Author:
- Introduction
- Methods
- Source of isolates of Leptospira.
- DNA extraction.
- Amplification of the 16S ribosomal gene using real-time PCR.
- HRM curve analysis.
- Sequencing.
- Species identification by phylogenetic analysis.
- Assessing the detection limit of PCR–HRM assay.
- Assessing the ability of PCR–HRM to detect genomic DNA of Leptospira in blood.
- Ethics statement.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Adler B, de la Pena Moctezuma A, 2010. Leptospira and leptospirosis. Vet Microbiol 140: 287–296.
-
2.
Costa F, Hagan JE, Calcagno J, Kane M, Torgerson P, Martinez-Silveira MS, Stein C, Abela-Ridder B, Ko AI, 2015. Global morbidity and mortality of leptospirosis: a systematic review. PLoS Negl Trop Dis 9: e0003898.
-
3.
McBride AJ, Athanazio DA, Reis MG, Ko AI, 2005. Leptospirosis. Curr Opin Infect Dis 18: 376–386.
-
4.
Levett PN, 2001. Leptospirosis. Clin Microbiol Rev 14: 296–326.
-
5.
Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, Levett PN, Gilman RH, Willig MR, Gotuzzo E, Vinetz JM, 2003. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 3: 757–771.
-
6.
Bourhy P, Collet L, Brisse S, Picardeau M, 2014. Leptospira mayottensis sp. nov., a pathogenic species of the genus Leptospira isolated from humans. Int J Syst Evol Microbiol 64: 4061–4067.
-
7.
Brenner DJ, Kaufmann AF, Sulzer KR, Steigerwalt AG, Rogers FC, Weyant RS, 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 Bacteriol 49: 839–858.
-
8.
Hookey JV, 1990. The detection of genetic variation in Leptospira interrogans serogroup ICTEROHAEMORRHAGIAE by ribosomal RNA gene restriction fragment patterns. FEMS Microbiol Lett 60: 329–335.
-
9.
Hookey JV, Bryden J, Gatehouse L, 1993. The use of 16S rDNA sequence analysis to investigate the phylogeny of Leptospiraceae and related spirochaetes. J Gen Microbiol 139: 2585–2590.
-
10.
Galloway RL, Levett PN, 2010. Application and validation of PFGE for serovar identification of Leptospira clinical isolates. PLoS Negl Trop Dis 4: e824.
-
11.
Slack AT, Dohnt MF, Symonds ML, Smythe LD, 2005. Development of a multiple-locus variable number of tandem repeat analysis (MLVA) for Leptospira interrogans and its application to Leptospira interrogans serovar Australis isolates from Far North Queensland, Australia. Ann Clin Microbiol Antimicrob 4: 10.
-
12.
Boonsilp S, Thaipadungpanit J, Amornchai P, Wuthiekanun V, Bailey MS, Holden MT, Zhang C, Jiang X, Koizumi N, Taylor K, Galloway R, Hoffmaster AR, Craig S, Smythe LD, Hartskeerl RA, Day NP, Chantratita N, Feil EJ, Aanensen DM, Spratt BG, Peacock SJ, 2013. A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species. PLoS Negl Trop Dis 7: e1954.
-
13.
Colebrander A, Terpstra W, Hartskeerl R, 1994. Detectie van pathogene leptospiren met behulp van NASBA. Ned Tijdschr Geneeskd 138: 436.
-
14.
Lin X, Chen Y, Lu Y, Yan J, 2009. Application of a loop-mediated isothermal amplification method for the detection of pathogenic Leptospira. Diagn Microbiol Infect Dis 63: 237–242.
-
15.
Ahmed A, Martin P, Paul R, Klaster A, Rudy A, 2012. Molecular approaches in the detection and characterization of Leptospira. J Bacteriol Parasitol 3: 133.
-
16.
Wagenaar JA, Segers RP, Van der Zeijst BA, 1994. Rapid and specific detection of pathogenic Leptospira species by amplification of ribosomal sequences. Mol Biotechnol 2: 1–14.
-
17.
Woo TH, Smythe LD, Symonds ML, Norris MA, Dohnt MF, Patel BK, 1997. Rapid distinction between Leptospira interrogans and Leptospira biflexa by PCR amplification of 23S ribosomal DNA. FEMS Microbiol Lett 150: 9–18.
