclusions:
This procedure could be useful for testing resistance mutations in patients undergoing highly active antiretroviral therapy, in which the viral load is commonly <400 copies/mL, and even if it is <20 RNA copies/mL.