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目的:为了保障蜂产品免受致病性单核细胞增生李斯特菌的威胁,对蜂产品中单核细胞增生李斯特菌进行检测。方法:采用培养和分子生物学的检测方法研究蜂产品中致病性单核细胞增生李斯特菌的污染状况。将高浓度的病原菌人工污染到蜂蜜和蜂王浆中,室温存放一定时间后,在选择性培养基上增菌培养;以毒力基因hly和16sRNA基因为靶序列,建立双重PCR检测病原菌的方法;以5’、3’端标记FAM、TAMRA的hly基因探针进行荧光定量PCR检测。结果:单增李斯特菌在蜂蜜中的存活时间为5d,而在蜂王浆中不能存活。对未经增菌的人工污染蜂产品采用溶菌酶+蛋白酶K的方法提取DNA,同时采用本实验中建立的双重PCR和荧光定量PCR方法检测,蜂蜜中单增李斯特菌含量均为102CFU/mL。采用这两种方法可在8h内完成蜂蜜中单核细胞增生李斯特菌的快速检测。结论:单增李斯特菌能够在蜂蜜中存活数天,几乎不继续繁殖,而在蜂王浆中不能存活。
Objective: In order to protect the bee products from the pathogenic Listeria monocytogenes, the detection of Listeria monocytogenes in bee products. Methods: The contamination of pathogenic Listeria monocytogenes in bee products was studied by culture and molecular biology methods. The high concentration of pathogens artificial contamination of honey and royal jelly, at room temperature for a certain period of time, the selective culture medium enrichment culture; virulence genes hly and 16sRNA gene as target sequence, the establishment of dual PCR detection of pathogens; 5 ’, 3’ end labeled FAM, TAMRA hly gene probe for quantitative PCR detection. Results: Listeria monocytogenes survived in honey for 5 days and did not survive in royal jelly. DNA was extracted by lysozyme + proteinase K from the uninfected artificially contaminated bee products. The results of double-PCR and real-time PCR showed that the content of Listeria monocytogenes in honey was both 102CFU / mL . Using both of these methods, rapid detection of Listeria monocytogenes in honey can be completed within 8 h. Conclusion: Listeria monocytogenes can survive in honey for several days, rarely continue to breed, and can not survive in royal jelly.