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Development and assessment of a multiplex real-time PCR assay for quantification of human immunodeficiency virus type 1 DNA.

https://arctichealth.org/en/permalink/ahliterature89109
Source
J Clin Microbiol. 2009 Jul;47(7):2194-9
Publication Type
Article
Date
Jul-2009
Author
Beloukas A.
Paraskevis D.
Haida C.
Sypsa V.
Hatzakis A.
Author Affiliation
Department of Hygiene, Epidemiology and Medical Statistics, Medical School, University of Athens, Athens, Greece.
Source
J Clin Microbiol. 2009 Jul;47(7):2194-9
Date
Jul-2009
Language
English
Publication Type
Article
Abstract
Previous studies showed that high levels of human immunodeficiency virus type 1 (HIV-1) DNA are associated with a faster progression to AIDS, an increased risk of death, and a higher risk of HIV RNA rebound in patients on highly active antiretroviral therapy. Our objective was to develop and assess a highly sensitive real-time multiplex PCR assay for the quantification of HIV-1 DNA (RTMP-HIV) based on molecular beacons. HIV-1 DNA quantification was carried out by RTMP in a LightCycler 2.0 apparatus. HIV-1 DNA was quantified in parallel with CCR5 as a reference gene, and reported values are numbers of HIV-1 DNA copies/10(6) peripheral blood mononuclear cells (PBMCs). The clinical sensitivity of the assay was assessed for 115 newly diagnosed HIV-1-infected individuals. The analytical sensitivity was estimated to be 12.5 copies of HIV-1 DNA per 10(6) PBMCs, while the clinical sensitivity was 100%, with levels ranging from 1.23 to 4.25 log(10) HIV-1 DNA copies/10(6) PBMCs. In conclusion, we developed and assessed a new RTMP-HIV assay based on molecular beacons, using a LightCycler 2.0 instrument. This multiplex assay has comparable sensitivity, reproducibility, and accuracy to single real-time PCR assays.
PubMed ID
19420173 View in PubMed
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Quantitative detection of the M204V hepatitis B virus minor variants by amplification refractory mutation system real-time PCR combined with molecular beacon technology.

https://arctichealth.org/en/permalink/ahliterature88584
Source
J Clin Microbiol. 2009 Aug;47(8):2544-50
Publication Type
Article
Date
Aug-2009
Author
Ntziora F.
Paraskevis D.
Haida C.
Magiorkinis E.
Manesis E.
Papatheodoridis G.
Manolakopoulos S.
Beloukas A.
Chryssoy S.
Magiorkinis G.
Sypsa V.
Hatzakis A.
Author Affiliation
Department of Hygiene, Epidemiology and Medical Statistics, Athens University, Greece.
Source
J Clin Microbiol. 2009 Aug;47(8):2544-50
Date
Aug-2009
Language
English
Publication Type
Article
Abstract
Mutations in the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif are frequently associated with resistance to antivirals and represent a major concern in the treatment of hepatitis B virus (HBV) infection. Conventional methods fail to detect minority populations of drug-resistant viral quasispecies if they represent less than 25% of the total sample virus population. The amplification refractory mutation system real-time PCR (ARMS RT-PCR) was combined with molecular beacon technology using the LightCycler system. The samples from HBV patients selected for assay evaluation included (i) 57 samples from treatment-na?ve patients for biological discriminatory ability (cutoff) estimation, (ii) 12 samples from patients with treatment failure that were M204V positive by sequencing, and (iii) 13 samples from patients with treatment failure that were negative for mutation at codon 204 by sequencing. The discriminatory ability of the assay was 0.25% when tested with laboratory-synthesized DNA target sequences. The median mutant-to-wild-type ratio for samples from naive patients tested positive for the wild type and for mutant variants was 0.01% (5th and 95th percentiles = 0.0001 and 0.04%, respectively). A value of 0.04% was selected as the biological cutoff of the assay of clinical samples. In all samples M204V positive by sequencing (12/12), the mutant variant was detected as the predominant population (range, 82.76 to 99.43%). Interestingly, in 5 (38%) of 13 samples negative by sequencing, the M204V variant was detected at a ratio above the biological cutoff (0.05 to 28%). The assay represents an efficient technique for the early detection and quantification of M204V variants before mutant strains emerge to dominate the population.
PubMed ID
19553583 View in PubMed
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