布鲁氏菌病与Q热检测方法的建立以及共感染研究

发布时间：2018-02-15 05:16

本文关键词： 布鲁氏菌病 Q热 IS711 高拷贝基因靶标基因　出处：《石河子大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:临床样本中细菌负荷相对较低,难以检出,尤其是寄生在宿主细胞内引起慢性感染的细菌,如本研究中布鲁氏菌和柯克斯体。检测灵敏度的改进将大大减少假阴性结果,有助于更准确的诊断。在本研究中,我们提出一种通过检测基因组中多拷贝基因以提高核酸检测灵敏度的新策略,并研究布鲁氏菌和柯克斯体是否存在共感染。方法:(1)布鲁氏菌作为一个测试案例。通过对羊种布鲁氏菌基因组序列进行筛选,确定几种高拷贝基因。定量PCR比较它们的检测效率,对IS711序列的分析及临床样品检测。(2)基于柯克斯体的多拷贝基因IS1111a,我们对其设计了引物和探针,针对检测过程中可能引起的污染,我们利用融合PCR的方法构建了假阳性质粒作为阳性对照,以此方法检测蜱的DNA样本。(3)对新疆地区和东北地区进行布鲁氏菌病和Q热的检测,了解两种病原体是否存在共感染。结果:(1)对羊种布鲁氏菌16M基因组序列分析,共发现38个多拷贝基因。用IS711、BMEI1001、BMEI0775、BMEI0027引物,定量PCR扩增梯度稀释的布鲁氏菌16M基因组DNA时,多拷贝基因表现出较好的灵敏度。检测20个血清学阳性的布氏样本,IS711和BMEI1001的靶浓度始终高于BMEI0775和BMEI0027。IS711的靶浓度范围从1.92到5.92(95%CI 2.88-3.94),其次为BMEI1001,范围从2.58到5.35(95%CI 2.91-3.94)。20个布鲁氏菌血清学阳性样本检测,IS711检测到17个阳性,其次是BMEI1001 14个,BMEI0775 6个和BMEI0027 5个。(2)多拷贝插入序列IS1111a作为Q热的靶标基因,荧光定量PCR对178份白城样本总核酸检测,4份Q热检测阳性,成功构建的假阳性质粒可以代替真阳性质粒标准品作对照,大大减少了污染问题。(3)库尔勒、伊犁、阿图什、吉木乃、哈巴河的布鲁氏菌病和Q热的共感染率分别为6.94%、10.98%、4.85%、7.46%、11.76%,东北地区共感染率为0。结论:临床样本中细菌病原体因浓度低难以检出。尽管高敏感的检测方法正运用于病原体检测,但仍然被细菌的数量低所限制。在这项研究中,我们提出了一个基于基因组中高拷贝数基因以提高检测灵敏度的新策略,敏感性的提高取决于细菌中靶标基因的拷贝数。因为几乎所有已知的细菌病原体的基因组已经测序和注释,所以可以筛选多拷贝基因作为靶标基因。所以,这种策略是通用的,可以扩展运用到其他细菌的检测。
[Abstract]:Objective: the bacterial load in clinical samples is relatively low and difficult to detect, especially for those bacteria that cause chronic infection in host cells, such as Brucella and Coxella in this study. Improved sensitivity will greatly reduce false negative results. In this study, we propose a new strategy to improve the sensitivity of nucleic acid detection by detecting multiple copies of genes in the genome. The co-infection of Brucella and Coxella was studied. Methods Brucella was used as a test case. The genomic sequence of Brucella was screened. Several high copy genes were identified. Quantitative PCR was used to compare their detection efficiency. The analysis of IS711 sequence and clinical sample detection. 2) based on Coxella multicopy gene IS1111a, we designed primers and probes for it. In view of the possible contamination in the detection process, we constructed a false positive plasmid as a positive control by using the method of fusion PCR to detect brucellosis and Q fever in Xinjiang and Northeast China by using this method to detect DNA samples of ticks. Results A total of 38 multicopy genes were found in 16M genome sequence analysis of Brucella sibiricus. The genomic DNA of 16M strain of Brucella was amplified by quantitative PCR using IS7111BMEI1001BMEI0775BMEI0027 primer. The target concentrations of IS711 and BMEI1001 in 20 seropositive samples were always higher than those of BMEI0775 and BMEI0027.IS711, followed by BMEI 1001, CI 2.91-3.940.Twenty brucellosis strains were found to be in the range of 1.92 to 5.9295 CI 2.88-3.94, followed by BMEI1001, with a range of 2.58 to 5.35995 CI 2.91-3.94. Seropositive samples detected 17 positive in IS711. Secondly, BMEI1001 14 BMEI0775 6 and BMEI0027 5. 2) IS1111a were used as the target genes of Q fever. The total nucleic acid of 178 samples of Baicheng was detected by fluorescence quantitative PCR. 4 of them were positive for Q fever. The false-positive plasmids constructed successfully can replace the standard plasmids of true-positive plasmids, and greatly reduce the pollution problem of Kurla, Yili, Atush, Jimenai. The co-infection rates of brucellosis and Q fever in Haba River were 6.940.98 and 4.857.46 and 11.766.Conclusion: it is difficult to detect bacterial pathogens in clinical samples because of their low concentration. But it is still limited by the low number of bacteria. In this study, we proposed a new strategy based on high copy number genes in the genome to improve detection sensitivity. The increase in sensitivity depends on the number of copies of target genes in bacteria. Because the genomes of almost all known bacterial pathogens have been sequenced and annotated, multiple copies of genes can be screened as target genes. Can be extended to other bacteria detection.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R516.7;R513.4

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