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 compe titive internal standard with the sandwich hybridization of the produc ts. The variability of the PCR efficiency was corrected using a specif ically designed internal standard competitive not only for the PCR amp lification, but also for the hybridization on capture probes fixed ont o microwells. The design of such standard gave a dynamic range extendi ng from 30-1 million copies of target DNA when the internal standard c opy number was fixed to 1000 using a simple colorimetric detection. Th e assay was independent from the number of PCR cycles, which indicates a true competition between the standard and the template DNA. The ass ay was developed for a cytomegalovirus (CMV) DNA sequence and is illus trated by the quantification of CMV in a culture sample.