【作者】 金大智；

【导师】 谢明杰； 曹际娟；

【作者基本信息】 辽宁师范大学 ， 细胞生物学， 2004， 硕士

【摘要】 食品中常见的致病菌是引起食物中毒的主要原因之一，可导致多种疾病的发生，严重威胁着人们的健康，而且这些细菌大多数都难培养。目前，食品检验检疫工作中的细菌鉴定仍然停留在生化水平上，少数用于PCR、免疫学、探针杂交等技术检测。快速、准确的检测食品中的致病菌是有效预防和控制病原菌感染的前提条件。本文运用实时荧光PCR、实时荧光RT-PCR和DNA芯片技术建立了快速检测食源性致病菌的方法。在实时荧光PCR方法中，设计了不同的引物和探针可以特异的检测八种食源性致病菌，分别是单增李氏菌、肠出血性大肠杆菌0157：H7、副溶血性弧菌、沙门氏菌、空肠弯曲菌、霍乱弧菌、创伤弧菌、志贺氏菌。以单增李氏菌为例，建立了基因组DNA、细胞、定量分子质粒三种标准曲线进行定量检测，检测的灵敏度可达10~1CFU／mL；并且运用实时荧光RT-PCR技术将单增李氏菌的死活状态进行区分；同时，本文运用DNA芯片技术建立了对常见食源性致病菌进行检测的方法，检测的目的菌株是霍乱弧菌、肠出血性大肠杆菌0157：H7、副溶血性弧菌、耶尔森氏菌、单增李氏菌、富氏志贺氏菌、鼠伤寒沙门氏菌、空肠弯曲菌。致病菌的DNA分别通过自行设计的三对在16S rDNA、23S rDNA序列上的通用引物在同一条件下扩增后与DNA芯片上的探针杂交，通过杂交结果可以看出设计的探针特异性强，并且相对于传统的方法操作简单、用时少，稳定性、重复性都非常好，灵敏度可达到155CFU／mL。上述实验结果表明运用实时荧光PCR、实时荧光RT-PCR和DNA芯片技术所建立的四种检测方法准确、可靠、实用性强，是检测食品中常见致病菌技术水平的一次飞跃。并且使用DNA芯片技术可一次实验操作同时检测运用实时荧光PcR和DNA芯片技术检测和鉴定食源性致病菌方法的研究/又种食源性致病菌。本文所建立的四种检测食源性致病菌的方法为今后在食源性致病菌检测、鉴定领域开展进一步的研究和更大规模的使用奠定可靠的理论与技术基础。更多还原

【Abstract】 The food borne pathogens are the primary reasons that lead to food poisoning and are capable of causing significant illness and represent a serious public heath threat worldwide. Most of food borne pathogens are difficult to be incubated in the testing laboratories. Current routine techniques for identification and detection of food borne pathogens are still based on biochemical and physiological methods. Several molecular methods are also used for the detection and identification of the pathogens such as real time PCR, ELISA, and southern blotting. The rapid and accurate methods for detection and identification of food borne pathogens are the precondition of preventing and controlling pathogens. In the paper, the rapid and accurate methods for detection and identification of food borne pathogens were established using real time PCR, RT-real time PCR and DNA chip. In the methods using real time PCR, the eight food borne pathogens were specially detected through different primers and probes, which were Listeria monocytogenes, Escherichia coli 0157:H7, Vibrio parahaemolyticus, Salmonella, Campy lobacter jejuni. Vibrio cholerae, vibrio vulnificus, Shigella. Listeria monocytogenes was regarded as a case, three kinds of stardard curves (genomic DNA, cell, plasmid) were appliedto quantitive detection, the sensitivity was approximately l0CFU/mL; At the same time, an assay using RT-real time PCR was applied to detect or distinguish between viable and nonviable Listeria monocytogenes. Furthermore, another assay for detection and identification of food borne pathogens was also established using DNA chip technique. The detected "targe" pathogens were Vibrio cholerae, Escherichia coli 0157 :H7, Vibrio parahaemolyticus, Yersinia enterocolitica, Listeria monocytogenes, Shigella flexneri, Salmonella typhimurium, Campylobacter j’ejuni. The DNA fragments of pathogens were amplified by 16S rDNA and 23S rDNA universal primers under the same conditions respectively. The PCR products were then applied to the DNA chip for hybridization. The results showed that the selected oligonucleotide probes were very specific. The process of whole operation was easier and more efficient than conventional methods. The repeatability and stability were eligible. The limit of detection was approximately 155CFU/mL. All above results indicated four assays for detection and identification of food borne pathogens were accurate, reliable, and feasible. Those assays would lead to an innovation in the field of detection and identification of food borne pathogens. Eight food borne pathogens were detected using DNA chip technique simultaneously in one operating process. The establishment of four methods in this paper provided a firm technical and theoretical basis for further research and use of wider scope in the field of detecting and identifying food borne pathogens.更多还原