中文摘要

    布鲁氏菌病(Brucellosis)是由布鲁氏菌引起的动物源性人畜共患病。易致使家畜流产，可造成人类周期性波浪热等症状，严重威胁全球畜牧业健康发展与公共卫生安全。近十年间新疆地区布病疫情较为严峻，布病阳性率逐年递增, 同时由于传统临床诊断方法无法鉴别布鲁氏菌流行株与疫苗株，致使新疆地区原本严峻的布鲁氏菌病疫情雪上加霜。建立简便快捷，实用性强的鉴别布鲁氏菌流行感染与疫苗免疫的诊断技术成为遏制新疆地区布病疫情的当务之急。LAMP 方法由于其简便快捷，适用范围广等优势被应用于传染病防控，食品安全检测及胚胎性别鉴定等领域。目前已建立基于布鲁氏菌外膜蛋白基因 Omp25、毒力基因 Pgm 、核糖体基因 IS711 等基因的布鲁氏菌 LAMP 快速检测方法。然而已建立的布鲁氏菌 LAMP 鉴定方法不能区分鉴别布鲁氏菌流行株与疫苗株，有关新疆布鲁氏菌流行株与疫苗株 LAMP 鉴别诊断方法未有相关的报道，因此本研究对布鲁氏菌新疆流行株与疫苗株进行比较基因组学研究及建立 LAMP鉴别诊断方法。主要开展的工作研究如下：

    1.布鲁氏菌新疆流行株与疫苗株间差异基因筛选鉴定及生物学功能预测。以在前期分离出的布鲁氏菌新疆流行株 027 株与实验室保存的疫苗株S2株与 M5 株为研究对象，通过比较基因组学研究方法筛选菌株间的差异基因，BLAST数据库验证基因同源性，设计引物进行PCR扩增与生物测序进一步验证其差异性；最后，运用生物信息学工具预测生物学功能。结果显示：共筛选出50个差异基因，其中24个027株存在而S2株缺失的基因，25个027株缺失而S2株存在的基因；1个027 株存在而M5 株缺失的基因；PCR鉴定结果与 BLAST数据库验证结果相符；经在线生物信息学软件分析后，41个差异基因可编码外膜蛋白，5个差异基因可编码膜内蛋白，4 个差异基因可编码跨膜蛋白。以上实验结果证实布鲁氏菌新疆流行株027株与疫苗S2株、M5 株间存在基因组学结构差异。

2.布鲁氏菌新疆流行株与疫苗株LAMP 鉴别方法的建立与初步应用。以已验证过的 027 株与 S2 株间差异基因为研究对象，通过优化 LAMP 反应各组分浓度与扩增条件，进行灵敏性实验与特异性实验；使用建立的 LAMP 方法与PCR 方法检测混匀布鲁氏菌 027 株、S2 株、 M5株及 PBS 的40 份模拟奶样与40 份模拟血样，计算两种检测方法的符合率；使用建立的 LAMP 方法，虎红平板凝集方法与 PCR 方法检测 32份昌吉地区采集的血液样本；使用LAMP方法与PCR方法检测 40 份伊犁地区采集的流产母羊的奶液样本与流产胎儿的肝脾组织样本。结果显示：LAMP 鉴别方法检测大肠杆菌、链球菌、沙门氏菌等均为阴性，灵敏性检测极限为 7.5fg/μL。在检测份模拟样本时，与 PCR检测方法符合率为100%。 检测32份采集的昌吉地区临床血样时， LAMP 方法可以鉴定布鲁氏菌新疆流行株。使用建立LAMP方法检测32份血样时，28份为阳性，其余6、7、12、14样本为阴性。经虎红平板凝集试验测试后，32份血液中，28份为阳性，其中14 , 15 ，24 , 25 号为阴性血样。使用PCR方法检测时，30、31样本份为阳性，剩余为阴性。经LAMP方法鉴定30、31号样本为阳性与PCR、虎红平板凝集方法检测结果一致。经LAMP方法鉴定 6、7、12、14样本为阴性与PCR检测结果一致，14号样本检测结果与PCR方法、虎红凝集实验检测结果一致。