-
18.
Kawabata H, Dancel LA, Villanueva SY, Yanagihara Y, Koizumi N, Watanabe H, 2001. flaB-polymerase chain reaction (flaB-PCR) and its restriction fragment length polymorphism (RFLP) analysis are an efficient tool for detection and identification of Leptospira spp. Microbiol Immunol 45: 491–496.
-
19.
Slack AT, Symonds ML, Dohnt MF, Smythe LD, 2006. Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene. BMC Microbiol 6: 95.
-
20.
Reitstetter RE, 2006. Development of species-specific PCR primer sets for the detection of Leptospira. FEMS Microbiol Lett 264: 31–39.
-
21.
Palaniappan RU, Chang YF, Chang CF, Pan MJ, Yang CW, Harpending P, McDonough SP, Dubovi E, Divers T, Qu J, Roe B, 2005. Evaluation of lig-based conventional and real time PCR for the detection of pathogenic leptospires. Mol Cell Probes 19: 111–117.
-
22.
Branger C, Blanchard B, Fillonneau C, Suard I, Aviat F, Chevallier B, Andre-Fontaine G, 2005. Polymerase chain reaction assay specific for pathogenic Leptospira based on the gene hap1 encoding the hemolysis-associated protein-1. FEMS Microbiol Lett 243: 437–445.
-
23.
Chandan S, Umesha S, Bhure S, Haraprasad N, Chandrashekar S, 2011. Development of PCR assay for targeting partial lipL21 and lipL41 gene of Leptospira. Nepal J Biotechnol 1: 22–30.
-
24.
Ahmed A, Engelberts MF, Boer KR, Ahmed N, Hartskeerl RA, 2009. Development and validation of a real-time PCR for detection of pathogenic Leptospira species in clinical materials. PLoS One 4: e7093.
-
25.
Theodoridis D, Bohmer J, Homuth M, Strutzberg-Minder K, 2005. Development of a novel ELISA for serodiagnosis of leptospirosis and additional detection of pathogenic Leptospira by polymerase chain reaction for veterinary routine diagnostics. Rev Cubana Med Trop 57: 49–50.
-
26.
Merien F, Portnoi D, Bourhy P, Charavay F, Berlioz-Arthaud A, Baranton G, 2005. A rapid and quantitative method for the detection of Leptospira species in human leptospirosis. FEMS Microbiol Lett 249: 139–147.
-
27.
Smythe LD, Smith IL, Smith GA, Dohnt MF, Symonds ML, Barnett LJ, McKay DB, 2002. A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp. BMC Infect Dis 2: 13.
-
28.
Thaipadungpanit J, Chierakul W, Wuthiekanun V, Limmathurotsakul D, Amornchai P, Boonslip S, Smythe LD, Limpaiboon R, Hoffmaster AR, Day NP, Peacock SJ, 2011. Diagnostic accuracy of real-time PCR assays targeting 16S rRNA and lipL32 genes for human leptospirosis in Thailand: a case-control study. PLoS One 6: e16236.
-
29.
Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ, 2003. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49: 853–860.
-
30.
Wittwer CT, 2009. High-resolution DNA melting analysis: advancements and limitations. Hum Mutat 30: 857–859.
-
31.
Vossen RH, Aten E, Roos A, den Dunnen JT, 2009. High-resolution melting analysis (HRMA): more than just sequence variant screening. Hum Mutat 30: 860–866.
-
32.
Reed GH, Wittwer CT, 2004. Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem 50: 1748–1754.
-
33.
Tulsiani SM, Craig SB, Graham GC, Cobbold RC, Dohnt MF, Burns MA, Jansen CC, Leung LK, Field HE, Smythe LD, 2010. High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, inter-serovar convergence, and evidence of attenuation in Leptospira reference collections. Ann Trop Med Parasitol 104: 427–437.
-
34.
Tulsiani SM, Craig SB, Graham GC, Cobbold RC, Dohnt MF, Burns MA, Leung LK, Field HE, Smythe LD, 2010. High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira. Ann Trop Med Parasitol 104: 151–161.
-
35.
