Rational design of HIV-1 fluorescent hydrolysis probes considering phylogenetic variation and probe performance.
Quantitative PCR (qPCR) using fluorescent hydrolysis probes (FH-probes; TaqMan-probes) of variable genomes, such as HIV-1, can result in underestimation of viral copy numbers due to mismatches in the FH-probe's target sequences. Therefore both target conservation and physical properties of FH-probes, such as melting temperature, baseline fluorescence and secondary structure, should be considered in design of FH-probes. Analysis of a database of 1242 near full-length HIV-1 sequences with a novel computational tool revealed that the probability of target and FH-probe identity decreases exponentially with FH-probe length. In addition, this algorithm allowed for identification of continuous sequence stretches of high conservation, from which FH-probes with global HIV-1 clade coverage could be chosen. To revise the prerequisites of physical FH-probe function, properties of 30 DNA and 21 chimeric DNA locked nucleic acid (DLNA) HIV-1 FH-probes were correlated with their performance in qPCR. This identified the presence of stable secondary structures within FH-probes and the base composition and thermal stability of the 5' proximal end as novel predictors of FH-probe performance. Thus, empirically validated novel principles of FH-probe design regarding conservation and qPCR-performance were identified, which complement and extend current rules for FH-probe design.
Althaus
Claudia F
CF
Division of Infectious Diseases, University Hospital Zurich, University of Zurich, Zurich, Switzerland. claudia.althaus@usz.ch
Gianella
Sara
S
Rieder
Philip
P
von Wyl
Viktor
V
Kouyos
Roger D
RD
Niederöst
Barbara
B
Schmid
Adrian
A
Metzner
Karin J
KJ
Joos
Beda
B
Günthard
Huldrych F
HF
Fischer
Marek
M
D001483:Base Sequence; D005456:Fluorescent Dyes / Q000737:chemistry*; D014644:Genetic Variation; D016679:Genome, Viral / Q000235:genetics*; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D009841:Oligonucleotides; C477371:locked nucleic acid