Quantification of Lawsonia intracellularis in porcine faeces by real-time PCR.
OBJECTIVE
To develop and to validate a method for the quantification of Lawsonia intracellularis in porcine faeces by real-time PCR.


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.


CONCLUSIONS
The quantification method described herein revealed consistent results for the quantitation of L. intracellularis in porcine faeces samples.


CONCLUSIONS
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.
Nathues
H
H
Field Station for Epidemiology, University of Veterinary Medicine, Foundation, Hannover, Germany. Heiko.Nathues@tiho-hannover.de
Holthaus
K
K
grosse Beilage
E
E
D000818:Animals; D015169:Colony Count, Microbial / Q000379:methods*; D005243:Feces / Q000382:microbiology*; D007410:Intestinal Diseases / Q000382:microbiology / Q000662:veterinary*; D020639:Lawsonia Bacteria / Q000235:genetics / Q000302:isolation & purification*; D057230:Limit of Detection; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D034421:Sus scrofa / Q000382:microbiology*; D013553:Swine Diseases / Q000175:diagnosis / Q000382:microbiology*