A single-run, real-time PCR for detection and identification of Borrelia burgdorferi sensu lato species, based on the hbb gene sequence

• Published in: FEMS microbiology letters 2006-06, Vol.259 (1), p.35-40
• Main Authors: Portnoï, Denis, Sertour, Natacha, Ferquel, Elisabeth, Garnier, Martine, Baranton, Guy, Postic, Danièle
• Format: Article
• Language: English
• Subjects: Amplification; Animals; Bacterial Proteins - genetics; Bacterial Typing Techniques; Bacteriology; Biological and medical sciences; Borrelia burgdorferi; Borrelia burgdorferi Group - classification; Borrelia burgdorferi Group - genetics; Borrelia burgdorferi Group - isolation & purification; Borreliosis; Correlation analysis; DNA Primers; DNA, Bacterial - analysis; DNA, Bacterial - isolation & purification; DNA-Binding Proteins - genetics; Energy transfer; Epidemiology; Fluorescein; Fluorescence; Fluorescence resonance energy transfer; Fundamental and applied biological sciences. Psychology; Gene polymorphism; Genes. Genome; Genetics; Genotyping; HBB gene; Infectious diseases; Melting; Melting curve; Microbiology; Molecular and cellular biology; Molecular genetics; Mutation; Polymerase Chain Reaction - methods; Polymorphism; Real time; real‐time PCR; Reproducibility of Results; Sensitivity and Specificity; Species; Spirochetes; tick extracts; Ticks; Ticks - microbiology; Transition Temperature; Vector-borne diseases
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.2006.00249.x

Abstract Lyme borreliosis is the most important vector-borne disease caused by spirochetes within the Borrelia burgdorferi sensu lato (B. burgdorferi sl) complex. There is strong evidence that different species of this group of genetically diverse spirochetes are involved in distinct clinical manifestations of the disease. In order to differentiate species within this bacterial complex, we developed a real-time-PCR protocol, which targets the hbb gene. We designed a fluorescein-labeled probe specific of a region of this gene harboring a polymorphism linked to species. An internally Red640 labeled primer allowed a fluorescence resonance energy transfer to occur. The sensitivity of this method was in the range of 10 bacteria per assay. After amplification, a melting curve was generated for genotyping. Analysis of these melting curves clearly allowed the distinction between the main European species of B. burgdorferi sl. One hundred seventy tick extracts were analysed by this hbb-based method and in parallel by amplification of the 5S–23S intergenic spacer and RFLP analyses. There was a good correlation between these two methods. We conclude that this hbb-based real-time-PCR is suitable for epidemiological studies on field-collected ticks, although rare mutations in the genomic sequence spanned by the probe could lead to misidentification.