【作者】 代敏；

【导师】 王红宁；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 本研究应用平板稀释法对规模化猪场分离的16株猪源致病性沙门氏菌进行药敏试验，测定了它们对四环素类抗菌素(四环素、土霉素、金霉素)的最低抑菌浓度(MIC)，参照美国临床实验室标准委员会(NCCLS)(1997)的标准，判定菌株的耐药性。结果表明，16株致病性沙门氏菌对四环素类抗菌素产生了普遍耐受性，耐药率达100％，其中MIC值＞128(μg／mL)的高耐药菌株13株，高耐药率81.25％，MIC为32(μg／mL)的低耐药菌株3株，低耐药率18.75％。且沙门氏菌对四环素类抗菌素存在交叉耐药性，即一旦沙门氏菌对四环素类中的一种抗生素产生耐药性，则将对同类其它品种抗生素也产生耐药性。 根据文献报道及GenBank中注册的tetC基因序列设计引物，在国内首次用PCR技术对16株猪源致病性沙门氏菌的四环素耐药基因tetC进行了扩增，结果在以染色体为模板的扩增体系中，未见有特异性产物；在以质粒为模板的扩增体系中，12株菌株获得特异性扩增产物，扩增产物大小约400bp，与药敏试验的阳性符合率达75％，具有较高的检出率，表明猪沙门氏菌四环素耐药基因tetC只存在于质粒上。对tetC与血清型、MIC值、地理来源之间的关系分析表明tetC的有无与血清型、MIC值、地理来源无明显相关性。对来源不同、MIC值分别为32μg／mL(DYPS1)和128μg／mL(SLPS1-3)的2株菌株的PCR扩增产物进行序列测定，并与pBR322(GenBank Accession No：J01749)的四环素耐药基因tetC进行序列比较，结果表明，菌株DYPS1、SLPS1-3和pBR322三者之间tetC的核苷酸同源率为97.7％～98.7％。 在国内首次采用光生物素对高耐药菌株(SLPS1-3)的PCR扩增产物进行标记，制备核酸探针，用菌落原位杂交对猪源致病性沙门氏菌的tetC基因进行了检测，并对杂交条件进行优化。结果表明，13株受试菌株呈阳性，3株受试菌株呈阴性，与PCR检测结果阳性符合率达93.75％，具有较好的重复性，制备的tetC探针具有较高的特异性和灵敏度。 本研究通过试验条件的筛选和优化，建立的猪源致病性沙门氏菌四环素耐药基因tetC的PCR和核酸探针检测技术，可用于规模化猪场沙门氏菌四环素耐药性的分子流行病学监测。更多还原

【Abstract】 The MIC values of tetracycline, oxytetracycline and chlortetracycline of the 16 pathogenic Salmonella strains from swine were detected by disc dilution method. Using NCCLS guidelines hi America hi 1990, all strains showed resistance. In the 16 strains, thirteen strains showed high level of resistance to tetracyclines, and three strains showed low level of resistance.According to the tetC sequences published, a pair of primers were designed and synthesized. The tetC of 16 salmonella strains was amplified by polymerase chain reaction. Twelve strains were positive, and the gene located on plasmids. There is no correlation between tetC and serotype, between tetC and MIC, between tetC and origin of strain. The PCR products of two strains (SLPS1-3 and DYPS1) were sequenced and analysed. Comparing the nucleotide sequences of tetC in Salmonella with pBR322 reported (GenBank accession No: J01749), the percent identity ranged from 97.7% to 98.7%.The PCR product of SLPS1-3 was labelled with photo-biotin. Colony hybridization with the probe was applied. The result indicated that 13 strains were positive, 3 strains were negative. Compared with the result of PCR, The method obtained 93.75% concordance in positive rate. The method can be used for the detection of tetC precisely, sensitively and quickly.In the study, two methods of PCR and nucleic acid probe were established on the base of optimized conditions. By them, the tetC of salmonella would be detected, and molecular epidemiology of tetracyclines-resistance would be investigated.更多还原