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

Summary
genome equivalent per reaction volume; the upper detection limit is 10 8 genome
equivalents / µl reaction volume. An absolute quantification is possible within the
range of 10 1 to 10 7 genome equivalents / µl reaction volume. The precision was
determined as repeatability and intermediate precision and was confirmed for all
concentration stages of the range (relative standard deviation ≤ 15% and ≤ 20%,
respectively, limit 30%).
The identification of a possible inhibition of the PCR reaction is ensured by an
internal amplification control. A possible influence of the amplification of this control
on the quantification was tested and excluded. The use of the QIAamp ® DNA Stool
Mini Kit for sample preparation and isolation of DNA is thus suitable for ensuring the
deletion of inhibitors from sample matrix. The recovery of genome equivalents from
L. intracellularis in faecal samples amounted between 1,8% and 3,5%.
In a comparative study of 300 faecal samples, which were characterized previously
by multiplex-PCR to detect specific target-DNA of L. intracellularis, the detection rate
was increased from 50% to 81,7%. According to these results, the real-time PCR is
considerably more sensitive than multiplex-PCR. The test stands out due to a high
robustness and is suitable for routine diagnostics.
The distribution of L. intracellularis in faecal samples is homogenous, if the sample is
stirred manually immediately prior to the initialisation of the test. The addition of a
liquid to the sample in order to decrease viscosity and thereby achieve a better
distribution, merely lead to a dilution of the sample.
The influence of the sample storage on the content of specific genome fragments of
L. intracellularis was examined at different temperatures (6 °C, -20 °C and -70 °C)
and duration periods. The content was constant independently of the temperature
until end of the examination (day 132), measured in GE by L. intracellularis.
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