Naze F, Desvars A, Picardeau M, Bourhy P, Michault A, 2015. Use of a new high resolution melting method for genotyping pathogenic Leptospira spp. PLoS One 10: e0127430.
-
36.
Hall T, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41: 95–98.
-
37.
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG, 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948.
-
38.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.
-
39.
Valverde M de L, Ramirez JM, Montes de Oca LG, Goris MG, Ahmed N, Hartskeerl RA, 2008. Arenal, a new Leptospira serovar of serogroup Javanica, isolated from a patient in Costa Rica. Infect Genet Evol 8: 529–533.
-
40.
Rivera P, Ticlla M, Balda L, Gonzalez D, Cespedes M, 2012. Genetic diversity of Peruvian isolates of Leptospira spp. through pulsed field gel electrophoresis [in Spanish]. Rev Peru Med Exp Salud Publica 29: 469–476.
-
41.
Bourhy P, Herrmann Storck C, Theodose R, Olive C, Nicolas M, Hochedez P, Lamaury I, Zinini F, Bremont S, Landier A, Cassadou S, Rosine J, Picardeau M, 2013. Serovar diversity of pathogenic Leptospira circulating in the French West Indies. PLoS Negl Trop Dis 7: e2114.
-
42.
Silvio A, 2001. Isolation of Leptospira santarosai, serovar guaricura from buffaloes (Bubalus bubalis) in Vale do Ribeira, Sao Paulo, Brazil. Braz J Microbiol 32: 298–300.
-
43.
Carmona G, 2011. Deteccion de Leptospira santarosai y L. kirschneri en bovinos: nuevos aislados con potencial impacto en producción bovina y salud publica. Vet Mex 42: 277–288.
-
44.
Levett PN, Morey RE, Galloway RL, Steigerwalt AG, 2006. Leptospira broomii sp. nov., isolated from humans with leptospirosis. Int J Syst Evol Microbiol 56: 671–673.
-
45.
Szonyi B, Agudelo-Florez P, Ramirez M, Moreno N, Ko AI, 2011. An outbreak of severe leptospirosis in capuchin (Cebus) monkeys. Vet J 188: 237–239.
-
46.
Romero-Vivas CM, Cuello-Perez M, Agudelo-Florez P, Thiry D, Levett PN, Falconar AK, 2013. Cross-sectional study of Leptospira seroprevalence in humans, rats, mice, and dogs in a main tropical sea-port city. Am J Trop Med Hyg 88: 178–183.
-
47.
Romero-Vivas CM, Thiry D, Rodriguez V, Calderon A, Arrieta G, Mattar S, Cuello M, Levett PN, Falconar AK, 2013. Molecular serovar characterization of Leptospira isolates from animals and water in Colombia. Biomedica 33 (Suppl 1): 179–184.
-
48.
Slack AT, Galloway RL, Symonds ML, Dohnt MF, Smythe LD, 2009. Reclassification of Leptospira meyeri serovar Perameles to Leptospira interrogans serovar Perameles through serological and molecular analysis: evidence of a need for changes to current procedures in Leptospira taxonomy. Int J Syst Evol Microbiol 59: 1199–1203.
-
49.
Smythe L, Adler B, Hartskeerl RA, Galloway RL, Turenne CY, Levett PN, 2013. Classification of Leptospira genomospecies 1, 3, 4, and 5 as Leptospira alstonii sp. nov., Leptospira vanthielii sp. nov., Leptospira terpstrae sp. nov. and Leptospira yanagawae sp. nov., respectively. Int J Syst Evol Microbiol 63: 1859–1862.
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High-Resolution Melting Curve Analysis of the 16S Ribosomal Gene to Detect and Identify Pathogenic and Saprophytic Leptospira Species in Colombian Isolates
Ronald G. Peláez Sánchez
Ronald G. Peláez Sánchez
School of Microbiology, Group of Primary Immunodeficiencies, University of Antioquia, Medellín, Antioquia, Colombia.
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Juan Álvaro López Quintero
Juan Álvaro López Quintero
School of Microbiology, Group of Primary Immunodeficiencies, University of Antioquia, Medellín, Antioquia, Colombia.
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Martha María Pereira
Martha María Pereira
World Health Organization Collaborating Center for Leptospirosis, Oswaldo Cruz Institute/FIOCRUZ, Rio de Janeiro, Brazil.
