an epidemiological study of listeriosis in dairy cattle

A single colony was picked off a culture plate and put into 5ml of sterile LEB
with no antibiotic supplement and incubated at 37 0 C overnight. The culture was
harvested by centrifugation for 5 minutes at 2500 g. The pellet was washed twice with
sterile distilled water and then suspended in 150μl sterile distilled water, boiled for 4
minutes and centrifuged at 1300xg for 30 seconds. The supernatant was used as
template DNA. No attempt was made to quantify the amount of DNA but the total
number of bacteria used to extract DNA was calculated as approximately 4x10 7 cfu/ml.
This was calculated as explained earlier. The same culture conditions and methods were
used prior to DNA extraction so as to standardise the number of bacteria used for
extraction.
(iv) DNA primers:
The primers (primer Universal, 2, 3, 4, 5 and 6) were obtained from the
Department of Medical Microbiology, Southmead Health Services NHS Trust, Bristol.
The primers and their DNA sequence are given below.
Primer -mer DNA Sequence
universal 21 TTATGTAAAACGACGGCCACT
primer 2 21 ATCTGCAGCTGAACGGTCTGG
primer 3 20 CAGAATTCATGCCACGTCCC
primer 4 20 GGGCGTTGTCGGTGTTCATG
primer 5 20 ACAGGTCCAACAAAAGCTGG
primer 6 19 AACAGCACTCTGTTCAGGC
(v) RAPD amplification conditions:
DNA amplification reactions were performed in a reaction mixture containing;
10mM Tris-HCl, pH 9.0, 1.5mM MgCl2, 50mM KCl, gelatin 0.1% w/v, Triton X100
0.1% w/v, 200μM each of dATP, dGTP, dTTP and dCTP (Pharmacia, Hertfordshire,
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