Brucellosis 2003 proceedings - PHIDIAS

Poster Session
44- DEVELOPMENT AND VALIDATION OF GENUS-SPECIFIC PCR FOR
DIAGNOSIS OF Brucella INFECTION IN ANIMALS.
Jafar A. Qasem, Sabah Al-Momin, Salwa Al-Mouqati. Kuwait Institute for Scientific Research, Food
Resources Division, Biotechnology Department. P.O.Box 24885, SAFAT, Kuwait 13109.
Brucellosis is a common zoonotic disease of worldwide distribution, which
infects mainly cattle, sheep, goats and swine, resulting in a decrease of reproduction
efficiency and abortion. The disease is caused by different species of Brucella
existing as Gram-negative coccobacillary rods.
This work was conducted to develop a diagnostic method using PCR. Two
primer sets were developed from RAPD generated fragment that was specific for
Brucella genus. The two sets of oligonucleotide primers KW1&KW2 and KW3&KW4
were optimized for direct PCR amplification. Primers KW3 and KW4 were used for
field test on samples from infected animals; 6 liver tissue samples, 6 kidney tissue
samples and 8 lymph node tissue samples were tested for Brucella by PCR. One
liver (16.6%), 3 kidney (50%) and 7 lymph (87.5%) tested positive for Brucella by
PCR. The PCR amplification was specific for Brucella genus, as these primers were
not able to amplify any non-Brucella organism associated with farm animals. The
results suggest that application of the established PCR techniques may be more
useful than the conventional tests in the specific diagnosis of animal Brucellosis.
45- CLONING AND SEQUENCING OF 1.3 Kb RAPD FRAGMENT FOR THE
DEVELOPMENT OF Brucella SPECIFIC PRIMERS.
Jafar Qasem, Sabah Al-Momin and Salwa Al-Mouqati. Kuwait Institute for Scientific Research, P. O.
Box 24885 SAFAT, Shwayik City, Kuwait.
Brucellosis is a common zoonotic disease of worldwide distribution, which
infects mainly cattle, sheep, goats and swine, resulting in a decrease of reproduction
efficiency and abortion. The disease is caused by different species of Brucella
existing as Gram-negative coccobailary rods. Transmission to human occurs by
exposure to infected animals or by ingestion of contaminated milk or milk products.
A Brucella abortus RAPD profile was generated by 10 mer primer OBP-01 5'-
GTTCGCTCC-3'. In order to develop primers for direct-PCR amplification we cloned
and sequenced the 1.3 kb fragment generated by OPB-01 random primer (Al-Momin
et al. 1998 WJMB 14, 415-420). The fragment was cloned using the T/A cloning
method relying on the terminal extendase activity of Ultima Taq polymerase into
pCR2.1 vector and transformed in E. coli TIOP10f'. The DNA sequence was
determined using the dideoxy ribonucleotide chain termination method. From the
sequence data two primer sets were developed primer KW-1 and KW-2, which gave
a 1050 bp amplification product. A second amplification product of 700 bp was
generated when primers KW-3 and KW-4 were used. Both segments were amplified
from Brucella genomic DNA. These generated PCR-fragments were specific for
Brucella spp. As these were not able to amplify any non-Brucella organisms
associated with farm animals. We hope to use these primer sets for species-specific
detection of Brucella organisms and Brucella infection.
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Brucellosis 2003 International Research Conference