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Updated: Jul 13, 2026
Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays
Published on: September 26, 2011
Detection and quantification of minority HIV isolates harbouring the D30N mutation by real-time PCR amplification
Maria G Detsika1, Becky Chandler, Saye H Khoo
1Department of Pharmacology and Therapeutics, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, UK.
Objectives:
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.
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