title: Development of quantitative real-time polymerase chain reaction for detection of and discrimination between Erysipelothrix rhusiopathiae and other Erysipelothrix species.
abstract: Quantitative real-time polymerase chain reaction (qPCR) assays were developed and validated in combination with enrichment culture for the detection and discrimination of Erysipelothrix rhusiopathiae and other Erysipelothrix species from tissue samples. The targets for SYBR green qPCR assays were the 16S ribosomal RNA gene for Erysipelothrix species and a gene involved in capsular formation for E. rhusiopathiae. The specificity of the assays was assessed with Erysipelothrix species and other related bacterial species. The limit of detection was found to be 5 colony-forming units per reaction. Amplification of DNA extracted from spleen and joint samples spiked with increasing quantities of Erysipelothrix cells was shown to be equally sensitive to DNA extracted from a pure bacterial culture. The assays were evaluated with 88 tissue samples from 3 experimentally infected pigs and 50 mice and with 36 tissue samples from 3 naturally infected pigs and 11 noninfected pigs. Results were compared with those of direct qPCR and conventional culture. The qPCR after enrichment increased the diagnostic sensitivity over that of culture and qPCR, thereby significantly reducing the total time taken for the detection of E. rhusiopathiae and other Erysipelothrix species. Therefore, this technique could be used for practical applications.
authors.affiliation: Nippon Institute for Biological Science, 9-2221-1 Shinmachi, Ome, Tokyo 198-0024, Japan. toho@nibs.or.jp
authors.forename: Ho
authors.initials: H
authors.lastname: To
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Koyama
authors.forename: Shinya
authors.initials: S
authors.lastname: Nagai
authors.forename: Kotaro
authors.initials: K
authors.lastname: Tuchiya
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nunoya
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000235:genetics; D004888:Erysipelothrix / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D004889:Erysipelothrix Infections / Q000175:diagnosis; D005784:Gene Amplification; D007596:Joints / Q000382:microbiology; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013154:Spleen / Q000382:microbiology; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S