导　　师： 吴德峰;孔繁德

学科专业： I0601

授予学位： 硕士

作　　者： ;

机构地区： 福建农林大学

摘　　要： 目的：建立一套快速，准确和简便的实验程序，应用于人畜共患致病菌沙门氏菌的检测。 方法：《1)建立胶体金免疫层析法(gold immunochromatography assay，gica)：利．用沙门氏菌免疫实验兔获得高免血清，提纯出沙门氏菌多克隆抗体：然后用柠檬酸三钠还原氯金酸法制备34nm的胶体金颗粒，加入沙门氏菌多克隆抗体15ug/ml，制备出金标抗体，再与硝酸纤维膜，羊抗兔抗体，玻璃纤维膜等材料结合制成试纸条，最后评价gica的灵敏度和特异性，并在不同储存条件下，测试gica的稳定性。(2)建立荧光定量pcr(fiuorescencer quantitive polymerasechain reaction，fq-pcr)方法：根据沙门氏菌保守基因序列fimy基因设计合成引物和taqman探针，然后对反应体系和条件进行优化，最后评价fq-pcr的灵敏度，特异性，和重复性。(3)运用gica和fq-pcr对饲料，猪粪，肉制品等样品进行临床检测，同时采用传统细菌分离鉴定方法比较结果。 结果：(1)gica的沙门氏菌最低检测量为2.3x10<'5>fu/ml，与大肠杆菌、变形杆菌、阴沟肠杆菌等常见肠道菌不发生交叉反应；放置4℃或37℃存放9个月，检测结果无差异；整个试验过程仅需10～15 min即可判断结果。12)fq-pcr的检测灵敏度达到4.5 cfu/反应体系，比普通pcr高100倍；检测非沙门氏菌均呈阴性，检测沙门氏菌均呈阳性：平行实验的结果一致性高，可重复性强。(3)通过检测临床样品，gica和fq-pcr相结合的方法与传统方法所得结果相符合，但操作程序较为简便，操作时间大大缩短。 结论：综合实验结果，最终确定优化的沙门氏菌检测程序即对大量样品采用gica筛检，除去大量阴性样品，阳性样品采用fq-pcr法作进一步鉴定：需要定型时则用传统方法。此法具有高度的特异性、敏感性和快速简便等优点，为常年需处理大量样品的动检、食品饲料监测和防疫等部门提供一种有效的检测手段。既可保证样品检测的准确性和可靠性，又可节省大量的人力、物力和财力，有较大的社会经济效益。 Objective: To develop a rapid and accurate protocol for the detection of Salmonella, which is important pathogenic agents for both human beings and animals. Methods: /(1/) Gold immunochromatography assay/(GICA/): At the beginning , the pure antiserum was obtained from rabbits immunized by several successive injections of cell suspensions of Salmonella and polyclonal antibody was extracted from the serum using Protein A Sepharose TMCL-4B. In the second place,the colloidal gold of 34nm for labeling Polyclonal antibody of the bacteria was produced through reducing or deoxygenating the gold chloride with trisodium citrate.Then the Polyclonal antibody was mixed with the 34nm colloidal gold with a concentration of 15ug //ml to produce gold-labeled Polyclonal antibody mixture.After that,a test strip for Salmonella was assembled with nitrocellulose membrane,the purified antiserum,the gold-labeled polyclonal antibody,sheep anti-rabbit immunoglobulinG/(Purchased/) and glass fibrous membrane /(Purchased/).Finally,the test strip was examined for its spccificity and sensitivity.The life of the test strip was also investigated under different storage conditions./(2/) Fluorescencer quantitive polymerase chain reaction/(FQ-PCR/): At first,the Taqman probe and primers were designed and sythesized according to the conserved gene fimY of salmonella available in GenBank.Second,the reaction parameters were optimized to develop FQ-PCR.Finally,the sensitivity, the specificity and the reproducibility was evaluated./(3/) GICA and FQ-PCR were used to detect Salmonella in feed, dung of swine,meat and so on, depending on the National Standard/(GB/) method to verify the results.Results: /(1/) GICA has a better specificity and no cross-reaction with other enterobacteria such as Escherichia coli,Proteus mirabilis,Enterobacter cloacae. Its sensitivity is 2．3×10~5 cfu //ml. It can be Stored at 4℃or 37℃for 9 months and maintain good detecting result. In addition, the whole test procedure could be finished in only 10~15minutes./(2/) FQ-PCR could detect 4．5 cfu per reaction, and its sensitivity was 100 times higher than that of the routine PCR,otherwise, the detection results of different strains of non-Salmonella and Salmonella showed high specificity of this assay and the reproducibility is good,too./(3/) The resuluts of detecting samples showed that the sensitivity and specificity of combination use of two methods /(GICA and FQ-PCR/) was 100/%, compared with the results of National Standard method. It also indicated that this combined methods is more quick and simple than National standard method. Conclusion:Based on all the results,an optimized protocol for the detection of Salmonella was developed,that is,to use GICA to screen the large number of sarnples first,and then to use FQ-PCR to test the GICA positive samples.The final step is,if needed, to use National Standard method to determine the serotypes of Salmonella.This combined method was highly specific sensitive and very quick.lt provides an efficient tool to handle many samples in animal quarantine,food and feed hygiene,diseases prevention.This procedure ensures the accuracy and reliability of detecting results and saves lots of material and manpower,which would create great social and economic interest.

关 键 词： 沙门氏菌 胶体金免疫层析法 荧光定量检测

分 类 号： [S852.612]

领　　域： [农业科学] [农业科学] [农业科学]