本文關鍵詞： 布魯氏菌病 Q熱 IS711 高拷貝基因 靶標基因　出處：《石河子大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:臨床樣本中細菌負荷相對較低,難以檢出,尤其是寄生在宿主細胞內引起慢性感染的細菌,如本研究中布魯氏菌和柯克斯體。檢測靈敏度的改進將大大減少假陰性結果,有助于更準確的診斷。在本研究中,我們提出一種通過檢測基因組中多拷貝基因以提高核酸檢測靈敏度的新策略,并研究布魯氏菌和柯克斯體是否存在共感染。方法:(1)布魯氏菌作為一個測試案例。通過對羊種布魯氏菌基因組序列進行篩選,確定幾種高拷貝基因。定量PCR比較它們的檢測效率,對IS711序列的分析及臨床樣品檢測。(2)基于柯克斯體的多拷貝基因IS1111a,我們對其設計了引物和探針,針對檢測過程中可能引起的污染,我們利用融合PCR的方法構建了假陽性質粒作為陽性對照,以此方法檢測蜱的DNA樣本。(3)對新疆地區(qū)和東北地區(qū)進行布魯氏菌病和Q熱的檢測,了解兩種病原體是否存在共感染。結果:(1)對羊種布魯氏菌16M基因組序列分析,共發(fā)現38個多拷貝基因。用IS711、BMEI1001、BMEI0775、BMEI0027引物,定量PCR擴增梯度稀釋的布魯氏菌16M基因組DNA時,多拷貝基因表現出較好的靈敏度。檢測20個血清學陽性的布氏樣本,IS711和BMEI1001的靶濃度始終高于BMEI0775和BMEI0027。IS711的靶濃度范圍從1.92到5.92(95%CI 2.88-3.94),其次為BMEI1001,范圍從2.58到5.35(95%CI 2.91-3.94)。20個布魯氏菌血清學陽性樣本檢測,IS711檢測到17個陽性,其次是BMEI1001 14個,BMEI0775 6個和BMEI0027 5個。(2)多拷貝插入序列IS1111a作為Q熱的靶標基因,熒光定量PCR對178份白城樣本總核酸檢測,4份Q熱檢測陽性,成功構建的假陽性質粒可以代替真陽性質粒標準品作對照,大大減少了污染問題。(3)庫爾勒、伊犁、阿圖什、吉木乃、哈巴河的布魯氏菌病和Q熱的共感染率分別為6.94%、10.98%、4.85%、7.46%、11.76%,東北地區(qū)共感染率為0。結論:臨床樣本中細菌病原體因濃度低難以檢出。盡管高敏感的檢測方法正運用于病原體檢測,但仍然被細菌的數量低所限制。在這項研究中,我們提出了一個基于基因組中高拷貝數基因以提高檢測靈敏度的新策略,敏感性的提高取決于細菌中靶標基因的拷貝數。因為幾乎所有已知的細菌病原體的基因組已經測序和注釋,所以可以篩選多拷貝基因作為靶標基因。所以,這種策略是通用的,可以擴展運用到其他細菌的檢測。
[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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R516.7;R513.4

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