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Establishing and Applying a Rapid Detection System of Yersinia Enterocolitica

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Tutor: ZhouGuangHong
School: Nanjing Agricultural College
Course: Of Food Science
Keywords: Yersinia enterocolitica,real-time PCR,pre-enrichment,primer
CLC: TS207.4
Type: Master's thesis
Year:  2011
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Abstract:
Yersinia enterocolitica is one kind of pathogen for both human and livestock which mainly parasitized in healthy pig¡¯s throats. It infectes the human through food and so on, causing human¡¯s food poisoning and posing the very big threat to the humanity. The study chose a pair of primer with high specificity by establishing a kind of PCR methods, then determined pre-enrichment protocol for Yersinia Enterocolitica by selecting a kind of enrichment broths. Finally, a real-time PCR for detection of pathogenic Yersinia enterocolitica in food combined with an universal internal amplitication control system was established, which could supply effective technological means for rapidly detecting Yersinia Enterocolitica in meat.1.Establishing a kind of PCR method for detecting pathogenic Yersinia enterocolitica.According to the ail gene of Yersinia Enterocolitica, a suitable pair of primers were selected from three pairs of primers, the annealing temperature¡¢the specificity and the sensitivity were determined by testing. Meanwhile, the duplex PCR was established with ail gene and 16s rDNA gene. At last, the specificity of these primer was further verified by real-time PCR. The results showed that the aill primer was the suitable primer for PCR of Yersinia Enterocolitica, because of the higher specificity and the better sensitivity.2.The Selection of pre-enrichment protocol for Yersinia enterocoliticaYersinia Enterocolitica has been paid more attention as a kind of foodborn pathogen. Yersinia Enterocolitica was cultivated in TSB¡¢PSB and ITC, and ultimately by real-time PCR the result was verified. The result showed that TSB was better than others in pure Yersinia Enterocolitica system, and the time of enrichment was 24h. The enrichment in PSB was more sensitive compared to enrichment in TSB and ITC in mix microbe system, the consuming time of enrichment could be shorten to 8h. Therefore, PSB could be used in the detection of Yersinia Enterocolitica of the samples.3. Establishing a kind of real-time PCR method for detecting pathogenic Yersinia Enterocolitica. A duplex 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 gos gene of rice as target for the internal amplification control, and detecting the 32 samples of lymphaden of the lower jaw and the 38 samples of tongue and tosil by the method. The specificity of the normal and duplex real-time PCR was pretty high,and the limits of detection were lOpg/¦ÌL and 100pg/¦ÌL seperately. 5.71%¡¢44.29% and 8.57% of pork samples were detected to be contaminated with traditional culture, nomal and duplex real-time PCR. There were no inhibitory substances in the reation, because all the samples had amplification curves of the internal amplification control gene. All the samples detected by the cultural method were also detected by the real-time PCR. It showed that the real-time PCR was highly effective and timesaving.Generally, this paper selected a pair of primers with high specificity of Yersinia Enterocolitica. The PSB broth was the best pre-enrichment culture medium for the actual meat samples. At last, the paper established a real-time PCR of Yersinia enterocolitica combined with an universal internal amplification control system successfully.
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