本文關鍵詞： SPF動物 病原菌檢測 傳統(tǒng)方法 PCR特異性 PCR敏感性　出處：《河北醫(yī)科大學》2010年碩士論文　論文類型：學位論文

【摘要】： 目的:實驗動物是生命科學研究的基礎和重要支撐條件,也是藥品生產(chǎn)檢測和新藥研究的基礎。實驗動物按其微生物、寄生蟲控制程度,可劃分為四個等級,即普通動物、清潔動物、無特定病原體動物(SPF動物)和無菌動物。SPF動物是國際上公認的標準級別的實驗動物,適用于所有科研實驗。國內的重點科研項目、GLP實驗室等要與國際接軌,就必須采用SPF動物。而實驗動物是否達到了SPF級別,其重要的評價手段就是微生物和寄生蟲質量控制,必須建立一套完整的微生物和寄生蟲檢測體系,來確保實驗動物不攜帶有應排除的病原體。SPF級實驗動物微生物和寄生蟲檢測標準是在普通級和清潔級實驗動物應排除的病原體基礎上,增加排除6種細菌、9種病毒和2種寄生蟲。 多年來,實驗動物病原菌的檢測主要是依靠傳統(tǒng)的分離培養(yǎng)鑒定方法,這些方法或耗時,或特異性低。與這些傳統(tǒng)的形態(tài)分析方法相比,病原菌的基因型比形態(tài)特征更具特異性和精確性,不會受外界因素(如溫度改變、化學藥物)影響而改變,并且針對其基因型的檢測技術--分子生物學技術(如PCR)敏感度高、快速、簡便,它不僅能檢測出活的病原菌,而且也能檢測出死亡的和難以培養(yǎng)的病原菌。因此,分子生物學技術已被越來越多地應用于實驗動物病原菌的鑒別、分類及種系發(fā)生學等研究中。本研究主要建立了SPF動物應排除的5種病原菌(肺炎克雷伯桿菌、金黃色葡萄球菌、肺炎鏈球菌、乙型溶血性鏈球菌、綠膿桿菌)的傳統(tǒng)分離培養(yǎng)鑒定方法,并對金黃色葡萄球菌和肺炎鏈球菌同時采用了PCR方法進行驗證,最后應用這些檢測方法對本單位實驗動物中心飼養(yǎng)的大鼠、小鼠進行檢測,從而評價檢測方法的可行性和可操作性。 方法: 1應用標準菌株建立傳統(tǒng)分離培養(yǎng)鑒定方法 取適量標準菌液分別接種在相應的選擇培養(yǎng)基上,次日觀察生長出的菌落特征,并對單個菌落進行純培養(yǎng)。對于純培養(yǎng)的單個菌落,以克氏雙糖鐵瓊脂實驗觀察細菌對乳糖、葡萄糖的利用及產(chǎn)酸、產(chǎn)氣情況;以細菌微量生化實驗觀察細菌的生化反應;以半固體動力實驗觀察細菌有無動力;以革蘭氏染色法觀察菌體形態(tài);以血漿凝固酶實驗觀察金黃色葡萄球菌是否產(chǎn)生血漿凝固酶;以氧化酶實驗觀察綠膿桿菌是否產(chǎn)生氧化酶;以42℃生長實驗觀察綠膿桿菌在高溫環(huán)境下能否生長。 2應用金黃色葡萄球菌和肺炎鏈球菌標準菌株建立PCR方法 應用細菌基因組DNA提取試劑盒來提取以上5種細菌和大腸桿菌的基因組DNA。測定提取的金黃色葡萄球菌DNA和肺炎鏈球菌DNA的濃度和純度。對于提取的金黃色葡萄球菌DNA和肺炎鏈球菌DNA,分別使用特異性引物進行PCR擴增,同時對PCR方法的特異性和敏感性進行測定。 3本單位實驗動物中心動物檢測 取實驗動物氣管分泌物和回盲部內容物,接種相應的選擇培養(yǎng)基,挑取純培養(yǎng)的單個菌落進行一系列傳統(tǒng)鑒定實驗,同時提取細菌基因組DNA,應用特異性引物進行PCR擴增,根據(jù)各項實驗結果判定該動物是否攜帶有相應的病原菌。 結果: 1肺炎克雷伯桿菌:淡粉色菌落,雙糖培養(yǎng)基上產(chǎn)酸、產(chǎn)氣,檸檬酸鹽利用、尿素酶、賴氨酸脫羧酶、葡萄糖和乳糖利用陽性,無動力。 2金黃色葡萄球菌:金黃色菌落,有β溶血現(xiàn)象,G+球菌,甘露醇發(fā)酵實驗陽性,血漿凝固酶實驗陽性。PCR產(chǎn)物經(jīng)瓊脂糖凝膠電泳檢測顯示:只有金黃色葡萄球菌PCR得到了一條約270bp大小、清晰可辨的DNA條帶。把不同細菌基因組DNA混合在一起作為模板進行PCR時,只有混有金黃色葡萄球菌基因組DNA的PCR得到了陽性的結果。PCR檢測金黃色葡萄球菌DNA的下限為0.3ng。 3肺炎鏈球菌:有α溶血現(xiàn)象,G+雙球菌。PCR產(chǎn)物經(jīng)瓊脂糖凝膠電泳檢測顯示:只有肺炎鏈球菌PCR得到了一條約682bp大小、清晰可辨的DNA條帶。把不同細菌基因組DNA混合在一起作為模板進行PCR時,只有混有肺炎鏈球菌基因組DNA的PCR得到了陽性的結果。PCR檢測肺炎鏈球菌DNA的下限為30pg。 4乙型溶血性鏈球菌:有β溶血現(xiàn)象,G+球菌,水楊素、蔗糖、蕈糖、乳糖利用陽性。 5綠膿桿菌:產(chǎn)生綠色色素,G-桿菌,木糖、枸櫞酸鹽利用陽性,明膠液化陽性,有動力,氧化酶實驗陽性,42℃生長實驗陽性。 6應用傳統(tǒng)的分離培養(yǎng)鑒定方法,檢測到一只KM小鼠攜帶有金黃色葡萄球菌,PCR方法驗證同樣金黃色葡萄球菌陽性,其它被檢動物五種病原菌均為陰性。 結論: 1應用《中華人民共和國國家標準實驗動物微生物學檢測方法》所規(guī)定的方法,建立了本單位實驗動物中心SPF級大鼠、小鼠五種病原菌的傳統(tǒng)分離培養(yǎng)鑒定方法。 2應用PCR方法檢測金黃色葡萄球菌和肺炎鏈球菌,結果與傳統(tǒng)方法的結果一致。PCR方法與傳統(tǒng)方法相比,更快速、簡便、且具有高度特異性、敏感性。 3利用傳統(tǒng)的分離培養(yǎng)鑒定方法和PCR方法,檢測到本單位實驗動物中心飼養(yǎng)的KM小鼠攜帶有金黃色葡萄球菌,不符合SPF級標準,兩種檢測方法的結果一致。
[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.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R-331

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本文編號：1504192