Detection and quantification of minority HIV isolates harbouring the D30N mutation by real-time PCR amplification.
OBJECTIVE
HIV drug resistance is a major concern as the emergence of resistant strains of virus results in failure of first-line therapies with an associated increase in the cost of subsequent regimens. Genotypic resistance is currently assessed by direct sequencing and cannot detect resistant species below 20%. Real-time PCR amplification was assessed for its ability to detect the signature mutation for nelfinavir, D30N.


METHODS
A real-time PCR assay was optimized for detection of low levels of D30N and tested on in vitro-generated nelfinavir-resistant isolates as well as 10 clinical isolates (which were also characterized by sequencing).


RESULTS
The sensitivity of the assay was 1% and quantification was possible as low as 4% of the total viral population. Furthermore, this methodology enabled quantification of the 30N mutation in two isolates shown to be negative by sequencing.


CONCLUSIONS
Real-time PCR is a promising tool for the detection of minority species of HIV but further studies are required to determine the specificity of the assay in a larger and thus more diverse set of clinical isolates.
Detsika
Maria G
MG
Department of Pharmacology and Therapeutics, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, UK.
Chandler
Becky
B
Khoo
Saye H
SH
Winstanley
Craig
C
Cane
Pat
P
Back
David J
DJ
Owen
Andrew
A
D019943:Amino Acid Substitution / Q000235:genetics*; D024882:Drug Resistance, Viral / Q000235:genetics*; D006678:HIV / Q000187:drug effects / Q000235:genetics*; D015658:HIV Infections / Q000821:virology*; D006801:Humans; D009154:Mutation; D019888:Nelfinavir / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D019888:Nelfinavir