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Quantification of Lawsonia intracellularis in porcine faeces by real‐time PCR
Author(s) -
Nathues H.,
Holthaus K.,
Grosse Beilage E.
Publication year - 2009
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2009.04389.x
Subject(s) - lawsonia intracellularis , detection limit , repeatability , chromatography , biology , dna extraction , real time polymerase chain reaction , feces , polymerase chain reaction , microbiology and biotechnology , chemistry , genetics , gene
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 ΔΔCt 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 μl 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.