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J Med Microbiol 56 (2007), 1309-1316; DOI: 10.1099/jmm.0.47160-0
© 2007 Society for General Microbiology
ISSN 1473-5644

Multiple genus-specific markers in PCR assays improve the specificity and sensitivity of diagnosis of brucellosis in field animals

Falguni Mukherjee1, Jainendra Jain1, Vipul Patel1 and Mrinalini Nair2

1 Research and Development, National Dairy Development Board, Anand 388 001, Gujarat, India

2 Biotechnology Program, Department of Microbiology and Biotechnology Centre, MS University, Baroda 390002, Gujarat, India

Correspondence
Mrinalini Nair
mnair{at}msubiotech.ac.in
or
mnair_in{at}yahoo.com

Received 9 January 2007
Accepted 18 April 2007

Brucella-specific nucleotide sequences encoding the BCSP 31 kDa protein, Omp2 and the 16S rRNA were employed in three independent diagnostic PCR assays. Results of the three PCR assays on six reference strains of Brucella were in complete agreement. The results of PCR assays based on bcsp and omp2 on 19 Indian field isolates (human, bovine and murine tissues) also agreed completely. However, when the 16S rRNA gene was employed as the diagnostic target in the PCR, only 14 out of these 19 isolates and 2 out of 7 bovine milk isolates were identified as the genus Brucella. The bovine blood samples were insensitive to 16S rRNA PCR. The antibody-detecting ELISA results of field samples (n=87) from a serologically positive herd in India were compared separately with omp2 and bcsp PCRs of blood (n=62). While the bcsp PCR was the most sensitive, the degree of association of ELISA with omp2 blood PCR ({kappa}=0.37 at P <0.05) was similar to that with the bcsp blood PCR ({kappa} =0.34 at P <0.05). An improvement in the correlation between ELISA and blood PCR was noticed ({kappa} =0.5 at P <0.05) when a consensus result of omp2 and bcsp blood PCR was considered for comparison with ELISA. The use of more than one marker-based PCR gave increased sensitivity and higher specificity and appears to be a more reliable molecular diagnostic approach for screening of field animals.

Abbreviations: OPA, overall proportion of agreement.






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Copyright © 2007 Society for General Microbiology.