Elsevier

Analytical Biochemistry

Volume 361, Issue 1, 1 February 2007, Pages 55-64
Analytical Biochemistry

Evaluation of quantification methods for real-time PCR minor groove binding hybridization probe assays

https://doi.org/10.1016/j.ab.2006.11.023Get rights and content

Abstract

Real-time PCR data analysis for quantification has been the subject of many studies aimed at the identification of new and improved quantification methods. Several analysis methods have been proposed as superior alternatives to the common variations of the threshold crossing method. Notably, sigmoidal and exponential curve fit methods have been proposed. However, these studies have primarily analyzed real-time PCR with intercalating dyes such as SYBR Green. Clinical real-time PCR assays, in contrast, often employ fluorescent probes whose real-time amplification fluorescence curves differ from those of intercalating dyes. In the current study, we compared four analysis methods related to recent literature: two versions of the threshold crossing method, a second derivative maximum method, and a sigmoidal curve fit method. These methods were applied to a clinically relevant real-time human herpes virus type 6 (HHV6) PCR assay that used a minor groove binding (MGB) Eclipse hybridization probe as well as an Epstein–Barr virus (EBV) PCR assay that used an MGB Pleiades hybridization probe. We found that the crossing threshold method yielded more precise results when analyzing the HHV6 assay, which was characterized by lower signal/noise and less developed amplification curve plateaus. In contrast, the EBV assay, characterized by greater signal/noise and amplification curves with plateau regions similar to those observed with intercalating dyes, gave results with statistically similar precision by all four analysis methods.

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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