發(fā)布時(shí)間：2018-02-11 22:52

  本文關(guān)鍵詞： SPF動(dòng)物 病原菌檢測(cè) 傳統(tǒng)方法 PCR特異性 PCR敏感性　出處：《河北醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:實(shí)驗(yàn)動(dòng)物是生命科學(xué)研究的基礎(chǔ)和重要支撐條件,也是藥品生產(chǎn)檢測(cè)和新藥研究的基礎(chǔ)。實(shí)驗(yàn)動(dòng)物按其微生物、寄生蟲控制程度,可劃分為四個(gè)等級(jí),即普通動(dòng)物、清潔動(dòng)物、無特定病原體動(dòng)物(SPF動(dòng)物)和無菌動(dòng)物。SPF動(dòng)物是國(guó)際上公認(rèn)的標(biāo)準(zhǔn)級(jí)別的實(shí)驗(yàn)動(dòng)物,適用于所有科研實(shí)驗(yàn)。國(guó)內(nèi)的重點(diǎn)科研項(xiàng)目、GLP實(shí)驗(yàn)室等要與國(guó)際接軌,就必須采用SPF動(dòng)物。而實(shí)驗(yàn)動(dòng)物是否達(dá)到了SPF級(jí)別,其重要的評(píng)價(jià)手段就是微生物和寄生蟲質(zhì)量控制,必須建立一套完整的微生物和寄生蟲檢測(cè)體系,來確保實(shí)驗(yàn)動(dòng)物不攜帶有應(yīng)排除的病原體。SPF級(jí)實(shí)驗(yàn)動(dòng)物微生物和寄生蟲檢測(cè)標(biāo)準(zhǔn)是在普通級(jí)和清潔級(jí)實(shí)驗(yàn)動(dòng)物應(yīng)排除的病原體基礎(chǔ)上,增加排除6種細(xì)菌、9種病毒和2種寄生蟲。 多年來,實(shí)驗(yàn)動(dòng)物病原菌的檢測(cè)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(shí),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(huì)受外界因素(如溫度改變、化學(xué)藥物)影響而改變,并且針對(duì)其基因型的檢測(cè)技術(shù)--分子生物學(xué)技術(shù)(如PCR)敏感度高、快速、簡(jiǎn)便,它不僅能檢測(cè)出活的病原菌,而且也能檢測(cè)出死亡的和難以培養(yǎng)的病原菌。因此,分子生物學(xué)技術(shù)已被越來越多地應(yīng)用于實(shí)驗(yàn)動(dòng)物病原菌的鑒別、分類及種系發(fā)生學(xué)等研究中。本研究主要建立了SPF動(dòng)物應(yīng)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(duì)金黃色葡萄球菌和肺炎鏈球菌同時(shí)采用了PCR方法進(jìn)行驗(yàn)證,最后應(yīng)用這些檢測(cè)方法對(duì)本單位實(shí)驗(yàn)動(dòng)物中心飼養(yǎng)的大鼠、小鼠進(jìn)行檢測(cè),從而評(píng)價(jià)檢測(cè)方法的可行性和可操作性。 方法: 1應(yīng)用標(biāo)準(zhǔn)菌株建立傳統(tǒng)分離培養(yǎng)鑒定方法 取適量標(biāo)準(zhǔn)菌液分別接種在相應(yīng)的選擇培養(yǎng)基上,次日觀察生長(zhǎng)出的菌落特征,并對(duì)單個(gè)菌落進(jìn)行純培養(yǎng)。對(duì)于純培養(yǎng)的單個(gè)菌落,以克氏雙糖鐵瓊脂實(shí)驗(yàn)觀察細(xì)菌對(duì)乳糖、葡萄糖的利用及產(chǎn)酸、產(chǎn)氣情況;以細(xì)菌微量生化實(shí)驗(yàn)觀察細(xì)菌的生化反應(yīng);以半固體動(dòng)力實(shí)驗(yàn)觀察細(xì)菌有無動(dòng)力;以革蘭氏染色法觀察菌體形態(tài);以血漿凝固酶實(shí)驗(yàn)觀察金黃色葡萄球菌是否產(chǎn)生血漿凝固酶;以氧化酶實(shí)驗(yàn)觀察綠膿桿菌是否產(chǎn)生氧化酶;以42℃生長(zhǎng)實(shí)驗(yàn)觀察綠膿桿菌在高溫環(huán)境下能否生長(zhǎng)。 2應(yīng)用金黃色葡萄球菌和肺炎鏈球菌標(biāo)準(zhǔn)菌株建立PCR方法 應(yīng)用細(xì)菌基因組DNA提取試劑盒來提取以上5種細(xì)菌和大腸桿菌的基因組DNA。