IDENTIFICATION OF LACTIC-ACID BACTERIA USING TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS FOR DNA FRAGMENTS AMPLIFIED BY POLYMERASE CHAIN-REACTION

A simple and rapid identification method for microorganisms was developed. The procedure involves temperature gradient gel electrophoretic analysis (TGGE) of DNA fragments amplified by polymerase chain reaction (PCR). Using consensus primers, 5S rRNA genes of nine species of lactic acid bacteria were amplified by PCR and analyzed by TGGE. The mobility of each fragment of the nine species of lactic acid bacteria was different. For a more sensitive analysis, we investigated whether or not the heteroduplexes were produced when the PCR-amplified fragments of two species were heat-denatured and annealed together. When a sample of the two same species was analyzed by TGGE, only a homoduplex band was observed, In addition to the homoduplexes, heteroduplexes were produced by samples of two different species. Thus, the identification of lactic acid bacteria is possible by investigating whether or not the heteroduplexes are produced or by the heteroduplexes' patterns. This method requires only about 14 hr for identification and makes it more possible to detect a larger fraction of all possible base changes in a DNA fragment than does the restriction fragment length polymorphism method and the arbitrary primer PCR without full sequence analysis method.