TY - JOUR KW - multiplex PCR KW - real-time PCR KW - swine KW - faeces KW - Brachyspira KW - Lawsonia AU - H Willems AU - G Reiner AB - A multiplex real-time PCR assay was developed to detect and quantify B. hyodysenteriae, B. pilosicoli, and L. intracellularis in pig faeces. Specific probes and primers were directed against the NADH oxidase (nox) gene of Brachyspira and the aspartate ... BT - Berliner und Münchener Tierärztliche Wochenschrift C1 - {"oldId":69648,"title":"A multiplex real-time PCR for the simultaneous detection and quantitation of Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis in pig faeces","teaserText":"A multiplex real-time PCR assay was developed to detect and quantify B. hyodysenteriae, B. pilosicoli, and L. intracellularis in pig faeces. Specific probes and primers were directed against the NADH oxidase (nox) gene of Brachyspira and the aspartate ...","content":"

Summary<\/span>
A multiplex real-time PCR assay was developed to detect and quantify B. hyodysenteriae, B. pilosicoli, <\/span>and L. intracellularis<\/span> in pig faeces. Specific probes and primers were directed against the NADH oxidase (nox) gene of Brachyspira and the aspartate ammonia lyase (aspA) gene of L. intracellularis<\/span>, respectively. The analytical sensitivity for the real-time PCR assay, expressed as limit of detection (LOD) was below 10 DNA copies for L. intracellularis<\/span>, 14 DNA copies for B. pilosicoli<\/span> and 26 DNA copies per PCR reaction for B. hyodysenteriae. The experimental sensitivity, expressed as limit of quantitation (LOQ) for the real-time PCR assay was set to 100 DNA copies per PCR reaction which equals 8 x 103 cells per gram of faeces. The multiplex real-time PCR was tested in parallel to conventional PCR on 749 faecal samples from 121 farms. 73 (9.7%), 30 (4%), and 30 (4%) faecal samples were positive for L. intracellularis, B. hyodysenteriae,<\/span> and B. pilosicoli<\/span>, respectively by conventional PCR and 59 (7.9%), 27 (3.6%), and 7 (0.9%) by multiplex real-time PCR. From the real-time PCR positive results 34 (4.5%), 25 (3.3%), and 4 (0.5%) were above the LOQ. The multiplex real-time PCR will allow rapid and quantitative detection of clinical relevant amounts of the three key porcine enteric pathogens simultaneously.

Keywords:<\/span>
multiplex PCR, real-time PCR, swine, faeces, Brachyspira, Lawsonia


Zusammenfassung<\/span>
Der Artikel beschreibt eine Multiplex Realtime-PCR zum Nachweis und zur Quantifizierung von B. hyodysenteriae, B. pilosicoli<\/span> und L. intracellularis<\/span> in Schweinekot. Primer und Sonden richten sich gegen das NADH-Oxidase-Gen (nox) von Brachyspira und die Aspartat-Ammonium-Lyase (aspA) von Lawsonia intracellularis. Die analytische Sensitivit\u00e4t der PCR, ausgedr\u00fcckt als LOD (limit of detection) lag je Ansatz unter 10 DNA-Kopien f\u00fcr L. intracellularis<\/span>, 14 DNA-Kopien f\u00fcr B. pilosicoli<\/span> und 26 DNA-Kopien f\u00fcr B. hyodysenteriae<\/span>. Die experimentelle Sensitivit\u00e4t zur Quantifizierung (LOQ; limit of quantitation) ergab sich als 100 DNA-Kopien je PCRAnsatz. Dies entspricht einem Grenzwert von 8 x 103 Zellen pro Gramm Kot. Die Multiplex Realtime PCR wurde auf Basis von 749 Kotproben aus 121 nicht n\u00e4her definierten Betrieben mit einer konventionellen PCR verglichen. In der konventionellen PCR waren jeweils 73 (9,7 %), 30 (4 %), und 30 (4 %) der Kotproben positiv f\u00fcr L. intracellularis, B. hyodysenteriae<\/span> und B. pilosicoli<\/span>. Jeweils 59 (7,9 %), 27 (3,6 %) und 7 (0,9 %) der Kotproben waren positiv mittels Multiplex Realtime PCR. Von den Realtime PCR positiven Proben lagen 34 (4,5 %), 25 (3,3 %) und 4 (0,5 %) innerhalb des LOQ. Die Multiplex Realtime PCR erm\u00f6glicht den raschen, synchronen und quantitativen Nachweis wichtiger enteropathogener Bakterien.

