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Development of a novel genus-specific real-time PCR assay for detection and differentiation of Bartonella species and genotypes.

ABSTRACT: The genus Bartonella includes numerous species with varied host associations, including several that infect humans. Development of a molecular diagnostic method capable of detecting the diverse repertoire of Bartonella species while maintaining genus specificity has been a challenge. We developed a novel real-time PCR assay targeting a 301-bp region of the ssrA gene of Bartonella and demonstrated specific amplification in over 30 Bartonella species, subspecies, and strains. Subsequent analysis of ssrA sequences was sufficient to discriminate Bartonella species and provided phylogenetic data consistent with that of gltA, a commonly used gene for differentiating Bartonella genotypes. Using this assay, we identified Bartonella DNA in 29% and 47% of blood specimens from elk in Wyoming and cattle in the Republic of Georgia, respectively. Sequence analysis of a subset of genotypes from elk specimens revealed a cluster most closely related to Bartonella capreoli, and genotypes from cattle were identified as Bartonella bovis, both Bartonella species commonly found in wild and domestic ruminants. Considering the widespread geographic distribution and infectivity potential to a variety of hosts, this assay may be an effective diagnostic method for identification of Bartonella infections in humans and have utility in Bartonella surveillance studies.

SUBMITTER: Diaz MH

PROVIDER: S-EPMC3347110 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Development of a novel genus-specific real-time PCR assay for detection and differentiation of Bartonella species and genotypes.

Diaz Maureen H MH Bai Ying Y Malania Lile L Winchell Jonas M JM Kosoy Michael Y MY

Journal of clinical microbiology 20120229 5

The genus Bartonella includes numerous species with varied host associations, including several that infect humans. Development of a molecular diagnostic method capable of detecting the diverse repertoire of Bartonella species while maintaining genus specificity has been a challenge. We developed a novel real-time PCR assay targeting a 301-bp region of the ssrA gene of Bartonella and demonstrated specific amplification in over 30 Bartonella species, subspecies, and strains. Subsequent analysis o ...[more]

PMID: 22378904

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Project description:BACKGROUND: A simple real-time PCR assay using one set of primer and probe for rapid, sensitive and quantitative detection of Plasmodium species, with simultaneous differentiation of Plasmodium falciparum from the three other Plasmodium species (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) in febrile returning travellers and migrants was developed and evaluated. METHODS: Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be perfect matches to the 18S rRNA gene of the fourth Plasmodium species, while the acceptor probe sequence was designed for P. falciparum over a region containing one mismatched, which allowed differentiation of the three other Plasmodium species. The performance characteristics of the real-time PCR assay were compared with those of conventional PCR and microscopy-based diagnosis from 119 individuals with a suspected clinical diagnostic of imported malaria. RESULTS: Blood samples with parasite densities less than 0.01% were all detected, and analytical sensitivity was 0.5 parasite per PCR reaction. The melt curve means Tms (standard deviation) in clinical isolates were 60.5 degrees C (0.6 degrees C) for P. falciparum infection and 64.6 degrees C (1.8 degrees C) for non-P. falciparum species. These Tms values of the P. falciparum or non-P. falciparum species did not vary with the geographic origin of the parasite. The real-time PCR results correlated with conventional PCR using both genus-specific (Kappa coefficient: 0.95, 95% confidence interval: 0.9 - 1) or P. falciparum-specific (0.91, 0.8 - 1) primers, or with the microscopy results (0.70, 0.6 - 0.8). The real-time assay was 100% sensitive and specific for differentiation of P. falciparum to non-P. falciparum species, compared with conventional PCR or microscopy. The real-time PCR assay can also detect individuals with mixed infections (P. falciparum and non-P. falciparum sp.) in the same sample. CONCLUSION: This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of P. falciparum to other Plasmodium species. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

Dataset Information

Development of a novel genus-specific real-time PCR assay for detection and differentiation of Bartonella species and genotypes.

Publications

Development of a novel genus-specific real-time PCR assay for detection and differentiation of Bartonella species and genotypes.

Similar Datasets

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