【作者】 肖西志；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2013， 博士

【摘要】 传统的细菌系统分类的主要依据是形态特征和生理生化性状，采取的主要方法是对细菌进行纯培养，然后从形态学、生理生化反应特征以及血清学特性等方面加以鉴定。但在实践中，细菌的分离过程往往出现假阳性的结果，直到最后的鉴定（生化和血清学鉴定），而且对于目前已经开发成熟的鉴定系统，如VITEK、BIOLOG,、脂肪酸分析系统等仍然存在无法对相似菌进行鉴定的情况。在沙门氏菌的日常检测过程中，经常在选择性培养基中分离到类似于沙门氏菌的细菌。这些细菌的分离给后续确定带来很大的干扰，不仅造成人力和物力的浪费，还大大推迟了得到确诊结果的时间。对于分离到的可疑沙门氏菌，通常会进行系统的生化鉴定以及血清学鉴定。生化鉴定可以通过商品化的微生物鉴定系统，如VITEK2、BIOLOG和PHOENIX-100，血清学鉴定则需要昂贵的分型血清逐一进行。细菌的生化鉴定成本高、检测周期长、特异性低。同时血清型分析在临床检测中存在假阳性的缺点，而且也是个耗时、费力的工作。因此，迫切需要改良并开发新的方法来弥补传统鉴定方法的不足。在炭疽杆菌的检测过程中，假阳性的反应经常出现，一些常见的芽胞杆菌是引起假阳性的真正原因。这些细菌包括苏云金芽胞杆菌、球形芽胞杆菌、环状芽胞杆菌和蜡样芽胞杆菌等。因此，需要研究更准确、快速的炭疽杆菌鉴定方法。本研究的目的，一是在沙门氏菌检测过程中经常分离到的铜绿假单胞菌和奇异变形杆菌为研究对象，分别制备了这两种菌的多克隆抗体，通过体外抑菌试验和临床应用，拟建立一种抑制并消除这两种干扰菌所带来干扰的沙门氏菌快速鉴定方法；二是以动物皮毛中的细菌16S rDNA为研究对象，用PCR方法对分离到的可疑培养物进行16S rDNA的扩增，然后用17种内切酶分别对PCR产物进行酶切，琼脂糖凝胶电泳后出现特异性的限制性片段长度多态性（RFLP）图谱，从而达到对炭疽杆菌准确、快速鉴定的目的。本研究获得了以下研究结果：（1）用铜绿假单胞菌免疫新西兰大白兔后，血清经纯化后所得到的IgG抗体在体外能够抑制并杀灭铜绿假单胞菌，同时对其他来源的这两种菌也具有抑制和杀灭活性，该作用通过扫描电镜和细菌培养试验得到证实，即在加入铜绿假单孢菌多克隆抗体后的10min，菌体开始膨胀、裂解，并于80min后全部裂解。阴沟肠杆菌作为对照，没有受到所制备的抗体的影响。同时，所制备的多克隆抗体对添加于RVS肉汤和MkTTn肉汤中的沙门氏菌也没有任何抑制作用。（2）将铜绿假单胞菌多克隆抗体应用于沙门氏菌检测过程中，即添加于选择性增菌液中，可明显抑制增菌液中铜绿假单胞菌的生长，从而避免了在随后的选择性分离过程中铜绿假单胞菌所带来的干扰。在实际应用过程中，用未经改良的方法检测沙门氏菌阴性样品时，铜绿假单胞菌的检出率很高，最高达19.8%（45/227），而用改良的方法，则铜绿假单胞菌的检出率仅有2.2%(5/227)，沙门氏菌分离的准确率提高了89.2%。因此，在沙门氏菌检测过程中，添加铜绿假单孢菌多克隆抗体可明显提高沙门氏菌分离鉴定的准确性，在降低了检测成本的同时，也缩短了检测周期。（3）用奇异变形杆菌免疫新西兰大白兔后，血清经抗体纯化柱纯化所得到的IgG抗体在体外能够抑制并杀灭奇异变形杆菌，同时对其他来源的奇异变形杆菌也具有抑制和杀灭活性，该作用通过扫描电镜和细菌培养试验得到证实，即奇异变形杆菌与制备的奇异变形杆菌抗体孵育2h后，在扫描电镜下形成网状结构。裂解物的培养显示，无细菌生长。而所制备的奇异变形杆菌多克隆抗体对沙门氏菌和其他肠杆菌科的细菌无抑制作用。（4）将奇异变形杆菌多克隆抗体应用于沙门氏菌检测过程中，即添加于沙门氏菌的选择性增菌液SC和MM中，可抑制增菌液中奇异变形杆菌的生长，从而避免了在随后的选择性分离过程中奇异变形杆菌所带来的干扰。在实际应用过程中，常规方法的检测结果显示，假阳性率最高达到57%（12/21），而应用改进的方法，假阳性率只有18%（2/11），准确率提高了68.4%。因此，作为一种特异性的抑制剂，奇异变形杆菌多克隆抗体的应用明显提高了沙门氏菌检测过程中的准确性，降低了检测成本，节约了检测时间。（5）针对细菌16S rDNA，建立了鸡尾酒式的内切酶分析方法，即应用17种内切酶分别对PCR扩增的细菌16S rDNA分别进行酶切，然后进行琼脂糖凝胶电泳。结果显示，所建立的方法能够在48h内对存在于动物皮毛中常见的细菌进行准确、快速的鉴定，同时将炭疽杆菌与其他细菌进行了准确的区分。因此，所建立的方法作为炭疽杆菌鉴定的补充方法，可应用于临床炭疽杆菌的快速鉴定中。综上所述，本研究首次将抗体作为抑制剂介入到沙门氏菌的分离培养过程中，成功地抑制并消除了相应的干扰菌所带来的干扰。在实际的应用过程中，不仅提高了沙门氏菌分离的准确性，而且也节省了大量的人力、物力，缩短了沙门氏菌鉴定的时间；成功建立了基于细菌16S rDNA的PCR-RFLP指纹图谱的炭疽杆菌鉴定方法，该方法在无须进行DNA序列测定的情况下，对从动物皮毛中分离到的可疑炭疽杆菌培养物进行准确的鉴定，为炭疽杆菌的快速鉴定提供了一种补充方法。更多还原

【Abstract】 Traditional bacterial classification methods depend on morphological character andbiochemical assay. Pure culture followed by morphological, biochemical and serologicalassays involved in the whole process. While in clinical cases, false positive results alwaysoccurred until the last step, biochemical and serological identification. Moreover, there aremany bacteria which can not be identified by VITEK, BIOLOG, and fatty acid assay system.In the process of Salmonella inspection, microbes which resemble Salmonella wereisolated on selective media. These non-target microbes always take troulbes to the