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Piedad Agudelo-Flórez
Piedad Agudelo-Flórez
Faculty of Medicine, CES University, Medellín, Colombia.
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It is important to identify the circulating Leptospira agent to enhance the performance of serodiagnostic tests by incorporating specific antigens of native species, develop vaccines that take into account the species/serovars circulating in different regions, and optimize prevention and control strategies. The objectives of this study were to develop a polymerase chain reaction (PCR)–high-resolution melting (HRM) assay for differentiating between species of the genus Leptospira and to verify its usefulness in identifying unknown samples to species level. A set of primers from the initial region of the 16S ribosomal gene was designed to detect and differentiate the 22 species of Leptospira. Eleven reference strains were used as controls to establish the reference species and differential melting curves. Twenty-five Colombian Leptospira isolates were studied to evaluate the usefulness of the PCR–HRM assay in identifying unknown samples to species level. This identification was confirmed by sequencing and phylogenetic analysis of the 16S ribosomal gene. Eleven Leptospira species were successfully identified, except for Leptospira meyeri/Leptospira yanagawae because the sequences were 100% identical. The 25 isolates from humans, animals, and environmental water sources were identified as Leptospira santarosai (twelve), Leptospira interrogans (nine), and L. meyeri/L. yanagawae (four). The species verification was 100% concordant between PCR–HRM and phylogenetic analysis of the 16S ribosomal gene. The PCR–HRM assay designed in this study is a useful tool for identifying Leptospira species from isolates.
Author Notes
Financial support: This work was supported by COLCIENCIAS, Code: 122865740423 (to Piedad Agudelo-Flórez).
Authors' addresses: Ronald G. Peláez Sánchez and Juan Álvaro López Quintero, School of Microbiology, Group of Primary Immunodeficiencies, University of Antioquia, Medellín, Antioquia, Colombia, E-mails: latarogui@yahoo.com and alvaro.lopez@udea.edu.co. Martha María Pereira, World Health Organization Collaborating Center for Leptospirosis, Oswaldo Cruz Institute/FIOCRUZ, Rio de Janeiro, Brazil, E-mail: mpereira@ioc.fiocruz.br. Piedad Agudelo-Flórez, Faculty of Medicine, CES University, Medellín, Colombia, E-mail: pagudelo@ces.edu.co.
- Introduction
- Methods
- Source of isolates of Leptospira.
- DNA extraction.
- Amplification of the 16S ribosomal gene using real-time PCR.
- HRM curve analysis.
- Sequencing.
- Species identification by phylogenetic analysis.
- Assessing the detection limit of PCR–HRM assay.
- Assessing the ability of PCR–HRM to detect genomic DNA of Leptospira in blood.
- Ethics statement.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Adler B, de la Pena Moctezuma A, 2010. Leptospira and leptospirosis. Vet Microbiol 140: 287–296.
-
2.
Costa F, Hagan JE, Calcagno J, Kane M, Torgerson P, Martinez-Silveira MS, Stein C, Abela-Ridder B, Ko AI, 2015. Global morbidity and mortality of leptospirosis: a systematic review. PLoS Negl Trop Dis 9: e0003898.
-
3.
McBride AJ, Athanazio DA, Reis MG, Ko AI, 2005. Leptospirosis. Curr Opin Infect Dis 18: 376–386.
-
4.
Levett PN, 2001. Leptospirosis. Clin Microbiol Rev 14: 296–326.
-
5.
Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, Levett PN, Gilman RH, Willig MR, Gotuzzo E, Vinetz JM, 2003. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 3: 757–771.
-
6.
Bourhy P, Collet L, Brisse S, Picardeau M, 2014. Leptospira mayottensis sp. nov., a pathogenic species of the genus Leptospira isolated from humans. Int J Syst Evol Microbiol 64: 4061–4067.
-
7.
Brenner DJ, Kaufmann AF, Sulzer KR, Steigerwalt AG, Rogers FC, Weyant RS, 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 Bacteriol 49: 839–858.
-
8.
Hookey JV, 1990. The detection of genetic variation in Leptospira interrogans serogroup ICTEROHAEMORRHAGIAE by ribosomal RNA gene restriction fragment patterns. FEMS Microbiol Lett 60: 329–335.