Schl\u00fcsselw\u00f6rter:<\/span>
Multiplex PCR, Realtime PCR, Schwein, Kotproben, Brachyspira, Lawsonia <\/p>","categories":["Tier\u00e4rztliche Wochenschrift","Abostufe BMTW","Fachartikel"],"fromDate":"May 10, 2010 12:00:00 AM","toDate":"Dec 31, 2050 12:00:00 AM","oldUrls":["http:\/\/vetline.de\/multiplex-pcr-real-time-pcr-swine-faeces-brachyspira-lawsonia\/150\/3130\/69648"],"doiLanguage":"englisch","doiProductFormat":"Online","doiPublisher":"Schl\u00fctersche Verlagsgesellschaft mbH & Co. KG","doiSerialWorkTitle":"Berl. M\u00fcnch. Tier\u00e4rztl. Wschr.","doiDocumentUri":"http:\/\/www.vetline.de\/multiplex-pcr-real-time-pcr-swine-faeces-brachyspira-lawsonia\/150\/3130\/69648","doiSource":"Berl. M\u00fcnch. Tier\u00e4rztl. Wschr. 123: 5-6, 205-209 (2010)","doiissn":"0005-9366","doiNr":"10.2376\/0005-9366-123-205","doiFirstPage":"205","doiLastPage":"209","doiTransmitted":true,"doiAuthor":"Willems H, Reiner G","pdf":{"path":"http:\/\/data\/bmtw_2010_05_0205.pdf","title":"bmtw_2010_05_0205.pdf","description":"A multiplex real-time PCR for the simultaneous detection and quantitation of Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis in pig faeces

"},"authors":[{"firstName":"H","middleName":"","lastName":"Willems"},{"firstName":"G","middleName":"","lastName":"Reiner"}],"contentOptimised":"

Summary<\/strong>
A multiplex real-time PCR assay was developed to detect and quantify B. hyodysenteriae, B. pilosicoli, <\/strong>and L. intracellularis<\/strong> in pig faeces. Specific probes and primers were directed against the NADH oxidase (nox) gene of Brachyspira and the aspartate ammonia lyase (aspA) gene of L. intracellularis<\/strong>, respectively. The analytical sensitivity for the real-time PCR assay, expressed as limit of detection (LOD) was below 10 DNA copies for L. intracellularis<\/strong>, 14 DNA copies for B. pilosicoli<\/strong> and 26 DNA copies per PCR reaction for B. hyodysenteriae. The experimental sensitivity, expressed as limit of quantitation (LOQ) for the real-time PCR assay was set to 100 DNA copies per PCR reaction which equals 8 x 103 cells per gram of faeces. The multiplex real-time PCR was tested in parallel to conventional PCR on 749 faecal samples from 121 farms. 73 (9.7%), 30 (4%), and 30 (4%) faecal samples were positive for L. intracellularis, B. hyodysenteriae,<\/strong> and B. pilosicoli<\/strong>, respectively by conventional PCR and 59 (7.9%), 27 (3.6%), and 7 (0.9%) by multiplex real-time PCR. From the real-time PCR positive results 34 (4.5%), 25 (3.3%), and 4 (0.5%) were above the LOQ. The multiplex real-time PCR will allow rapid and quantitative detection of clinical relevant amounts of the three key porcine enteric pathogens simultaneously.

