ANALYSIS OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC PROFILES OF THE 16S rRNA GENE TO IDENTIFY GENERA OF BACTERIA

Abstract

Denaturing high performance liquid chromatography (DHPLC) is used in a wide variety of genetic applications. This report introduces a new application for this technique, the identification of bacteria. We combined the capability of DHPLC to detect sequence variation and the principles of rRNA genotyping analysis to develop a high throughput method of identifying microorganisms. Forty-six bacterial species from a broad spectrum of genera were tested to determine if DHPLC could be used for identification. Thirty-six of the 46 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 capability of this method to specifically identify Yersinia pestis and Bacillus anthracis. Ten of 10 Y. pestis samples and 12 of 14 B. anthracis 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 amplicons spanning regions of genetic variability will be a useful application for bacterial identification.