Touchdown digital polymerase chain reaction for quantification of highly conserved sequences in the HIV-1 genome

被引：15

作者：

De Spiegelaere, Ward ^{[1
,2
]}

Malatinkova, Eva ^{[1
]}

Kiselinova, Maja ^{[1
]}

Bonczkowski, Pawel ^{[1
]}

Verhofstede, Chris ^{[3
]}

Vogelaers, Dirk ^{[1
,2
]}

Vandekerckhove, Linos ^{[1
,2
,3
]}

机构：

[1] Univ Ghent, Dept Internal Med, Fac Med & Hlth Sci, B-9000 Ghent, Belgium

[2] Ghent Univ Hosp, Dept Gen Internal Med, HIV Translat Res Unit, B-9000 Ghent, Belgium

[3] Univ Ghent, Dept Clin Chem Microbiol & Immunol, AIDS Reference Lab, B-9000 Ghent, Belgium

来源：

ANALYTICAL BIOCHEMISTRY | 2013年 / 439卷 / 02期

关键词：

HIV-1; Digital PCR; Total HIV DNA; Touchdown PCR; DNA COPY NUMBER; ABSOLUTE QUANTITATION; EXPRESSION ANALYSIS; PCR; SAMPLES; ASSAY;

D O I：

10.1016/j.ab.2013.04.024

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Digital polymerase chain reaction (PCR) is an emerging absolute quantification method based on the limiting dilution principle and end-point PCR. This methodology provides high flexibility in assay design without influencing quantitative accuracy. This article describes an assay to quantify HIV DNA that targets a highly conserved region of the HIV-1 genome that hampers optimal probe design. To maintain high specificity and allow probe binding and hydrolysis of a probe with low melting temperature, a two-stage touchdown PCR was designed with a first round of amplification at high temperature and a subsequent round at low temperature to allow accumulation of fluorescence. (C) 2013 Elsevier Inc. All rights reserved.

引用

页码：201 / 203

页数：3

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