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. 2015 Jul 8;10(7):e0127430.
doi: 10.1371/journal.pone.0127430. eCollection 2015.

Use of a New High Resolution Melting Method for Genotyping Pathogenic Leptospira spp

Florence Naze  1 , Amélie Desvars  1 , Mathieu Picardeau  2 , Pascale Bourhy  2 , Alain Michault  1
Affiliations

Use of a New High Resolution Melting Method for Genotyping Pathogenic Leptospira spp

Florence Naze et al. PLoS One. .

Abstract

Background: Leptospirosis is a worldwide zoonosis that is endemic in tropical areas, such as Reunion Island. The species Leptospira interrogans is the primary agent in human infections, but other pathogenic species, such as L. kirschner and L. borgpetersenii, are also associated with human leptospirosis.

Methods and findings: In this study, a melting curve analysis of the products that were amplified with the primer pairs lfb1 F/R and G1/G2 facilitated an accurate species classification of Leptospira reference strains. Next, we combined an unsupervised high resolution melting (HRM) method with a new statistical approach using primers to amplify a two variable-number tandem-repeat (VNTR) for typing at the subspecies level. The HRM analysis, which was performed with ScreenClust Software, enabled the identification of genotypes at the serovar level with high resolution power (Hunter-Gaston index 0.984). This method was also applied to Leptospira DNA from blood samples that were obtained from Reunion Island after 1998. We were able to identify a unique genotype that is identical to that of the L. interrogans serovars Copenhageni and Icterohaemorrhagiae, suggesting that this genotype is the major cause of leptospirosis on Reunion Island.

Conclusions: Our simple, rapid, and robust genotyping method enables the identification of Leptospira strains at the species and subspecies levels and supports the direct genotyping of Leptospira in biological samples without requiring cultures.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Determination of the Leptospira species and genotypes with reference strains.
Fig 2
Fig 2. The materials and methods implemented in this study for the genotyping of Leptospira at the species level, and practical applications.
Fig 3
Fig 3. Melting curve analysis of pathogenic Leptospira strains after real-time PCR amplification using the G1/G2 and LFb1F/R primer sets.
Fig 4
Fig 4. The materials and methods implemented in this study for the genotyping of Leptospira at the subspecies level, and practical applications.
Fig 5
Fig 5. Cluster plot obtained after the HRM analysis using the VNTR-4bis and VNTR-Lb5.
See this image and copyright information in PMC

References

    1. Adler B, de la Peña Moctezuma A (2010) Leptospira and leptospirosis. VetMicrobiol 140: 287–296. 10.1016/j.vetmic.2009.03.012 - DOI - PubMed
    1. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA et al. (2003) Leptospirosis: a zoonotic disease of global importance. Lancet Infectious Dis 3: 757–771. - PubMed
    1. Ko AI, Goarant C, Picardeau M (2009) Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol 7: 736–747. 10.1038/nrmicro2208 - DOI - PMC - PubMed
    1. Picardeau M (2013) Diagnosis and epidemiology of leptospirosis. Med Mal Infect 43: 1–9. 10.1016/j.medmal.2012.11.005 - DOI - PubMed
    1. Brenner DJ, Kaufmann AF, Sulzer KR, Steigerwalt AG, Rogers FC, Weyant RSl (1999) Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies. Int J Syst Microbiol 49: 839–858. - PubMed

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