A real-time PCR for Detection of Pathogenic Yersinia enterocolitica in food combined with an Universal Internal Amplification Control System

Abstract

Abbreviations: bp = base pairs; CIN agar = cefsulodin irgasan novobiocin agar; ITC = irgasan ticarcillin sodim chlorate broth; PCR = polymerase chain reaction; PSB = peptone sorbit bile broth; PW = peptone water; SSCD agar = Salmonella Shigella agar with sodium deoxycholate and calcium chloride; TSB = tryptic soya broth.Summary: A real-time PCR system with an internal amplification control was developed for detection of pathogenic Yersinia) enterocolitica in food samples. The chromosomally encoded ail gene was chosen as PCR target. Sequences of plasmid pUC19 served as target for the internal amplification control. The method was validated in combination with sample enrichment in PSB and TSB broth using different food matrices spiked with Y. enterocolitica and naturally contaminated slaughterhouse samples. The results of the real-time PCR with internal control were verified by the cultural method according to EN ISO 10273:2003. The sensitivity of the real-time PCR with internal control is about 5 genome copies per reaction. Artificial contamination of food samples resulted in a detection level of 5 cfu per 25 g Y. enterocolitica in food samples. 100 % of porcine tonsils and about 22 % meat from pig heads were contaminated. The screening of samples by PCR prior to cultural analysis allows focusing on positive samples in routine analysis. This could result in a higher detection rate by cultural analysis.Zusammenfassung: Für den Nachweis von pathogenen Yersinia enterocolitica wurde ein real-time PCR System mit interner Amplifikationskontrolle entwickelt. Das Nachweissystem für pathogene Y. enterocolitica basiert auf dem chromosomal kodierten ail-Gen. Als Zielsequenz der internen Amplifikationskontrolle dient eine Sequenz aus dem Plasmid pUC19. Zur Validierung der Methode wurden sowohl natürlich kontaminierte Proben aus einem Schlachthof als auch künstlich kontaminierte Proben verschiedener Lebensmittelmatrices verwendet. Die Anreicherung der Proben vor der molekularbiologischen Untersuchung erfolgte parallel in Tryptikase Soja-Bouillon (TSB) und in Pepton-Sorbit-Gallensalz-Bouillon (PSB). Die Ergebnisse der molekularbiologischen Untersuchungen wurden anschließend kulturell in Anlehnung an das Standardverfahren nach EN ISO 10273:2003 verifiziert. Die real-time PCR mit interner Amplifikationskontrolle weist eine Sensitivität von 5 Genomkopien pro Reaktionsansatz auf. Die Nachweisgrenze des Verfahrens, bestimmt anhand künstlich kontaminierter Proben, beträgt etwa 5 KbE Y. enterocolitica pro 25 g Lebensmittel. Von den natürlich kontaminierten Proben aus einem Schlachthof waren die Tonsillen vom Schwein zu 100 % mit pathogenen Y. enterocolitica kontaminiert, Schweinefleischabschnitte aus dem Kopfbereich wiesen einen Kontaminationsgrad von 22 % auf.Ein Screening von Proben durch PCR erlaubt in der Routineanalytik die Fokussierung der kulturellen Analyse auf positive Proben. Dies könnte zu einer höheren Nachweisrate durch das kulturelle Verfahren führen.

Microbiological survey of raw and ready-to-eat leafy green vegetables marketed in Italy

“…RT-PCR positive results for Salmonella spp., L. monocytogenes, and thermotolerant Campylobacter, were confirmed using the corresponding reference analytical microbiological method, respectively the Conversely, the presence of pathogenic Y. enterocolitica was assayed simultaneously using both the microbiological ISO 10273:2003 reference method and an "in house" RT-PCR method targeting the virulence ail gene (Mäde et al, 2008). The same procedure was followed for E. coli O157:H7, for which magnetic immune-separation and ISO 16654:2001 were applied in parallel with the E. coli O157:H7PCR Adiafood AES Chemunex® alternative method.…”

Yersinia enterocolitica in Diagnostic Fecal Samples from European Dogs and Cats: Identification by Fourier Transform Infrared Spectroscopy and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…Molecular detection of the ail gene by real-time PCR was performed according to a protocol described previously by Mäde et al (36) on a Rotor Gene 6000 real-time PCR machine (Corbett Research, Australia), using Brilliant QPCR Master Mix with Sure Start Taq DNA polymerase (Stratagene, Heidelberg, Germany). The temperature profile included 10 min of predenaturation at 95°C followed by 45 cycles of 10 s at 95°C and 30 s at 60°C (data acquisition at the end of the 60°C step) and subsequent cooling to 40°C.…”

“…The temperature profile included 10 min of predenaturation at 95°C followed by 45 cycles of 10 s at 95°C and 30 s at 60°C (data acquisition at the end of the 60°C step) and subsequent cooling to 40°C. Y. enterocolitica strain SZ5108/01 was used as a positive control (36). For confirmation of four isolates, the 16S rRNA gene was partially amplified according to a modified protocol described previously and employing primers 27f and 1522rN (37,38).…”

Yersinia enterocolitica Isolates from Wild Boars Hunted in Lower Saxony, Germany

“…For this, the FD1mod and r533 primer pair was used, and the cycling conditions were those used in a previously described protocol (24). For the detection of the chromosomal gene ail, a real-time PCR assay described by Maede et al (25) was performed on a LightCycler 480 II instrument (Roche Diagnostics, Mannheim, Germany). In addition, the presence of further chromosomal virulence genes (ystA and ystB) and the plasmid-borne virulence genes yadA, virF, and yopT was investigated by PCR amplification as described earlier (26,27).…”