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

Naze F, Desvars A, Picardeau M, Bourhy P, Michault A - PLoS ONE (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, CHU de La Reunion, Saint-Pierre, La RĂ©union, France.

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.

No MeSH data available.


Related in: MedlinePlus

Melting curve analysis of pathogenic Leptospira strains after real-time PCR amplification using the G1/G2 and LFb1F/R primer sets.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496072&req=5

pone.0127430.g003: Melting curve analysis of pathogenic Leptospira strains after real-time PCR amplification using the G1/G2 and LFb1F/R primer sets.

Mentions: Our results are shown in Table 1 and Figs 1, 2 and 3. In all the runs, the negative control, a DNA extract from an in vitro culture of L. biflexa sv. Patoc, did not show amplification with lfb1F/R nor G1/G2. Seven primers were tested on 16 strains to characterize each species, including L. interrogans, L. borgpetersenii, L kirschneri, and L. noguchii. Only the association of the primer pairs lfb1F/R and G1/G2 resulted in a species-specific Tm (except L kirschneri, which had no amplification with G1/G2) and allowed for the discrimination of the four species (p<0.001); thus, these two primer pairs were selected for further analysis.


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

Naze F, Desvars A, Picardeau M, Bourhy P, Michault A - PLoS ONE (2015)

Melting curve analysis of pathogenic Leptospira strains after real-time PCR amplification using the G1/G2 and LFb1F/R primer sets.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496072&req=5

pone.0127430.g003: Melting curve analysis of pathogenic Leptospira strains after real-time PCR amplification using the G1/G2 and LFb1F/R primer sets.
Mentions: Our results are shown in Table 1 and Figs 1, 2 and 3. In all the runs, the negative control, a DNA extract from an in vitro culture of L. biflexa sv. Patoc, did not show amplification with lfb1F/R nor G1/G2. Seven primers were tested on 16 strains to characterize each species, including L. interrogans, L. borgpetersenii, L kirschneri, and L. noguchii. Only the association of the primer pairs lfb1F/R and G1/G2 resulted in a species-specific Tm (except L kirschneri, which had no amplification with G1/G2) and allowed for the discrimination of the four species (p<0.001); thus, these two primer pairs were selected for further analysis.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, CHU de La Reunion, Saint-Pierre, La RĂ©union, France.

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.

No MeSH data available.


Related in: MedlinePlus