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Calibration Curves for Real-Time PCR

Departments of¹ Laboratory Medicine and² Medicine, University of Washington Medical Center, Seattle, WA.
³ Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, Seattle, WA.

^aAddress correspondence to this author at: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N, D3-100, Seattle, WA 98109. Fax 206-667-4411; e-mail kjerome{at}fhcrc.org.

Background: Despite the increasing use of real-time PCR in the diagnosis and management of viral infections, there are no published studies adequately addressing the optimum number of calibrators, the number of replicates of each calibrator, and the frequency with which calibration needs to be repeated. This study was designed to address these issues.

Methods: Cycle threshold data (ABI 7700) was collected from >50 consecutive real-time PCR runs for hepatitis B and Epstein–Barr viruses. Our routine calibration curve made from serial 10-fold dilutions run in duplicate was compared with alternative options, including duplicate 100-fold dilutions, inclusion of a low-copy calibrator, and omission of the duplicate determination. Control data were used to examine the use of an average calibration curve made from multiple runs.

Results: Use of duplicate serial 10-fold dilutions led to the least imprecision, duplicate 100-fold dilutions had slightly higher imprecision, and calibration curves obtained with singlet measurements showed the greatest imprecision. For patient data, the duplicate 100-fold dilution calibration curve produced results that best matched those from the routine calibration curve. Use of singlet dilutions or inclusion of a low-copy calibrator produced poorer agreement. Variability in controls was lower with a daily calibration curve than with an average calibration curve.

Conclusions: Duplicate 100-fold dilution calibration curves produced equivalent results and the same imprecision as curves with more calibrators, and thus are a valid alternative. Laboratories should carefully evaluate the variability resulting from the use of average calibration curves before adopting this approach.

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