測(cè)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(duì)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(jìn)行PCR擴(kuò)增,同時(shí)對(duì)PCR方法的特異性和敏感性進(jìn)行測(cè)定。 3本單位實(shí)驗(yàn)動(dòng)物中心動(dòng)物檢測(cè) 取實(shí)驗(yàn)動(dòng)物氣管分泌物和回盲部?jī)?nèi)容物,接種相應(yīng)的選擇培養(yǎng)基,挑取純培養(yǎng)的單個(gè)菌落進(jìn)行一系列傳統(tǒng)鑒定實(shí)驗(yàn),同時(shí)提取細(xì)菌基因組DNA,應(yīng)用特異性引物進(jìn)行PCR擴(kuò)增,根據(jù)各項(xiàng)實(shí)驗(yàn)結(jié)果判定該動(dòng)物是否攜帶有相應(yīng)的病原菌。 結(jié)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(yáng)性,無動(dòng)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(shí)驗(yàn)陽(yáng)性,血漿凝固酶實(shí)驗(yàn)陽(yáng)性。PCR產(chǎn)物經(jīng)瓊脂糖凝膠電泳檢測(cè)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(xì)菌基因組DNA混合在一起作為模板進(jìn)行PCR時(shí),只有混有金黃色葡萄球菌基因組DNA的PCR得到了陽(yáng)性的結(jié)果。PCR檢測(cè)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(cè)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(xì)菌基因組DNA混合在一起作為模板進(jìn)行PCR時(shí),只有混有肺炎鏈球菌基因組DNA的PCR得到了陽(yáng)性的結(jié)果。PCR檢測(cè)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(yáng)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(yáng)性,明膠液化陽(yáng)性,有動(dòng)力,氧化酶實(shí)驗(yàn)陽(yáng)性,42℃生長(zhǎng)實(shí)驗(yàn)陽(yáng)性。 6應(yīng)用傳統(tǒng)的分離培養(yǎng)鑒定方法,檢測(cè)到一只KM小鼠攜帶有金黃色葡萄球菌,PCR方法驗(yàn)證同樣金黃色葡萄球菌陽(yáng)性,其它被檢動(dòng)物五種病原菌均為陰性。 結(jié)論: 1應(yīng)用《中華人民共和國(guó)國(guó)家標(biāo)準(zhǔn)實(shí)驗(yàn)動(dòng)物微生物學(xué)檢測(cè)方法》所規(guī)定的方法,建立了本單位實(shí)驗(yàn)動(dòng)物中心SPF級(jí)大鼠、小鼠五種病原菌的傳統(tǒng)分離培養(yǎng)鑒定方法。 2應(yīng)用PCR方法檢測(cè)金黃色葡萄球菌和肺炎鏈球菌,結(jié)果與傳統(tǒng)方法的結(jié)果一致。PCR方法與傳統(tǒng)方法相比,更快速、簡(jiǎn)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(cè)到本單位實(shí)驗(yàn)動(dòng)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(jí)標(biāo)準(zhǔn),兩種檢測(cè)方法的結(jié)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(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R-331

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李君文,晁福寰;致病微生物PCR檢測(cè)方法研究進(jìn)展[J];中國(guó)衛(wèi)生檢驗(yàn)雜志;1997年05期

，

本文編號(hào)：1504192