Keywords:<\/strong>
multiplex PCR, real-time PCR, swine, faeces, Brachyspira, Lawsonia


Zusammenfassung<\/strong>
Der Artikel beschreibt eine Multiplex Realtime-PCR zum Nachweis und zur Quantifizierung von B. hyodysenteriae, B. pilosicoli<\/strong> und L. intracellularis<\/strong> in Schweinekot. Primer und Sonden richten sich gegen das NADH-Oxidase-Gen (nox) von Brachyspira und die Aspartat-Ammonium-Lyase (aspA) von Lawsonia intracellularis. Die analytische Sensitivit\u00e4t der PCR, ausgedr\u00fcckt als LOD (limit of detection) lag je Ansatz unter 10 DNA-Kopien f\u00fcr L. intracellularis<\/strong>, 14 DNA-Kopien f\u00fcr B. pilosicoli<\/strong> und 26 DNA-Kopien f\u00fcr B. hyodysenteriae<\/strong>. Die experimentelle Sensitivit\u00e4t zur Quantifizierung (LOQ; limit of quantitation) ergab sich als 100 DNA-Kopien je PCRAnsatz. Dies entspricht einem Grenzwert von 8 x 103 Zellen pro Gramm Kot. Die Multiplex Realtime PCR wurde auf Basis von 749 Kotproben aus 121 nicht n\u00e4her definierten Betrieben mit einer konventionellen PCR verglichen. In der konventionellen PCR waren jeweils 73 (9,7 %), 30 (4 %), und 30 (4 %) der Kotproben positiv f\u00fcr L. intracellularis, B. hyodysenteriae<\/strong> und B. pilosicoli<\/strong>. Jeweils 59 (7,9 %), 27 (3,6 %) und 7 (0,9 %) der Kotproben waren positiv mittels Multiplex Realtime PCR. Von den Realtime PCR positiven Proben lagen 34 (4,5 %), 25 (3,3 %) und 4 (0,5 %) innerhalb des LOQ. Die Multiplex Realtime PCR erm\u00f6glicht den raschen, synchronen und quantitativen Nachweis wichtiger enteropathogener Bakterien.

