SPF级大鼠、小鼠五种病原菌检测方法的建立和应用

发布时间：2018-02-11 22:52

本文关键词： SPF动物病原菌检测传统方法 PCR特异性 PCR敏感性　出处：《河北医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的:实验动物是生命科学研究的基础和重要支撑条件,也是药品生产检测和新药研究的基础。实验动物按其微生物、寄生虫控制程度,可划分为四个等级,即普通动物、清洁动物、无特定病原体动物(SPF动物)和无菌动物。SPF动物是国际上公认的标准级别的实验动物,适用于所有科研实验。国内的重点科研项目、GLP实验室等要与国际接轨,就必须采用SPF动物。而实验动物是否达到了SPF级别,其重要的评价手段就是微生物和寄生虫质量控制,必须建立一套完整的微生物和寄生虫检测体系,来确保实验动物不携带有应排除的病原体。SPF级实验动物微生物和寄生虫检测标准是在普通级和清洁级实验动物应排除的病原体基础上,增加排除6种细菌、9种病毒和2种寄生虫。多年来,实验动物病原菌的检测主要是依靠传统的分离培养鉴定方法,这些方法或耗时,或特异性低。与这些传统的形态分析方法相比,病原菌的基因型比形态特征更具特异性和精确性,不会受外界因素(如温度改变、化学药物)影响而改变,并且针对其基因型的检测技术--分子生物学技术(如PCR)敏感度高、快速、简便,它不仅能检测出活的病原菌,而且也能检测出死亡的和难以培养的病原菌。因此,分子生物学技术已被越来越多地应用于实验动物病原菌的鉴别、分类及种系发生学等研究中。本研究主要建立了SPF动物应排除的5种病原菌(肺炎克雷伯杆菌、金黄色葡萄球菌、肺炎链球菌、乙型溶血性链球菌、绿脓杆菌)的传统分离培养鉴定方法,并对金黄色葡萄球菌和肺炎链球菌同时采用了PCR方法进行验证,最后应用这些检测方法对本单位实验动物中心饲养的大鼠、小鼠进行检测,从而评价检测方法的可行性和可操作性。方法: 1应用标准菌株建立传统分离培养鉴定方法取适量标准菌液分别接种在相应的选择培养基上,次日观察生长出的菌落特征,并对单个菌落进行纯培养。对于纯培养的单个菌落,以克氏双糖铁琼脂实验观察细菌对乳糖、葡萄糖的利用及产酸、产气情况;以细菌微量生化实验观察细菌的生化反应;以半固体动力实验观察细菌有无动力;以革兰氏染色法观察菌体形态;以血浆凝固酶实验观察金黄色葡萄球菌是否产生血浆凝固酶;以氧化酶实验观察绿脓杆菌是否产生氧化酶;以42℃生长实验观察绿脓杆菌在高温环境下能否生长。 2应用金黄色葡萄球菌和肺炎链球菌标准菌株建立PCR方法应用细菌基因组DNA提取试剂盒来提取以上5种细菌和大肠杆菌的基因组DNA。测定提取的金黄色葡萄球菌DNA和肺炎链球菌DNA的浓度和纯度。对于提取的金黄色葡萄球菌DNA和肺炎链球菌DNA,分别使用特异性引物进行PCR扩增,同时对PCR方法的特异性和敏感性进行测定。 3本单位实验动物中心动物检测取实验动物气管分泌物和回盲部内容物,接种相应的选择培养基,挑取纯培养的单个菌落进行一系列传统鉴定实验,同时提取细菌基因组DNA,应用特异性引物进行PCR扩增,根据各项实验结果判定该动物是否携带有相应的病原菌。结果: 1肺炎克雷伯杆菌:淡粉色菌落,双糖培养基上产酸、产气,柠檬酸盐利用、尿素酶、赖氨酸脱羧酶、葡萄糖和乳糖利用阳性,无动力。 2金黄色葡萄球菌:金黄色菌落,有β溶血现象,G+球菌,甘露醇发酵实验阳性,血浆凝固酶实验阳性。PCR产物经琼脂糖凝胶电泳检测显示:只有金黄色葡萄球菌PCR得到了一条约270bp大小、清晰可辨的DNA条带。把不同细菌基因组DNA混合在一起作为模板进行PCR时,只有混有金黄色葡萄球菌基因组DNA的PCR得到了阳性的结果。PCR检测金黄色葡萄球菌DNA的下限为0.3ng。 3肺炎链球菌:有α溶血现象,G+双球菌。PCR产物经琼脂糖凝胶电泳检测显示:只有肺炎链球菌PCR得到了一条约682bp大小、清晰可辨的DNA条带。把不同细菌基因组DNA混合在一起作为模板进行PCR时,只有混有肺炎链球菌基因组DNA的PCR得到了阳性的结果。PCR检测肺炎链球菌DNA的下限为30pg。 4乙型溶血性链球菌:有β溶血现象,G+球菌,水杨素、蔗糖、蕈糖、乳糖利用阳性。 5绿脓杆菌:产生绿色色素,G-杆菌,木糖、枸橼酸盐利用阳性,明胶液化阳性,有动力,氧化酶实验阳性,42℃生长实验阳性。 6应用传统的分离培养鉴定方法,检测到一只KM小鼠携带有金黄色葡萄球菌,PCR方法验证同样金黄色葡萄球菌阳性,其它被检动物五种病原菌均为阴性。结论: 1应用《中华人民共和国国家标准实验动物微生物学检测方法》所规定的方法,建立了本单位实验动物中心SPF级大鼠、小鼠五种病原菌的传统分离培养鉴定方法。 2应用PCR方法检测金黄色葡萄球菌和肺炎链球菌,结果与传统方法的结果一致。PCR方法与传统方法相比,更快速、简便、且具有高度特异性、敏感性。 3利用传统的分离培养鉴定方法和PCR方法,检测到本单位实验动物中心饲养的KM小鼠携带有金黄色葡萄球菌,不符合SPF级标准,两种检测方法的结果一致。
