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Published on: December 3, 2011
Quantification of Lawsonia intracellularis in porcine faeces by real-time PCR
H Nathues1, K Holthaus, E grosse Beilage
1Field Station for Epidemiology, University of Veterinary Medicine, Foundation, Hannover, Germany. Heiko.Nathues@tiho-hannover.de
Aim:
To develop and to validate a method for the quantification of Lawsonia intracellularis in porcine faeces by real-time PCR.
Methods And Results:
A real-time PCR including a calibrator based on plasmid DNA for quantification by means of DeltaDeltaCt method was evaluated. The parameters specificity, detection limit, quantification limit, linearity, range, repeatability, precision and recovery were validated. The detection limit of the agent was 1 copy per reaction, and quantification was reliable between 10(1) and 10(7) copies per microl reaction volume. The linearity calculated by logistic regression revealed a slope of -3.329 reflecting an efficiency of 99.7% for the assay. Moreover, it was shown that storage of samples and repetition of tests including DNA isolation by same or other investigators did not influence the outcome.
Conclusion:
The quantification method described herein revealed consistent results for the quantitation of L. intracellularis in porcine faeces samples.
Significance And Impact Of The Study:
In contrast to common PCR in combination with gel electrophoresis, this validated quantification method based on real-time PCR enhances a reliable quantification and is even more sensitive.