Schl\u00fcsselw\u00f6rter:<\/strong>
Multiplex PCR, Realtime PCR, Schwein, Kotproben, Brachyspira, Lawsonia <\/p>","primaryLanguage":"englisch","summary":"A multiplex real-time PCR assay was developed to detect and quantify B. hyodysenteriae, B. pilosicoli, <\/strong>and L. intracellularis<\/strong> in pig faeces. Specific probes and primers were directed against the NADH oxidase (nox) gene of Brachyspira and the aspartate ammonia lyase (aspA) gene of L. intracellularis<\/strong>, respectively. The analytical sensitivity for the real-time PCR assay, expressed as limit of detection (LOD) was below 10 DNA copies for L. intracellularis<\/strong>, 14 DNA copies for B. pilosicoli<\/strong> and 26 DNA copies per PCR reaction for B. hyodysenteriae. The experimental sensitivity, expressed as limit of quantitation (LOQ) for the real-time PCR assay was set to 100 DNA copies per PCR reaction which equals 8 x 103 cells per gram of faeces. The multiplex real-time PCR was tested in parallel to conventional PCR on 749 faecal samples from 121 farms. 73 (9.7%), 30 (4%), and 30 (4%) faecal samples were positive for L. intracellularis, B. hyodysenteriae,<\/strong> and B. pilosicoli<\/strong>, respectively by conventional PCR and 59 (7.9%), 27 (3.6%), and 7 (0.9%) by multiplex real-time PCR. From the real-time PCR positive results 34 (4.5%), 25 (3.3%), and 4 (0.5%) were above the LOQ. The multiplex real-time PCR will allow rapid and quantitative detection of clinical relevant amounts of the three key porcine enteric pathogens simultaneously.","keywords":["multiplex PCR","real-time PCR","swine","faeces","Brachyspira","Lawsonia"],"zusammenfassung":"Der Artikel beschreibt eine Multiplex Realtime-PCR zum Nachweis und zur Quantifizierung von B. hyodysenteriae, B. pilosicoli<\/strong> und L. intracellularis<\/strong> in Schweinekot. Primer und Sonden richten sich gegen das NADH-Oxidase-Gen (nox) von Brachyspira und die Aspartat-Ammonium-Lyase (aspA) von Lawsonia intracellularis. Die analytische Sensitivit\u00e4t der PCR, ausgedr\u00fcckt als LOD (limit of detection) lag je Ansatz unter 10 DNA-Kopien f\u00fcr L. intracellularis<\/strong>, 14 DNA-Kopien f\u00fcr B. pilosicoli<\/strong> und 26 DNA-Kopien f\u00fcr B. hyodysenteriae<\/strong>. Die experimentelle Sensitivit\u00e4t zur Quantifizierung (LOQ; limit of quantitation) ergab sich als 100 DNA-Kopien je PCRAnsatz. Dies entspricht einem Grenzwert von 8 x 103 Zellen pro Gramm Kot. Die Multiplex Realtime PCR wurde auf Basis von 749 Kotproben aus 121 nicht n\u00e4her definierten Betrieben mit einer konventionellen PCR verglichen. In der konventionellen PCR waren jeweils 73 (9,7 %), 30 (4 %), und 30 (4 %) der Kotproben positiv f\u00fcr L. intracellularis, B. hyodysenteriae<\/strong> und B. pilosicoli<\/strong>. Jeweils 59 (7,9 %), 27 (3,6 %) und 7 (0,9 %) der Kotproben waren positiv mittels Multiplex Realtime PCR. Von den Realtime PCR positiven Proben lagen 34 (4,5 %), 25 (3,3 %) und 4 (0,5 %) innerhalb des LOQ. Die Multiplex Realtime PCR erm\u00f6glicht den raschen, synchronen und quantitativen Nachweis wichtiger enteropathogener Bakterien.","schluesselwoerter":["Multiplex PCR","Realtime PCR","Schwein","Kotproben","Brachyspira","Lawsonia"],"translatedTitle":"","abstractE":"A multiplex real-time PCR assay was developed to detect and quantify B. hyodysenteriae, B. pilosicoli, and L. intracellularis in pig faeces. Specific probes and primers were directed against the NADH oxidase (nox) gene of Brachyspira and the aspartate ...","date":{"year":2010,"date":"05\/2010","accepted":"2010-05-10"},"volume":"123","openAccess":false,"journal":"Berliner und M\u00fcnchener Tier\u00e4rztliche Wochenschrift","titleImageId":944,"pages":"205-209","redirects":["multiplex-pcr-real-time-pcr-swine-faeces-brachyspira-lawsonia\/150\/3130\/69648"],"tierartCategories":[],"artikelartCategories":["Tier\u00e4rztliche Wochenschrift","Abostufe BMTW","Fachartikel"]} CY - Hannover DA - 05/2010 DO - 10.2376/0005-9366-123-205 LA - English N2 - A multiplex real-time PCR assay was developed to detect and quantify B. hyodysenteriae, B. pilosicoli, and L. intracellularis in pig faeces. Specific probes and primers were directed against the NADH oxidase (nox) gene of Brachyspira and the aspartate ... PB - Schlütersche Verlagsgesellschaft mbH & Co. KG PP - Hannover PY - 2010 SP - 205 EP - 209 T1 - A multiplex real-time PCR for the simultaneous detection and quantitation of Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis in pig faeces T2 - Berliner und Münchener Tierärztliche Wochenschrift TI - A multiplex real-time PCR for the simultaneous detection and quantitation of Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis in pig faeces VL - 123 SN - 0005-9366 ER -