[Abstract]:Objective: animal experiment is the foundation of life science research and the important supporting conditions, is also the basis for drug production testing and drug research. According to the experimental animal microbe, parasite control degree, can be divided into four grades, namely ordinary animal, clean animal, specific pathogen free animal (SPF animal) and sterile animal animal is.SPF the experimental animal level of the internationally recognized standards, applicable to all scientific experiments. The key scientific research project in China, the GLP laboratory with international standards, we must use SPF. But whether the animal experimental animal reached SPF level, the important evaluation means of microorganisms and parasites in quality control, we must establish a set of complete microorganism and parasite detection system, to ensure that the animal does not carry.SPF animal pathogen detection of microorganisms and parasites in the standard should be excluded from the ordinary and clean level. On the basis of the pathogens that should be excluded from animals, 6 kinds of bacteria, 9 viruses and 2 parasites are eliminated.
Over the years, the detection of animal pathogenic bacteria mainly rely on traditional methods of isolation and identification of these methods, or time-consuming, or low specificity. Compared with the traditional morphological analysis method, genotypes of pathogenic bacteria is more specific and accurate than morphological characteristics, not by external factors (such as temperature change, chemical drug) to change, and the detection technology of molecular biology techniques for its genotype (such as PCR) with high sensitivity, fast and simple, it can not only pathogen detection of live, but also can detect the death and difficult to cultivate bacteria. Therefore, molecular biology identification technology has been more and more used in the experiment of animal pathogenic bacteria, classification and phylogeny of 5 species of pathogenic bacteria. This study established SPF animal should be excluded (Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, soluble hepatitis Bloody Streptococcus, Pseudomonas aeruginosa) method for the identification of the traditional culture, and the Staphylococcus aureus and Streptococcus pneumoniae and PCR method was used to verify the last application of these detection methods on the experimental animal center fed rats, mice were detected from the feasibility and evaluation of the detection methods and operability.
