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Nested polymerase chain reaction for detection of pathogenic leptospires

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Abstract:

Leptospirosis is a widespread zoonosis caused by pathogenic members of the genus Leptospira that has a great impact on human and veterinary public health. Early diagnosis of leptospirosis is important because severe lepto spiral infection can have a fulminant course. The available serological techniques for the diagnosis of leptospirosis have low sensitivity during the early stage of the disease. Efforts are being made to develop simpler, effective, efficient, and inexpensive diagnostic methods. In this work, we first evaluate a polymerase chain reaction (PCR) based method for diagnosis of leptospirosis. Primers were designed to amplify a 264 bp region within the lipL32 gene that is conserved among pathogenic Leptospira and absent in nonpathogenic species. The sensitivity and specificity of the assay were evaluated using 7 saprophytic serovars, 37 pathogenic serovars, and 15 other microorganisms. The method was very specific for pathogenic serovars, however, it lacked sensitivity. To enhance the sensitivity, another primer pair was designed to amplify a 183 bp region within the 264 bp region of the lipL32 gene and was used in a nested PCR assay. This approach was much more sensitive than conventional PCR.Key words: leptospirosis, diagnosis, nested PCR, lipL32.

La leptospirose est une zoonose répandue causée par les membres pathogènes du genre Leptospira qui a un impact important sur la santé publique humaine autant que vétérinaire. Un diagnostic précoce de la leptospirose est important parce qu'une infection leptospirale grave peut avoir une progression fulgurante. Les techniques sérologiques disponibles pour le diagnostic de la leptospirose démontrent une basse sensibilité lors du stade précoce de la maladie. Des efforts sont déployés pour élaborer des méthodes de diagnostic plus simples, plus efficaces et plus abordables. Nous avons d'abord évalué dans ce travail une méthode basée sur le PCR pour le diagnostic de la leptospirose. Des amorces ont été conçues afin d'amplifier une région de 264 pb à l'intérieur du gène lipL32, qui est conservé parmi les Leptospira pathogènes et absent d'espèces non pathogènes. La sensibilité et la spécificité du test ont été évaluées à l'aide de 7 sérovars saprophytes, 37 sérovars pathogènes et 15 autres micro-organismes. La méthode fut très spécifique aux sérovars pathogènes mais a toutefois démontré une sensibilité médiocre. Afin d'améliorer la sensibilité, une autre paire d'amorces fut conçue qui amplifie une région de 183 pb à l'intérieur de la région de 264 pb du gène lipL32 et fut utilisée dans un test de PCR nidifié. Cette approche était beaucoup plus sensible que le PCR conventionnel.Mots clés : leptospirose, diagnostics, PCR nidifié, lipL32.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: August 1, 2006

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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