Authors: Tulsiani, S. M.¹; Craig, S. B.²; Graham, G. C.³; Cobbold, R. C.⁴; Dohnt, M. F.⁵; Burns, M.-A.⁵; Leung, L. K.-P.⁶; Field, H. E.⁷; Smythe, L. D.⁵

Source: Annals of Tropical Medicine and Parasitology, Volume 104, Number 2, March 2010 , pp. 151-161(11)

Publisher: Maney Publishing

Abstract:

A new test for pathogenic Leptospira isolates, based on RAPD–PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used to define serovars or detect genotypes. Ten serovars, of leptospires from the species Leptospira interrogans (serovars Australis, Robinsoni, Hardjo, Pomona, Zanoni, Copenhageni and Szwajizak), L. borgpetersenii (serovar Arborea), L. kirschneri (serovar Cynopteri) and L. weilii (serovar Celledoni), were typed against 13 previously published RAPD primers, using a real-time cycler (the Corbett Life Science RotorGene 6000) and the optimised reagents from a commercial kit (Quantace SensiMix). RAPD–HRM at specific temperatures generated defining amplicon melt profiles for each of the tested serovars. These profiles were evaluated as difference-curve graphs generated using the RotorGene software package, with a cut-off of at least 8 'U' (plus or minus). The results demonstrated that RAPD–HRM can be used to measure serovar diversity and establish identity, with a high degree of stability. The characterisation of Leptospira serotypes using a DNA-based methodology is now possible. As an objective and relatively inexpensive and rapid method of serovar identification, at least for cultured isolates, RAPD–HRM assays show convincing potential.

Document Type: Research Article

DOI: http://dx.doi.org/10.1179/136485910X12607012374037

Affiliations: 1: The University of Queensland, School of Veterinary Science, St Lucia, Queensland, 4072, Australia; WHO/OIE/FAO Collaborating Centre for Reference and Research on Leptospirosis, Communicable Diseases Unit, Queensland Health Forensic and Scientific Service, P.O. Box 594, Archerfield, Queensland, 4108, Australia; Australian Bio-security Cooperative Research Centre, The University of Queensland, St. Lucia, Queensland, 4072, Australia 2: WHO/OIE/FAO Collaborating Centre for Reference and Research on Leptospirosis, Communicable Diseases Unit, Queensland Health Forensic and Scientific Service, P.O. Box 594, Archerfield, Queensland, 4108, Australia; Faculty of Science, Health and Education, University of the Sunshine Coast, Sippy Downs Drive, Sippy Downs, Queensland, 4556, Australia 3: Faculty of Science, Health and Education, University of the Sunshine Coast, Sippy Downs Drive, Sippy Downs, Queensland, 4556, Australia; Organic Chemistry, Food Chemistry, Queensland Health Forensic and Scientific Services, P.O. Box 594, Archerfield, Queensland, 4108, Australia 4: The University of Queensland, School of Veterinary Science, St Lucia, Queensland, 4072, Australia 5: WHO/OIE/FAO Collaborating Centre for Reference and Research on Leptospirosis, Communicable Diseases Unit, Queensland Health Forensic and Scientific Service, P.O. Box 594, Archerfield, Queensland, 4108, Australia 6: The University of Queensland, School of Animal Studies, Gatton, Queensland, 4343, Australia 7: Animal Research Institute, Department of Primary Industries and Fisheries, Yeerongpilly, Queensland, 4105, Australia

Publication date: 2010-03-01

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• In 2012 Annals of Tropical Medicine and Parasitology changed its name to Pathogens and Global Health to reflect changes and developments in the subject area. View the issues of Pathogens and Global Health available online..
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