Method:
1 Application of standard strains to establish a traditional isolation and culture identification method
Take proper amount of standard strains were inoculated in the culture medium of choice, the next day to observe the growth characteristics of colonies, and pure culture of single colonies. For single colonies of pure culture, in order to Kligler iron agar experimental observation of bacteria on lactose, glucose utilization and acid production, gas production and biochemical reactions were observed; bacteria in the micro bacteria biochemical experiment; semi solid dynamic experimental observation of bacteria have no power; observation of cell morphology by Gram staining; observe whether Staphylococcus aureus produces coagulase in coagulase test; to observe whether Pseudomonas aeruginosa produced by oxygen oxidase enzyme experiment; experimental observation to 42 degrees the growth of Pseudomonas aeruginosa in under the high temperature environment can grow.
2 the establishment of PCR method for the application of Staphylococcus aureus and Streptococcus pneumoniae
Application of bacterial genomic DNA extraction kit to extract more than 5 kinds of bacteria and Escherichia coli genomic DNA. extraction of Staphylococcus aureus and Streptococcus pneumoniae DNA the concentration and purity of DNA. For the extraction of DNA from Staphylococcus aureus and Streptococcus pneumoniae DNA, specific primers were used for PCR amplification respectively, while the PCR method is specific and the sensitivity was determined.
3 unit laboratory animal center animal test
Taking the experimental animal tracheal secretions and ileocecal contents, select the corresponding inoculation medium, single colonies of pure culture of a series of traditional identification experiments, the bacterial genome DNA extracted using specific primers for PCR amplification, according to the experimental results to determine whether the animal is carrying pathogens accordingly.
Result:
1 Klebsiella pneumoniae: pink colonies, disaccharide medium acid production, gas production, citrate utilization, urease, lysine decarboxylase, glucose and lactose by positive, no power.
2: golden yellow staphylococcus aureus colony, beta hemolysis, G+ aureus, mannitol fermentation test positive, coagulase test positive.PCR agarose gel electrophoresis showed that only Staphylococcus aureus PCR got a treaty 270bp size, clear DNA bands. The different bacterial genome DNA mixed together as the template for PCR, only mixed with Staphylococcus aureus genomic DNA PCR obtained positive results limit.PCR detection of Staphylococcus aureus DNA 0.3ng.
3: alpha Hemolytic Streptococcus pneumoniae, G+ diplococcus.PCR agarose gel electrophoresis showed that only Streptococcus pneumoniae PCR got a treaty 682bp size, clear DNA bands. The different bacterial genome DNA mixed together as a template for PCR, only mixed with Streptococcus pneumoniae genomic DNA was obtained by PCR positive results limit.PCR detection of Streptococcus pneumoniae DNA 30pg.
4 hemolytic streptococcus: beta hemolytic phenomenon, G+ coccus, salicine, sucrose, mushroom sugar, and lactose use positive.
5 Pseudomonas aeruginosa: green pigment, G- bacilli, xylose, citrate positive, gelatin liquefaction positive, dynamic, oxidase test positive, 42 degrees of growth test positive.
6, using traditional isolation and culture methods, a KM mouse was found to have Staphylococcus aureus. PCR method also showed the same Staphylococcus aureus positive. The other five animals were negative.
Conclusion:
1, based on the method stipulated in the "People's Republic of China national standard laboratory animal microbiological examination method", we established the traditional isolation and culture identification method of five animal pathogenic bacteria in the laboratory animal center of SPF.
2 the detection of Staphylococcus aureus and Streptococcus pneumoniae by PCR method is consistent with the results of traditional methods..PCR method is more rapid, simple and highly sensitive than traditional methods.
3, by using the traditional methods of isolation, culture and identification and PCR, we found that the KM mice bred in our laboratory animal center had Staphylococcus aureus, which did not meet the SPF level standard. The results of the two methods were consistent.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R-331

【参考文献】