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

Tulsiani, S.M. and Craig, S.B. and Graham, G.C. and Cobbold, R.C. and Dohnt, M.F. and Burns, M.A. and Leung, L.K.P. and Field, H.E. and Smythe, L.D. (2010) High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira. Annals of Tropical Medicine and Parasitology, 104 (2). pp. 151-161.

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Article Link(s): http://dx.doi.org/10.1179/136485910X12607012374037

Publisher URL: http://www.maney.co.uk

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 potentia.

Item Type:Article
Corporate Creators:QPIF
Additional Information:© The Liverpool School of Tropical Medicine
Keywords:Polymerase chain reaction; arbitrarily primed PCR; real-time PCR; identification; differentiation; serovars; region; SPP.; genotypes; DNA markers.
Subjects:Science > Science (General)
Science > Biology > Genetics
Science > Microbiology > Bacteria
Deposited On:24 Jun 2010 05:07
Last Modified:07 Jun 2011 23:05

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