-
9.
Hookey JV, Bryden J, Gatehouse L, 1993. The use of 16S rDNA sequence analysis to investigate the phylogeny of Leptospiraceae and related spirochaetes. J Gen Microbiol 139: 2585–2590.
-
10.
Galloway RL, Levett PN, 2010. Application and validation of PFGE for serovar identification of Leptospira clinical isolates. PLoS Negl Trop Dis 4: e824.
-
11.
Slack AT, Dohnt MF, Symonds ML, Smythe LD, 2005. Development of a multiple-locus variable number of tandem repeat analysis (MLVA) for Leptospira interrogans and its application to Leptospira interrogans serovar Australis isolates from Far North Queensland, Australia. Ann Clin Microbiol Antimicrob 4: 10.
-
12.
Boonsilp S, Thaipadungpanit J, Amornchai P, Wuthiekanun V, Bailey MS, Holden MT, Zhang C, Jiang X, Koizumi N, Taylor K, Galloway R, Hoffmaster AR, Craig S, Smythe LD, Hartskeerl RA, Day NP, Chantratita N, Feil EJ, Aanensen DM, Spratt BG, Peacock SJ, 2013. A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species. PLoS Negl Trop Dis 7: e1954.
-
13.
Colebrander A, Terpstra W, Hartskeerl R, 1994. Detectie van pathogene leptospiren met behulp van NASBA. Ned Tijdschr Geneeskd 138: 436.
-
14.
Lin X, Chen Y, Lu Y, Yan J, 2009. Application of a loop-mediated isothermal amplification method for the detection of pathogenic Leptospira. Diagn Microbiol Infect Dis 63: 237–242.
-
15.
Ahmed A, Martin P, Paul R, Klaster A, Rudy A, 2012. Molecular approaches in the detection and characterization of Leptospira. J Bacteriol Parasitol 3: 133.
-
16.
Wagenaar JA, Segers RP, Van der Zeijst BA, 1994. Rapid and specific detection of pathogenic Leptospira species by amplification of ribosomal sequences. Mol Biotechnol 2: 1–14.
-
17.
Woo TH, Smythe LD, Symonds ML, Norris MA, Dohnt MF, Patel BK, 1997. Rapid distinction between Leptospira interrogans and Leptospira biflexa by PCR amplification of 23S ribosomal DNA. FEMS Microbiol Lett 150: 9–18.
-
18.
Kawabata H, Dancel LA, Villanueva SY, Yanagihara Y, Koizumi N, Watanabe H, 2001. flaB-polymerase chain reaction (flaB-PCR) and its restriction fragment length polymorphism (RFLP) analysis are an efficient tool for detection and identification of Leptospira spp. Microbiol Immunol 45: 491–496.
-
19.
Slack AT, Symonds ML, Dohnt MF, Smythe LD, 2006. Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene. BMC Microbiol 6: 95.
-
20.
Reitstetter RE, 2006. Development of species-specific PCR primer sets for the detection of Leptospira. FEMS Microbiol Lett 264: 31–39.
-
21.
Palaniappan RU, Chang YF, Chang CF, Pan MJ, Yang CW, Harpending P, McDonough SP, Dubovi E, Divers T, Qu J, Roe B, 2005. Evaluation of lig-based conventional and real time PCR for the detection of pathogenic leptospires. Mol Cell Probes 19: 111–117.
-
22.
Branger C, Blanchard B, Fillonneau C, Suard I, Aviat F, Chevallier B, Andre-Fontaine G, 2005. Polymerase chain reaction assay specific for pathogenic Leptospira based on the gene hap1 encoding the hemolysis-associated protein-1. FEMS Microbiol Lett 243: 437–445.
-
23.
Chandan S, Umesha S, Bhure S, Haraprasad N, Chandrashekar S, 2011. Development of PCR assay for targeting partial lipL21 and lipL41 gene of Leptospira. Nepal J Biotechnol 1: 22–30.
-
24.
Ahmed A, Engelberts MF, Boer KR, Ahmed N, Hartskeerl RA, 2009. Development and validation of a real-time PCR for detection of pathogenic Leptospira species in clinical materials. PLoS One 4: e7093.
