United States of America, as Represented by the Secretary of Agriculture

National Agriculture and Food Research Organization

Prima Meat Packers, Ltd.

Susumu Kawasaki

Shinichi Kawamoto

Pina M Fratamico

Kazuko Takeshita

Naoko Horikoshi

Takashi Sameshima

Yukio Okada

Kenji Isshiki

The phylogeny of twelvespecies was determined based on partial (1020-bp) gyrB gene sequences. Methods have been described for detection and speciation of, including 16S rRNA sequence analysis. However, gyrB provides a better resolution than the 16S rDNA gene forspecies with interspecies sequence similarities ranging from 58.3 to 89.2% compared to those reported for the 16S rRNA gene (ranging from 89 to 99%). A universal primer set, designed to amplify a 960-bp fragment of the gyrB gene inspp., was developed and used for (PCR-RFLP) of 19 strains representing twelvespecies and resulted in unique digest patterns for all twelvespecies. PCR assays for amplification of regions of the gyrB gene specific for eachspecies were also developed. Using these PCR and PCR-RFLP methods results in unambiguous identification of the majority ofspecies.

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The phylogeny of twelvespecies was determined based on partial (1020-bp) gyrB gene sequences. Methods have been described for detection and speciation of, including 16S rRNA sequence analysis. However, gyrB provides a better resolution than the 16S rDNA gene forspecies with interspecies sequence similarities ranging from 58.3 to 89.2% compared to those reported for the 16S rRNA gene (ranging from 89 to 99%). A universal primer set, designed to amplify a 900-bp fragment of the gyrB gene inspp., was developed and used for PCR-RFLP of 19 strains representing twelvespecies and resulted in unique digest patterns for all twelvespecies PCR assays for amplification of regions of the gyrB gene specific for eachspecies were also developed. Using these PCR and PCR-RFLP methods results in unambiguous identification of the majority ofspecies.

The present invention is to provide a multiple detection method that can detect contaminating microorganisms existing in foods, including pathogenicO157,andspp., with high sensitivity comparable or even superior to official methods, comprising the steps of amplifying a plural number of target genes with a single PCR reaction tube and analyzing the same. The following steps are performed consecutively: (A) a step of extracting DNA of the target microorganisms to be detected by treating with at least a lytic enzyme such as Achromopepidase and Lysozyme and/or bacteriocin having lytic activity such as Enterolysine, a surfactant and a protein denaturing agent; and (B) a step of mixing a specific primer to the target microorganisms to be detected to perform multiplex PCR. Further, it is preferable to add a step of culturing with a culture condition where 1 CFU/100 g microorganisms becomes 10.sup.3 CFU/ml or more after 18 to 48 h of culture, for example that the pH after culture becomes 5.1 or more, before the step of extracting DNA of the target microorganisms to be detected.

Method of multiplex microorganism detection

Growing community of inventors

Tsukuba, Japan

Susumu Kawasaki