检测40份伊犁地区采集的奶液及组织样本时，建立的LAMP方法检测40份伊犁地区样本时，31份为阳性，其余2、3、8、10、15、19、20、23、31样本为阴性。而PCR方法鉴定27、28、29、34、36为阳性，其余为阴性。其中2、3、8、10、15、19、23、31号样本为阴性，27、28、29、34、36样本为阳性均与PCR检测结果一致。结果表明建立了一种可以鉴别区分布鲁氏菌新疆流行株与疫苗株 S2株的环介导恒温扩增方法。

 Abstract

     Brucellosis is a zoonotic zoonotic disease caused by brucella. Livestock abortion, human cyclical wave heat and other symptoms were caused by the disease. It was a serious threat to the healthy development of global animal husbandry and public health. In recent years, the prevalence of brucellosis in Xinjiang has been more severe. The positive rate of brucellosis has been increasing year by year. At the same time, the traditional clinical diagnosis method coud not identify Brucella epidemic strains and vaccine strains, resulting in the severe epidemic of brucellosis in Xinjiang area. The establishment of the identification technology of Brucella infection and vaccine immunization diagnosiste  was the key to curb the epidemic in Xinjiang epidemic situation of the top priority. LAMP method was widely used in the fields of infectious disease prevention and control, food safety detection and embryo sex identification because of its advantages (wide application range and so on) and disadvantages. At present, Brucella LAMP identification method based on Brucella outer membrane protein gene Omp25, virulence gene Pgm and ribosomal gene IS711 gene has been established. However, the established Brucella LAMP identification method failed to identify different serotypes of brucella, such as epidemic strains and vaccine strains. In addition, the epidemic strains of Brucella in Xinjiang were not related to the differential diagnosis of LAMP in vaccine strains. Therefore, this study studied the comparative genomics of Brucella Xinjiang strain and vaccine strains and established LAMP identification method. The main work were carried out is as follows:

     1 Screening and identificating of different strains of Bacteria strain 027 and Vaccine strain S2 and M5 in Xinjiang, and predicting the biological function. The strains of strain Brucella 027 was isolated  in Xinjiang and the strain of strain M5 and.S2 were saved in Lab.The BLAST database was used to identify the gene homologous by comparing the genomic research method. And the primers were used to confirm the difference geng between PCR amplification and biological sequencing. Finally, the bioinformatics tools were used to predict the biological function. The results showed that a total of 24 genes were screened and the genes lacked in S2 strain, 25 genes were deleted from the 027 strain. The one gene was found in the 027 strain and the deletion of M5 strain. The results of PCR identification were Consistent with BLAST database .41 differential genes could encode outer membrane proteins, 5 differential encoded inner proteins, and 4 differential transgenic encoded proteins. The results of the above experiments confirmed that there were genomic differences between strain 027 strain and S2 、M5 strain of Brucella Xinjiang.

    2 Establishment and preliminary application of LAMP identification method for strain 027 and vaccine strain S2 of Brucella in Xinjiang. The LAMP method were optimized The sensitivity and specificity of the LAMP tested . The LAMP method and the PCR method were used to detect the 40 milk samples and 40 blood samples containing the Brucella 027 strain, S2 strain, M5 strain and PBS .After the teset ,the coincidence rate of the two detection methods were calculated . The established LAMP method, tiger red plate agglutination method and PCR method were used to detect 32 samples collected from Changji area. LAMP method 、RBPT method and PCR method were used to detect the samples of 40 tissue samples of aborted fetal collected from Yili area.The LAMP method was used to identify the epidemic strains of Brucella in Xinjiang when 32 blood samples collected from Changji area of clinical . The LAMP method was used to detect 32 blood samples.28 were positive and the remaining 6, 7, 12 and 1

 Abstract

     Brucellosis is a zoonotic zoonotic disease caused by brucella. Livestock abortion, human cyclical wave heat and other symptoms were caused by the disease. It was a serious threat to the healthy development of global animal husbandry and public health. In recent years, the prevalence of brucellosis in Xinjiang has been more severe. The positive rate of brucellosis has been increasing year by year. At the same time, the traditional clinical diagnosis method coud not identify Brucella epidemic strains and vaccine strains, resulting in the severe epidemic of brucellosis in Xinjiang area. The establishment of the identification technology of Brucella infection and vaccine immunization diagnosiste  was the key to curb the epidemic in Xinjiang epidemic situation of the top priority. LAMP method was widely used in the fields of infectious disease prevention and control, food safety detection and embryo sex identification because of its advantages (wide application range and so on) and disadvantages. At present, Brucella LAMP identification method based on Brucella outer membrane protein ge

参考文献

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