鹦鹉热嗜衣原体禽鸟株的分离、鉴定与基因分型

发布时间：2018-04-05 07:26

本文选题：鹦鹉热嗜衣原体（C.psittaci　切入点：Cps）　出处：《南华大学》2012年硕士论文

【摘要】：目的：优化鹦鹉热嗜衣原体（Chlamydophila psittaci，C.psittaci，Cps）禽鸟株分离鉴定培养技术，通过测序及进化树分析ompA基因的多态性，研究Cps的种系发育史及获得可靠Cps感染的流行病学资料，掌握衡阳地区Cps的流行病学特征，从而为Cps感染的预防、诊断、治疗措施提供依据。方法：收集衡阳地区可疑的禽鸟标本，利用分子生物学技术，提取禽鸟肝组织全基因组DNA，设计MOMP特异性引物，PCR扩增约264bp的基因片段，用1.7%琼脂糖凝胶电泳分离。将PCR阳性禽鸟标本肝组织匀浆液接种到单层敏感细胞株人喉癌上皮细胞（Hep-2）细胞和非洲绿猴肾细胞(Vero)中培养，免疫荧光染色法（IF）鉴定Cps包涵体，免疫荧光显微镜下观察两种细胞内包涵体形态的变化。按照文献设计另一对MOMP特异性引物，PCR扩增约1000bp的基因片段，用1.2%琼脂糖凝胶电泳分离，将阳性PCR产物进行纯化后送往测序公司进行测序，同时从Genebank中获得Cps各型参考株的ompA基因序列，利用软件ClustalX2和MEGA3构建系统进化树分析临床株与各参考株之间的亲缘关系。通过BLAST分析8例标本基因间的相似性。结果：通过PCR扩增及测序的方法从591例禽鸟标本（其中鹦鹉425只，鸽子23只及其它类型禽鸟143只）中检测到8例阳性，其中7例来源于鹦鹉，染率约为1.64%，1例来源于相思鸟。鸽子的染率为0%，其它类型禽鸟的染率约为0.69%；8例阳性标本在Hep-2及Vero细胞中成功培养出7株，成功率为87.5%；通过Cps在Hep-2细胞与Vero细胞中培养生长情况的比较，发现Hep-2细胞在原代培养时其胞内包涵体形成单位（IFU）数量大于Vero细胞，培养三代后免疫荧光染色发现，Vero细胞内的IFU数量较Hep-2细胞大；对8例阳性禽鸟标本PCR产物ompA基因序列进行测序，测序结果与Cps各型参考株ompA序列进行软件分析，系统进化树显示其中7例为A型，另外一例暂未能分型；将8例阳性禽鸟标本PCR产物ompA基因序列与C. psittaci6BC标准株ompA基因进行BLAST分析，Cps6BC与其中7例的相似度在92%～99%之间，另外一例与Cps6BC的相似度仅为58%；将E9的ompA基因与Genebank中其它已知菌株基因进行比对也未能对其进行确定。结论：（1）成功分离Cps禽鸟株7株，并完善了Cps阳性禽鸟株分离鉴定培养技术；（2）衡阳地区Cps可能主要以鹦鹉为其宿主，以基因型A为主要型别，，且存在一定的变异。
[Abstract]:Objective: to optimize the isolation, identification and culture techniques of Chlamydophila psittaciae (C. psittacii) avian strains, and analyze the polymorphism of ompA gene by sequencing and phylogenetic tree analysis, and to study the phylogenetic history of Cps and to obtain reliable epidemiological data of Cps infection.To grasp the epidemiological characteristics of Cps in Hengyang area, so as to provide basis for the prevention, diagnosis and treatment of Cps infection.Methods: the samples of birds in Hengyang were collected, and the whole genome DNA of bird liver tissue was extracted by molecular biology technique. About 264bp gene fragments were amplified by MOMP specific primer and separated by 1.7% agarose gel electrophoresis.The liver tissue homogenate of PCR positive birds was inoculated into human laryngeal carcinoma epithelial cells (Hep-2) and African green monkey kidney cells (PCR). The inclusion bodies of Cps were identified by immunofluorescence staining.The morphological changes of two kinds of inclusion bodies were observed under immunofluorescence microscope.According to the literature, another pair of MOMP specific primers were designed to amplify the 1000bp gene fragments. The positive PCR products were purified by 1.2% agarose gel electrophoresis and sent to the sequencing company for sequencing.At the same time, the ompA gene sequences of Cps reference strains were obtained from Genebank, and phylogenetic tree was constructed by software ClustalX2 and MEGA3 to analyze the relationship between clinical strains and reference strains.The genetic similarity of 8 specimens was analyzed by BLAST.Results:Among the 591 bird samples (425 parrots, 23 pigeons and 143 other birds), 8 were positive by PCR amplification and sequencing, of which 7 were from parrots and 1 from Acacia.The staining rate of pigeons was 0, and that of other types of birds was about 0.69. 7 strains were successfully cultured in Hep-2 and Vero cells, and the success rate was 87.5%.It was found that the number of Hep-2 cells was larger than that of Vero cells in primary culture, and the number of IFU in Vero cells was larger than that in Hep-2 cells after three passages.The ompA gene sequence of PCR product from 8 positive bird samples was sequenced. The results were analyzed by software with ompA sequences of Cps reference strains. The phylogenetic tree showed that 7 of them were type A, and the other one could not be typed.The PCR product ompA gene sequence of 8 positive bird samples and the ompA gene of C. psittaci6BC standard strain were analyzed by BLAST. The similarity between Cps6BC and 7 of them was between 92% and 99%.In the other case, the similarity between E9 and Cps6BC was only 58 and the ompA gene of E9 could not be determined by comparing with other known genes in Genebank.Conclusion:1) 7 Cps bird strains were isolated successfully, and the isolation and culture techniques of Cps positive bird strains were improved.2) in Hengyang area, parrot is the main host of Cps, genotype A is the main type, and there is some variation.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R374.2

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