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Zum Nachweis von Salmonella spp. aus Fleisch wurde eine IMS-real-time PCR-Methode etabliert und anhand des kulturellen Referenzverfahrens nach §35 LMBG durch artefiziell sowie nativ kontaminierte Fleischproben validiert. Die Verfahrensschritte der Schnellmethode schließen eine sechsstündige nicht selektive Voranreicherung, eine immunomagnetische Separation, Lysispuffer-Proteinase-K DNA-Extraktion sowie DNA-Säulenaufreinigung mit anschließender real-time PCR (Detektions-format: Hybridisierungssonden) ein. Die Nachweisgrenze für beide Methoden lag bei 10 KBE/25 g. Für die Schnellmethode ergab sich im Hinblick auf artefiziell kontaminierte Fleischproben (n = 36) eine Spezifität von 80 % (Falsch-Positiv-Rate: 20 %) sowie eine Sensitivität von 100 % (Falsch-Negativ-Rate: 0 %). Die relative Richtigkeit lag bei 94 %. Die durch den Konkordanzindex κ definierte statistische Übereinstimmung lag bei 0,85, so dass eine Übereinstimmung beider Methoden nach statistischen Kriterien gegeben ist. Für natürlich kontaminierte Fleischproben (n = 491) ergab sich für die Schnellmethode im Vergleich zur Referenzmethode eine Spezifität von 99,3 % (Falsch-Positive Rate: 0,7 %) und eine Sensitivität von 84 % (Falsch-Negativ-Rate: 16 %). Die relative Richtigkeit lag bei 98 %. Der Konkordanzindex κ zeigte mit einem Wert von 0,87 die Übereinstimmung beider Methoden nach statistischen Kriterien an. Im Vergleich zum kulturellen Verfahren, das ein Ergebnis nach 5-7 tagen liefert, errecht dies die Schnellmethode bereits nach 12-13 h. Da die IMS-real-time PCR-Methode jedoch kein lebendes Isolat zur epidemiologischen Typisierung liefert, wird die parallele Anwendung des kulturellen Verfahrens empfohlen For the detection of Salmonella spp. in meat an IMS-real-time PCR-method was established and validated with the method L 00.00-20 of the Official Collection of Methods of Analysis according to §35 of the German Law on Food and Commodities. The validation was carried out with artificially and naturally contaminated meat samples.The rapid method includes a non-selective preenrichment step for 6 hours, an immunomagnetic separation, lysis-buffer proteinase K DNA extraction as well as DNA purification followed by real-time PCR analysis (detection format: hybridisation probes). The detection limit of both methods was 10 cfu/25 g. Compared with the reference method and regarding aritificially contaminated meat samples (n = 36) the IMS-real-time PCR-method achieved a specificity of 80 % (false-positive-rate: 20 %) and a sensitivity of 100 % (false-negative-rate: 0 %). The relative accuracy was 94 %. The concordance index κ showed 0,85 what indicates that the IMS-real-time PCR-method and the cultural reference method agree on statistical criteria. Regarding naturally contaminated meat samples (n = 491) the real-time PCR showed a specificity of 99,3 % (false-positive-rate: 0,7 %) and a sensitivity of 84 % (false-negative-rate: 16,3 %). The relative accuracy was 98 %. The concordance index κ indicated with a value of 0,87 that both methods statistically agree. The cultural reference method provides a result after 5-7 days unlike the IMS-real-time PCR method, which gains a result after 12-13 h. The rapid method does not provide a viable isolate for epidemiological typing. Therefore the parallel application of the cultural reference method is recommended.

The detection of micrometastases in the bone marrow or peripheral blood of cancer patients is increasingly used for a more sensitive tumor staging and prognostication. The potential value of the currently used techniques for the detection of epithelial antigens by RT-PCR or immunohistochemistry in respect of specificity is currently controversially discussed. In the present study we demonstrate a new approach which enables the direct visualization of the tumor specific alteration of chromosome 8 in circulating tumor cells. We have therefore studied breast cancer patients with various tumor stages and tried to determine the frequency of circulating tumor cells in the peripheral blood by using interphase cytogenetics for chromosome 7 and 8. Imprints of primary breast cancers and cytospins with circulating tumor cells of corresponding patients were studied in a blinded fashion. The blood samples were generated by immunomagnetic enrichment of circulating tumor cells from peripheral blood by ferrofluid and centrifugation onto cover slips. These cytospins were then hybridized with centromer probes 7 and 8. After analyzing 27 patients with benign as well as malignant breast tumors we can demonstrate that the chromosomal pattern between malignant tumor and corresponding circulating tumor cells is identical. Furthermore, the detection of circulating tumor cells directly correlates with the primary tumor stage. We did not find any cells with chromosome 8 alterations in the patients with benign disease. Surprisingly, even in early breast cancers (T1N0) interphase cytogenetics identified circulating tumor cells in 2 out of 4 patients. In conclusion, interphase cytogenetics represent a non-invasive, sensitive and specific assay for the direct visualization of circulating tumor cells in the peripheral blood. The prognostic value of these findings remains to be further evaluated in larger prospective studies.

Coupling of immunomagnetic separation and polymerase chain reaction was used after preenrichment for the rapid detection of salmonellae in minced meat and chicken giblets. The primers used are specific to the origin of DNA replication (oriC) on the Salmonella chromosome and produce a 163 bp DNA fragment. The method allowed a screening of Salmonella free samples within 24 hours. It detected 5 serovars when inoculated in minced meat at a contamination level of 1 to 4 cfu/g. Compared with the method L 00.00-20 of the Official Collection of Methods of Analysis according to section 35 of the German Law on Food and Commodities it showed a sensitivity of 94% and a specificity of 87.5% for the examination of chicken giblets (n = 45) and a specificity of 100% for the examination of minced meat (n = 63).