followingidentification and waste much consumable materials, labour force and delayed inspcetion timeas well. Biochemical identification by instuments such as VITEK2, BIOLOG andPHOENIX-100, and serological identification would be carried out for suspect isolatedSalmonella.Biochemical identification is characterized by high-cost, long inspection cycleand low specificity. False positive results were occurred in serological assay which itself is atime wasting and labor exhausting. So, modification and development of new mothod isurgent to make up the deficiency of traditional method.In the process of inspection of B. anthrax, false-positive results always occurred. Somefamiliar bacillus which include B. thuringiensis, B. sphaericus, B. circulans, and B. Cereus,caused this result. So, more accurate and fast method was needed to be developed forinspcetion of B. anthrax.There are two goals in this research. Study on improved inspection method ofSalmonella based on the application of antisera against interference bacteria is the first goal.The targets were Proteus mirabilis and P. aeruginosa which were familiar in Salmonellainspection and cuase false positive results in our laboratory were used as research targets.Poly clonal antibodies against the two microbes was prepared through inoculating NewZealand rabbits respectively. Fast and accurate identification method for Salmonnella wasdeveloped through antimicrobial assay in vitro and clinical application. Secondly, PCR-RFLPwas applied to bacterial identification based on16S rDNA. PCR was used to amplify the pureculture and followed by enzyme digestion by17endonucleases respectively. Finger print ofRFLP was shown by agarose gel electrophoresis. The method developed could identify B.anthrax accurately and fastly. The research results were: (1) The sera of New Zealand rabbits which were inoculated by P. aeruginosa werecolleted. The purified IgG could inhibit and kill P. aeruginosa in vitro. The microbicidalactividy was confirmed by scanning eletron microscopy(SEM) and bacterial culture assay, i.e,P. aeruginosa became swolen and began to lyse when antibody of P. aeruginosa was addedinto broth in10minutes and lysed absolutely in80minutes. There was no bacterial growthfor lysate by culture assay. While, as control, E. cloacae was not affected by the antibody. Atthe same time, the prepared poly clonal antibody against P. aeruginosa could not inhibitSalmonella which was added into RVS broth and MkTTn broth.(2) The poly clonal antibody against P. aeruginosa was introduced into the process ofSalmonella, i.e. was added into the selective enrichment media. The growth of P. aeruginosawas inhibited obviously and so, P. aeruginosa was excluded in the process of Salmonella. Inclinical application, the rate of P. aeruginosa was19.8%（45/227）for Salmonella negativesamples with normal method. While, with modified method, the rate was only2.2%(5/227)which the accurate rate for Salmonella increased by89.2%. So, the introduction of antibodyagainst P. aeruginosa increased the accuracy of Salmonella isolation obviously coupled withthe decrease of cost and time of inspection.