Quantitative determination of CMV DNA using a combination of competitive PCR amplification and sandwich hybridization.
A quantitative PCR method is proposed that combines the use of a competitive internal standard with the sandwich hybridization of the products. The variability of the PCR efficiency was corrected using a specifically designed internal standard, competitive not only for the PCR amplification, but also for the hybridization on capture probes fixed onto microwells. The design of such standard gave a dynamic range extending from 30-1 million copies of target DNA when the internal standard copy number was fixed to 1000 using a simple colorimetric detection. The assay was independent from the number of PCR cycles, which indicates a true competition between the standard and the template DNA. The assay was developed for a cytomegalovirus (CMV) DNA sequence and is illustrated by the quantification of CMV in a culture sample.
Alexandre
I
I
University of Namur, UK.
Zammatteo
N
N
Ernest
I
I
Ladriere
J M
JM
Le
L
L
Hamels
S
S
Chandelier
N
N
Vipond
B
B
Remacle
J
J
D001483:Base Sequence; D001667:Binding, Competitive; D019921:Biotinylation; D003124:Colorimetry; D003587:Cytomegalovirus / Q000235:genetics*; D003586:Cytomegalovirus Infections / Q000175:diagnosis / Q000235:genetics; D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005347:Fibroblasts / Q000821:virology; D006801:Humans; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral