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Denaturing HPLC for identifying bacteria.

William Hurtle·David Shoemaker·Erik Henchal·David Norwood
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Abstract

Denaturing HPLC (DHPLC) is used in a wide variety of genetic applications. Here we introduce a new application for this technique, the identification of bacteria. We combined the capability of DHPLC to detect sequence variation with the principles of rRNA genotyping analysis to develop a high-throughput method of identifying microorganisms. Thirty-nine bacterial species from a broad spectrum of genera were tested to determine if DHPLC could be usedfor identification. Most (36 of 39) species of bacteria had a unique peak profile that could be used as a molecular fingerprint. Furthermore, a blind panel of 65 different bacterial isolates was analyzed to demonstrate the diagnostic capability of this method to specifically identify Yersinia pestis and Bacillus anthracis. All the Y. pestis samples (10 of 10) and the majority of B. anthracis samples (12 of 14) were correctly identified. The procedure had an overall specificity of 100%, overall sensitivity of 91.7%, and a predictive value of 96.9%. The data suggest that DHPLC of products spanning regions of genetic variability will be a useful application for bacterial identification.

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BacteriaBase SequenceChromatography, High Pressure LiquidDNA FingerprintingDNA Mutational AnalysisDNA, BacterialFalse Negative ReactionsFalse Positive ReactionsMolecular Sequence DataMutationNucleic Acid DenaturationNucleic Acid HybridizationPolymerase Chain ReactionReproducibility of ResultsSensitivity and SpecificitySpecies SpecificityEvaluation StudyJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.

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William Hurtle, David Shoemaker, Erik Henchal, David Norwood. 2002. Denaturing HPLC for identifying bacteria.. https://doi.org/10.2144/02332rr05

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