Allele-specific real-time PCR testing for minor HIV-1 drug resistance mutations: assay preparation and application to reveal dynamic of mutations in vivo / 中华医学杂志(英文版)
Chinese Medical Journal
;
(24): 3389-3395, 2010.
Article
in English
| WPRIM
| ID: wpr-319096
ABSTRACT
<p><b>BACKGROUND</b>It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20% - 30%. The aim of this study was to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions 103, 184 and 215.</p><p><b>METHODS</b>We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140 clinical samples using this method.</p><p><b>RESULTS</b>The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M184I, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained.</p><p><b>CONCLUSIONS</b>Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Y emerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Reproducibility of Results
/
Sensitivity and Specificity
/
HIV-1
/
Drug Resistance, Viral
/
Alleles
/
Real-Time Polymerase Chain Reaction
/
Genetics
/
Methods
/
Mutation
Type of study:
Diagnostic study
/
Practice guideline
/
Prognostic study
Limits:
Humans
Language:
English
Journal:
Chinese Medical Journal
Year:
2010
Type:
Article
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