Quantitativer Nachweis von Lawsonia intracellularis mittels real-time ...

7 Summary
Kerstin Holthaus
Summary
„Quantitative detection of Lawsonia intracellularis by real-time PCR in faecal
samples from pigs“
Lawsonia (L.) intracellularis is the etiologic agent of proliferative enteropathy in swine.
This intestinal disease is common worldwide and occurs in different forms. The
infection with L. intracellularis is verified intra vitam either indirectly via serology
and/or directly via the detection of the causative organism by PCR resp. IFA in faecal
samples. However, current available methods of direct detection do not offer the
possibility of distinguishing between a natural infection with the wild type and an
active immunization. Furthermore, the results of a qualitative PCR can only
distinguish between „yes“ or „no“, i.e. if the animal is infected or not. No evidence is
gained about the amount of L. intracellularis within the sample.
The aim of this study was to develop and to validate a sensitive and highly specific
detection method, which allows the quantification of the amount of L. intracellularis in
porcine faecal samples. Additionally, the possible influences of storage temperature
and duration on the sample as to the detection of L. intracellularis were tested. The
distribution of the agent in faecal samples and the influence of the method of sample
homogenization were also analysed.
This real-time PCR was validated comprehensively according to national and
international guidelines. The analytical specificity of primer and probe was tested on
several bacterial strains, all of which can occur in porcine faecal samples in
significant quantities. In this study no „false positive“-results could be detected.
Sequences of PCR-products agree with the sequence of the type strain of L.
intracellularis to 100%.
A standard, used for quantification by ∆∆Ct-method, was made from plasmids which
included the PCR-product containing sequence from L. intracellularis. A dilution
series of these plasmids was also employed to establish the standard curve. The
slope of the standard curve was -3,329. The efficiency of the real-time PCR therefore
is 99,7%. The range contains concentration stages from 10 0 to 10 7 genome
equivalents / µl reaction unit. The lower detection limit of the real-time PCR is 1
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