An inter-platform repeatability study investigating real-time amplification of plasmid DNA

• Published in: BMC biotechnology 2005-05, Vol.5 (1), p.15-15, Article 15
• Main Authors: Donald, Carol E, Qureshi, Fizza, Burns, Malcolm J, Holden, Marcia J, Blasic, Jr, Joseph R, Woolford, Alison J
• Format: Article
• Language: English
• Subjects: Analysis of Variance; Base Sequence; Biotechnology - methods; Blotting, Southern; copy number; Data processing; DNA; DNA - chemistry; DNA, Bacterial - genetics; exonuclease; Fluorescent Dyes - pharmacology; Gene amplification; Models, Statistical; Molecular Sequence Data; Plasmids; Plasmids - metabolism; Polymerase Chain Reaction; Q1; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction - instrumentation; Reverse Transcriptase Polymerase Chain Reaction - methods; Statistical analysis
• ISSN: 1472-6750; 1472-6750
• DOI: 10.1186/1472-6750-5-15

The wide variety of real-time amplification platforms currently available has determined that standardisation of DNA measurements is a fundamental aspect involved in the comparability of results. Statistical analysis of the data arising from three different real-time platforms was conducted in order to assess inter-platform repeatability. On three consecutive days two PCR reaction mixes were used on each of the three amplification platforms - the LightCycler, ABI PRISM 7700 and Rotor Gene 3000. Real-time PCR amplification using a fluorogenic 5' exonuclease assay was performed in triplicate on negative controls and DNA plasmid dilutions of 108-102 copies to give a total of 24 reactions per PCR experiment. The results of the statistical analyses indicated that the platform with the most precise repeatability was the ABI PRISM 7700 when coupled with the FastStart PCR reaction mix. It was also found that there was no obvious relationship between plasmid copy number and repeatability. An ANOVA approach identified the factors that significantly affected the results, in descending order of magnitude, as: plasmid copy number, platform, PCR reaction mix and day (on which the experiment was performed). In order to deliver useful, informative genetic tests, standardisation of real-time PCR detection platforms to provide repeatable, reliable results is warranted. In addition, a better understanding of inter-assay and intra-assay repeatability is required.