(3) The sera of New Zealand rabbits which were inoculated by Proteus mirabilis werecolleted. The purified IgG could inhibit and kill Proteus mirabilis in vitro. The activity wasalso effective to other strains of Proteus mirabilis. The microbicidal actividy was confirmedby scanning eletron microscopy(SEM) and bacterial culture assay, i.e, the obvious networkwas formed when Proteus mirabilis was treated by the antibody in2hours. The cultureassay showed that there was no bacterial growth for lysate. While, the prepared poly clonalantibody against Proteus mirabilis could not inhibit Salmonella and other enteric bacteria.(4) The poly clonal antibody against Proteus mirabilis was introduced into the processof Salmonella, i.e. was added into the selective enrichment media, SC and MM. The growthof Proteus mirabilis was inhibited obviously and so, Proteus mirabilis was excluded in theprocess of Salmonella. In clinical application, the false positive rate of Salmonella was57%(12/21) with normal method. While, with modified method, the rate was only18%(2/21)which the accurate rate for Salmonella increased by68.4%. So, as a specific inhibitant, theintroduction of antibody against P. aeruginosa increased the accuracy of Salmonella isolationobviously coupled with the decrease of cost and time of inspection.(5) Based on bacterial16S rDNA, method of finger print of PCR-RFLP was developed,i.e.17endonucleases were adopted to cut the amplificants of16S rDNA of bacterium andthen argarose electrophoresis was used to analyse the products. The results showed thatdeveloped method could identify the bacteria which lied in animal hair and wool fastly and accurately in48hours. At the same time, developed method could classify B. anthrax fromother baceria, escipecially bacillus. So, developed method could be an alternative method forB. anthrax identification.In conclusion, specific antibody which act as inhibitor was introduced into the process ofSalmonella isolation first time. The interference caused by relative bacteria was excludedsuccessfully. The application of specific antibody increased the accuracy of Salmonella inclinical samples obviously. So, this could save much money and labour force. At the sametime, finger print of PCR-RFLP based on bacterial16S rDNA was developed successfully.The method could identify bacteria isolated from animal hair and wool accurately withoutDNA sequencing. So, this assay could be applied to B. anthrax identification as an alternativemethod.更多还原