Evaluation of quantification methods for real-time PCR minor groove binding hybridization probe assays
Section snippets
HHV6 assay procedure
The standard test solutions used to compare data analysis methods were prepared by the reconstitution of a lyophilized HHV6 plasmid clone that was formulated as an assay standard for a clinical HHV6 assay. The lyophilized standard was reconstituted in deionized water before it was combined with HHV6 primers, FAM-labeled HHV6 MGB Eclipse hybridization probe, and bulk master mix. The final dilutions of the standards contained 260,000, 26,000, 2600, 260, and 26 copies per the 10-μl aliquots of
Results and discussion
A representative HHV6 amplification plot is depicted in Fig. 2A in an uncorrected form. The key features of the MGB Eclipse hybridization probe PCR data, in contrast to those of intercalating dyes, are apparent in this figure. These features include a sloped baseline, a gradual transition toward (but not reaching) a plateau, and significant amplification curve noise. An amplification plot from the second assay analyzed, EBV, is depicted in Fig. 2B. In this assay, the MGB Pleiades probe was
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