an epidemiological study of listeriosis in dairy cattle

UK), 0.5μM DNA primer, 1 unit Supertaq DNA polymerase (Stratech Scientific,
Cambridge, UK) and 10μl of cell supernatant (final volume 50μl). Single primer
(Cruachem Ltd., Glasgow UK), at concentration of 25μM, was added to each reaction
mixture which was then overlaid with 3-4 drops of liquid paraffin. The mixture was
then placed in a PCR Thermal Reactor (Hybaid, Middlesex, UK) with temperature
programming as follows;
-one cycle at 94 0 C for 3 minutes to denature template DNA,
-four low stringency cycles at 94 0 C for 45 seconds, 26 0 C for 2 minutes, and 72 0 C for 2
minutes with ramp setting of 2, to anneal template DNA and primer,
-thirty cycles at 94 0 C for 45 seconds, 36 0 C for 1 minute, 72 0 C for 2minutes with ramp
default setting,
-finally one cycle at 72 0 C for 5 minutes to extend the reaction with Taq polymerase.
(vi) Test repeatability:
The repeatability of the test was evaluated by using 2 isolates of L.
monocytogenes and 1 isolate of L. innocua in the test on two occasion using the same
procedure.
(vii) Analysis of PCR product:
The PCR products were analysed by electrophoresis in 1% w/v agarose with
TBE buffer (0.089M Tris base, 0.089M orthoboric acid, 0.002M EDTA, pH8.0). 2μl of
loading buffer (containing bromophenol blue 0.25% w/v and sucrose 40% w/v in
distilled water) was added to 10μl of final PCR product and mixed well. 10μl of this
mixture was loaded onto the gel and run at 100-115 volts for about 2-3 hours. The gel
was then soaked in ethidium bromide (0.5mg/L) for about 30 minutes the bands
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