title: Touchdown digital polymerase chain reaction for quantification of highly conserved sequences in the HIV-1 genome.
abstract: 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.
authors.affiliation: Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium. ward.despiegelaere@ugent.be
authors.forename: Ward
authors.initials: W
authors.lastname: De Spiegelaere
authors.forename: Eva
authors.initials: E
authors.lastname: Malatinkova
authors.forename: Maja
authors.initials: M
authors.lastname: Kiselinova
authors.forename: Pawel
authors.initials: P
authors.lastname: Bonczkowski
authors.forename: Chris
authors.initials: C
authors.lastname: Verhofstede
authors.forename: Dirk
authors.initials: D
authors.lastname: Vogelaers
authors.forename: Linos
authors.initials: L
authors.lastname: Vandekerckhove
mesh_terms: D001483:Base Sequence; D017124:Conserved Sequence; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics*
chemical_list: D012367:RNA, Viral
keywords: Digital PCR; HIV-1; Total HIV DNA; Touchdown PCR