Improved conditions for extraction and amplification of human immunodeficiency virus type 1 RNA from plasma samples with low viral load.
OBJECTIVE
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.
Villahermosa
M L
ML
Instituto de Salud Carlos III, Centro Nacional de Biología Fundamental, Area de Patogenia Viral, Madrid, Spain.
Thomson
M
M
Vázquez de Parga
E
E
Cuevas
M T
MT
Contreras
G
G
Pérez-Alvarez
L
L
Delgado
E
E
Manjón
N
N
Medrano
L
L
Nájera
R
R
D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D015658:HIV Infections / Q000097:blood / Q000188:drug therapy / Q000821:virology*; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D009154:Mutation; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D019562:Viral Load
D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase