Quantitative Determination of CMV DNA Using a Combination of Competitive PCR Amplification and Sandwich Hybridization. (October 1998)

• Record Type: Journal Article
• Authors: Alexandre, I.; Zammatteo, N.; Ernest, I.; Ladriere, J.-M.; Le, L.; Hamels, S.; Chandelier, N.; Vipond, B.; Remacle, J.
• Is Part Of: Biotechniques. Volume 25:Number 4(1998)
• Page Start: 676
• Page End: 683
• Publication Date: 1998-10
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/98254dt07
• Languages: English
• ISSNs: 0736-6205

Abstract

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.