Evaluation of uncertainty in quantitative real-time PCR

• Published in: Journal of microbiological methods Vol. 67; no. 2; pp. 349 - 356
• Main Authors: Love, John L., Scholes, Paula, Gilpin, Brent, Savill, Marion, Lin, Susan, Samuel, Laly
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 01.11.2006; Elsevier Science
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; C. jejuni; Campylobacter jejuni; Campylobacter jejuni - chemistry; Campylobacter jejuni - genetics; Chemistry Techniques, Analytical - methods; Chemistry Techniques, Analytical - standards; DNA; DNA, Bacterial - analysis; Fundamental and applied biological sciences. Psychology; Microbiology; Polymerase Chain Reaction - methods; Quantitation; Real-time PCR; Reproducibility of Results; Taq Polymerase - metabolism; Uncertainty; Uncertainty; Quantitation; Real-time PCR; DNA; C. jejuni; Polymerase chain reaction; Evaluation; Campylobacteraceae; Bacteria; Real time; Quantitative analysis; Campylobacter jejuni
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2006.04.005

Quantitative real-time PCR is one of the newer methods for measurement of the amount of nucleic material in biological systems. However, reliable measurement requires an appropriate estimation of uncertainty and this paper has developed the uncertainty budget associated with this procedure using as an example, data from a quantitative real-time PCR method for the enumeration of Campylobacter jejuni. This uncertainty is relatively large and for instance, a measured result of 151 units of DNA would have a 95% confidence interval of ± 84 units of DNA with the main sources of uncertainty being the measurement of the threshold cycle (Ct) value, the predicted DNA content of the unknown sample from the calibration line and the molar absorbance value for DNA.