Improved Diagnosis of Brucella in human serum samples by double PCR assay

Brucellae are gram-negative intracellular pathogens which cause zoonotic disease in humans. Clinical manifestations of brucellosis in human are variable and often are non-specific,and the diagnosis requires fast and accurate confirmation. Since the use of serum instead of whole-blood samples offers several advantages for nucleic acid amplification methods, in this study we developed an improved PCR assay for the rapid and specific laboratory diagnosis of human brucellosis directly from serum specimens. DNA was extracted from 100 μL of serum from 30 patients with acute serologic brucellosis. The PCR reaction was carried out with Specific primers. Second PCR reaction for reamplification of the first reaction products was designed. a 223 bp conserved region on the sequence encoding the 31-KDa immunogenic outer membrane protein which is specific to the genus Brucella (BCSP31) and present in all its biovars was amplified in all serum samples. For confirmation and efficient amplification of the specific target, reamplification of the first PCR products had a sharper banding patterns with high sensitivity and specificity that might be considered as a new useful method for diagnosis of human brucellosis in serum specimens.