Evaluation of 3 PCR techniques for detection of brucella DNA in peripheral human blood

ABSTRACT

Brucellosis is a widespread zoonosis transmittable to humans. Currently the diagnosis of this zoonosis is based on microbiological and serological laboratory tests. PCR has been used to detect DNA from Brucella. Different target genes, primer pairs, PCR techniques and extraction procedures have been previously published for Brucella detection. But only a few of these primers have been used in human samples, and only study has been carried out to compare sensitivity between them. In the present study, 3 sets of primers and 3 different PCR protocols amplifying 3 different regions of the Brucella genome were compared for detection of Brucella DNA in a peripheral-blood PCR assay to conclude which is most suitable for the clinical diagnostic laboratory. These three pairs of primers amplify three different fragments included in i] a gene encoding a 31-kDa B. abortus antigen [B4/B5], ii] a sequence 16S rRNA of B. abortus [F4/R2], and [iii] a gene encoding an outer membrane protein [omp-2] [JPF/JPR]. Some modifications on the reported techniques were applied during the present work to improve the outcome. Results showed that B4/B5 primer pair had the highest sensitivity for detection of positive samples [98%], JPF/JPR primer pair detected 88.4% of positive samples while F4/R2 primer pair was the least sensitive being able to detect only 53.1% of positive samples. The specificity of the 3 techniques was 100%. B4/B5 primers were also able to detect the smallest number of bacteria [700 cfu/ml] while JPF/JPR were able to detect 7x10[5] cfu/ml and F4/R2 primers were able to detect 7x10[7] cfu/ml. It is thus concluded that using the B4/B5 primer PCR with the suggested modifications is a robust assay, which meets the sensitivity requirements to be used for testing of human blood samples for brucellosis in the diagnostic laboratory