-
25.
Theodoridis D, Bohmer J, Homuth M, Strutzberg-Minder K, 2005. Development of a novel ELISA for serodiagnosis of leptospirosis and additional detection of pathogenic Leptospira by polymerase chain reaction for veterinary routine diagnostics. Rev Cubana Med Trop 57: 49–50.
-
26.
Merien F, Portnoi D, Bourhy P, Charavay F, Berlioz-Arthaud A, Baranton G, 2005. A rapid and quantitative method for the detection of Leptospira species in human leptospirosis. FEMS Microbiol Lett 249: 139–147.
-
27.
Smythe LD, Smith IL, Smith GA, Dohnt MF, Symonds ML, Barnett LJ, McKay DB, 2002. A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp. BMC Infect Dis 2: 13.
-
28.
Thaipadungpanit J, Chierakul W, Wuthiekanun V, Limmathurotsakul D, Amornchai P, Boonslip S, Smythe LD, Limpaiboon R, Hoffmaster AR, Day NP, Peacock SJ, 2011. Diagnostic accuracy of real-time PCR assays targeting 16S rRNA and lipL32 genes for human leptospirosis in Thailand: a case-control study. PLoS One 6: e16236.
-
29.
Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ, 2003. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49: 853–860.
-
30.
Wittwer CT, 2009. High-resolution DNA melting analysis: advancements and limitations. Hum Mutat 30: 857–859.
-
31.
Vossen RH, Aten E, Roos A, den Dunnen JT, 2009. High-resolution melting analysis (HRMA): more than just sequence variant screening. Hum Mutat 30: 860–866.
-
32.
Reed GH, Wittwer CT, 2004. Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem 50: 1748–1754.
-
33.
Tulsiani SM, Craig SB, Graham GC, Cobbold RC, Dohnt MF, Burns MA, Jansen CC, Leung LK, Field HE, Smythe LD, 2010. High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, inter-serovar convergence, and evidence of attenuation in Leptospira reference collections. Ann Trop Med Parasitol 104: 427–437.
-
34.
Tulsiani SM, Craig SB, Graham GC, Cobbold RC, Dohnt MF, Burns MA, Leung LK, Field HE, Smythe LD, 2010. High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira. Ann Trop Med Parasitol 104: 151–161.
-
35.
Naze F, Desvars A, Picardeau M, Bourhy P, Michault A, 2015. Use of a new high resolution melting method for genotyping pathogenic Leptospira spp. PLoS One 10: e0127430.
-
36.
Hall T, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41: 95–98.
-
37.
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG, 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948.
-
38.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.
-
39.
Valverde M de L, Ramirez JM, Montes de Oca LG, Goris MG, Ahmed N, Hartskeerl RA, 2008. Arenal, a new Leptospira serovar of serogroup Javanica, isolated from a patient in Costa Rica. Infect Genet Evol 8: 529–533.
-
40.
Rivera P, Ticlla M, Balda L, Gonzalez D, Cespedes M, 2012. Genetic diversity of Peruvian isolates of Leptospira spp. through pulsed field gel electrophoresis [in Spanish]. Rev Peru Med Exp Salud Publica 29: 469–476.
-
41.
Bourhy P, Herrmann Storck C, Theodose R, Olive C, Nicolas M, Hochedez P, Lamaury I, Zinini F, Bremont S, Landier A, Cassadou S, Rosine J, Picardeau M, 2013. Serovar diversity of pathogenic Leptospira circulating in the French West Indies. PLoS Negl Trop Dis 7: e2114.
-
42.
Silvio A, 2001. Isolation of Leptospira santarosai, serovar guaricura from buffaloes (Bubalus bubalis) in Vale do Ribeira, Sao Paulo, Brazil. Braz J Microbiol 32: 298–300.
-
43.
Carmona G, 2011. Deteccion de Leptospira santarosai y L. kirschneri en bovinos: nuevos aislados con potencial impacto en producción bovina y salud publica. Vet Mex 42: 277–288.
-
44.
Levett PN, Morey RE, Galloway RL, Steigerwalt AG, 2006. Leptospira broomii sp. nov., isolated from humans with leptospirosis. Int J Syst Evol Microbiol 56: 671–673.
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45.
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