Development a set of reagents for real-time PCR diagnostics and surveillance of infections caused by B. pertussis , B. parapertussis and B. bronchiseptica

Pertussis is a Vaccine-preventable disease, but the goal to reduce the incidences of whooping cough by 2010 to 1 case cases per 100,000, has not been achieved in any country. Causative agent of whooping cough is B.pertussis , but B.parapertussis , cause of about 45% of clinical whooping cough cases. Cross-immunity against B.parapertussis is absent. Our goal was to develop a set of reagents for Real-Time PCR differential identification of B.pertussis , B.parapertussis and B.bronchiseptica . The complexity is that genomes of these species are very similar. To solve this problem specific primers and probes have been chosen for detection of four targets in one tube. One of the targets is pertussis toxin (ptx) gene, which presents in genomes of B.pertussis, B.parapertussis and B.bronchiseptica. The second one is gene unique for Bordetella pertussis, the third one is gene unique for B.bronchiseptica. Fourth target is an exogenous internal control for assessment the efficacy of all stages of PCR analysis. Analytical sensitivity of developed set of reagents is 5*10 2 genomic units/ml of the sample. 51 Bordetella cultures isolated from children clinical samples with whooping cough in Russia in 1967 - 2011 and 6 Bordetella strains from animal were tested. The specificity of Bordetella species identification has been proved by sequencing of pertussis toxin gene promoter region. Preliminary results on 188 clinical samples have revealed high diagnostic efficacy of developed set of reagents.