Improved detection of human immunodeficiency virus type 1 variants by analysis of replicate amplification reactions: relevance to studies of human immunodeficiency virus type 1 vertical transmission.
BACKGROUND
Human immunodeficiency virus type 1 (HIV-1)-infected individuals typically harbor mixtures of HIV-1 variants. For HIV-1 transmission studies, methods used for genotypic analysis should reliably detect variant mixtures. Such studies typically analyze complementary DNAs (cDNAs) from a single polymerase chain reaction (PCR) amplification. This approach may fail to detect variant mixtures in some samples because of analytic bias.


METHODS
To evaluate the impact of analytic bias on the detection of HIV-1 variants, we analyzed samples from a mother and infant known to contain both subtypes A and D HIV-1. The env third variable region of HIV-1 gp120 (V3 region) was amplified and cloned in five replicate experiments using a single plasma sample from each individual. Ten cDNAs from each experiment were analyzed.


RESULTS
The subtype mixture was detected in only four of 10 amplification experiments (three of five for the mother and one of five for the infant). Sequencing of uncloned PCR products showed that a single subtype, either A or D, was preferentially amplified in each experiment. However, the subtype mixture was detected for each sample by analyzing the five replicate experiments as a group.


CONCLUSIONS
This shows that mixtures of HIV-1 variants may be more readily detected when replicate amplification reactions are analyzed. This approach may be useful for characterizing HIV-1 variants for studies of HIV-1 transmission.
Becker-Pergola
G
G
Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Mellquist
J L
JL
Eshleman
J R
JR
Jackson
J B
JB
Eshleman
S H
SH
D000163:Acquired Immunodeficiency Syndrome / Q000635:transmission*; D000595:Amino Acid Sequence; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D005260:Female; D014644:Genetic Variation; D005838:Genotype; D015699:HIV Envelope Protein gp120 / Q000032:analysis / Q000235:genetics; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007223:Infant; D018445:Infectious Disease Transmission, Vertical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D004279:DNA, Viral; D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D010446:Peptide Fragments