{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T03:50:29Z","timestamp":1788580229504,"version":"build-2803163510"},"reference-count":34,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2007,5,1]],"date-time":"2007-05-01T00:00:00Z","timestamp":1177977600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2007,5]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            Botulinum toxins (BoNTs) are classically produced by\n            <jats:italic>Clostridium botulinum<\/jats:italic>\n            but rarely also from neurotoxigenic strains of\n            <jats:italic>Clostridium baratii<\/jats:italic>\n            and\n            <jats:italic>Clostridium butyricum<\/jats:italic>\n            . BoNT type A (BoNT\/A), BoNT\/B, BoNT\/E, and very rarely BoNT\/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of\n            <jats:italic>C. botulinum<\/jats:italic>\n            . The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting\n            <jats:italic>C. botulinum<\/jats:italic>\n            type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of\n            <jats:italic>C. botulinum<\/jats:italic>\n            type A, 4 strains of\n            <jats:italic>C. botulinum<\/jats:italic>\n            type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 \u00d7 10\n            <jats:sup>1<\/jats:sup>\n            copies of\n            <jats:italic>C. botulinum<\/jats:italic>\n            type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of\n            <jats:italic>C. botulinum<\/jats:italic>\n            types A and Ab (inclusivity and exclusivity, 100%).\n          <\/jats:p>","DOI":"10.1128\/aem.02234-06","type":"journal-article","created":{"date-parts":[[2007,3,17]],"date-time":"2007-03-17T01:22:02Z","timestamp":1174094522000},"page":"2891-2896","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":39,"title":["SYBR Green Real-Time PCR Method To Detect\n            <i>Clostridium botulinum<\/i>\n            Type A"],"prefix":"10.1128","volume":"73","author":[{"given":"Lucia","family":"Fenicia","sequence":"first","affiliation":[{"name":"National Reference Centre for Botulism"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabrizio","family":"Anniballi","sequence":"additional","affiliation":[{"name":"National Reference Centre for Botulism"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dario","family":"De Medici","sequence":"additional","affiliation":[{"name":"Unit of Microbiological Food-Borne Hazards, National Centre for Food Quality and Risk Assessment, Istituto Superiore di Sanita\u0300, Viale Regina Elena 299, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elisabetta","family":"Delibato","sequence":"additional","affiliation":[{"name":"Unit of Microbiological Food-Borne Hazards, National Centre for Food Quality and Risk Assessment, Istituto Superiore di Sanita\u0300, Viale Regina Elena 299, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paolo","family":"Aureli","sequence":"additional","affiliation":[{"name":"National Reference Centre for Botulism"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0167-7012(01)00305-0"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1006\/mcpr.2002.0431"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1089\/fpd.2004.1.247"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.4315\/0362-028X-65.8.1267"},{"key":"e_1_3_2_6_2","unstructured":"Microbiology of food and animal feeding stuffs. 2005"},{"key":"e_1_3_2_7_2","unstructured":"Microbiology of food and animal feeding stuffs. 2003"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.2807\/esm.04.01.00047-en"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/154.2.207"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.38.5.1753-1757.2000"},{"key":"e_1_3_2_11_2","first-page":"1899","year":"1998","unstructured":"Centers for Disease Control and Prevention. 1998. Botulism in the United States, 1899-1966. Handbook for epidemiologists, clinical and laboratory workers. Centers for Disease Control and Prevention, Atlanta, GA.","journal-title":"Handbook for epidemiologists, clinical and laboratory workers."},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.3201\/eid0403.980347"},{"key":"e_1_3_2_13_2","doi-asserted-by":"publisher","DOI":"10.1055\/s-2004-830901"},{"key":"e_1_3_2_14_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.69.6.3456-3461.2003"},{"key":"e_1_3_2_15_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.68.12.5870-5876.2002"},{"key":"e_1_3_2_16_2","doi-asserted-by":"publisher","DOI":"10.1086\/313497"},{"key":"e_1_3_2_17_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.41.11.5134-5146.2003"},{"key":"e_1_3_2_18_2","first-page":"95","volume":"41","year":"1999","unstructured":"Hall, T. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95\/98\/NT. 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