High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira

S M Tulsiani; Scott B Craig; Glenn C Graham; R C Cobbold; M F Dohnt; M A Burns; L K-P Leung; H E Field; Lee D Smythe

doi:10.1179/136485910X12607012374037

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High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira

Journal article

Peer reviewed

High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira

S M Tulsiani, Scott B Craig, Glenn C Graham, R C Cobbold, M F Dohnt, M A Burns, L K-P Leung, H E Field and Lee D Smythe

Annals of Tropical Medicine and Parasitology, Vol.104(2), pp.151-161

2010

DOI: https://doi.org/10.1179/136485910X12607012374037

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Abstract

Leptospira

high-resolution melt analysis

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.

Details

Title: High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira
Authors: S M Tulsiani (Author) - University of Queensland
Scott B Craig (Author) - University of the Sunshine Coast - Faculty of Science, Health and Education
Glenn C Graham (Author) - University of the Sunshine Coast - Faculty of Science, Health and Education
R C Cobbold (Author) - University of Queensland
M F Dohnt (Author) - Queensland Health Forensic and Scientific Services
M A Burns (Author) - Queensland Health Forensic and Scientific Services
L K-P Leung (Author) - University of Queensland
H E Field (Author) - University of Queensland
Lee D Smythe (Author) - Queensland Health Forensic and Scientific Services
Publication details: Annals of Tropical Medicine and Parasitology, Vol.104(2), pp.151-161
Publisher: Maney Publishing
Date published: 2010
DOI: 10.1179/136485910X12607012374037
ISSN: 0003-4983
Organisation Unit: School of Science and Engineering - Legacy; University of the Sunshine Coast, Queensland
Language: English
Record Identifier: 99449456202621
Output Type: Journal article

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Collaboration types: Domestic collaboration
Web Of Science research areas: Parasitology; Public, Environmental & Occupational Health; Tropical Medicine

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