本文選題：鸚鵡熱嗜衣原體（C.psittaci　切入點：Cps）　出處：《南華大學(xué)》2012年碩士論文

【摘要】：目的：優(yōu)化鸚鵡熱嗜衣原體（Chlamydophila psittaci，C.psittaci，Cps）禽鳥株分離鑒定培養(yǎng)技術(shù)，通過測序及進(jìn)化樹分析ompA基因的多態(tài)性，研究Cps的種系發(fā)育史及獲得可靠Cps感染的流行病學(xué)資料，掌握衡陽地區(qū)Cps的流行病學(xué)特征，從而為Cps感染的預(yù)防、診斷、治療措施提供依據(jù)。 方法：收集衡陽地區(qū)可疑的禽鳥標(biāo)本，利用分子生物學(xué)技術(shù)，提取禽鳥肝組織全基因組DNA，設(shè)計MOMP特異性引物，PCR擴(kuò)增約264bp的基因片段，用1.7%瓊脂糖凝膠電泳分離。將PCR陽性禽鳥標(biāo)本肝組織勻漿液接種到單層敏感細(xì)胞株人喉癌上皮細(xì)胞（Hep-2）細(xì)胞和非洲綠猴腎細(xì)胞(Vero)中培養(yǎng)，免疫熒光染色法（IF）鑒定Cps包涵體，免疫熒光顯微鏡下觀察兩種細(xì)胞內(nèi)包涵體形態(tài)的變化。按照文獻(xiàn)設(shè)計另一對MOMP特異性引物，PCR擴(kuò)增約1000bp的基因片段，用1.2%瓊脂糖凝膠電泳分離，將陽性PCR產(chǎn)物進(jìn)行純化后送往測序公司進(jìn)行測序，同時從Genebank中獲得Cps各型參考株的ompA基因序列，利用軟件ClustalX2和MEGA3構(gòu)建系統(tǒng)進(jìn)化樹分析臨床株與各參考株之間的親緣關(guān)系。通過BLAST分析8例標(biāo)本基因間的相似性。 結(jié)果： 通過PCR擴(kuò)增及測序的方法從591例禽鳥標(biāo)本（其中鸚鵡425只，鴿子23只及其它類型禽鳥143只）中檢測到8例陽性，其中7例來源于鸚鵡，染率約為1.64%，1例來源于相思鳥。鴿子的染率為0%，其它類型禽鳥的染率約為0.69%；8例陽性標(biāo)本在Hep-2及Vero細(xì)胞中成功培養(yǎng)出7株，成功率為87.5%；通過Cps在Hep-2細(xì)胞與Vero細(xì)胞中培養(yǎng)生長情況的比較，發(fā)現(xiàn)Hep-2細(xì)胞在原代培養(yǎng)時其胞內(nèi)包涵體形成單位（IFU）數(shù)量大于Vero細(xì)胞，培養(yǎng)三代后免疫熒光染色發(fā)現(xiàn)，Vero細(xì)胞內(nèi)的IFU數(shù)量較Hep-2細(xì)胞大；對8例陽性禽鳥標(biāo)本PCR產(chǎn)物ompA基因序列進(jìn)行測序，測序結(jié)果與Cps各型參考株ompA序列進(jìn)行軟件分析，系統(tǒng)進(jìn)化樹顯示其中7例為A型，另外一例暫未能分型；將8例陽性禽鳥標(biāo)本PCR產(chǎn)物ompA基因序列與C. psittaci6BC標(biāo)準(zhǔn)株ompA基因進(jìn)行BLAST分析，Cps6BC與其中7例的相似度在92%～99%之間，另外一例與Cps6BC的相似度僅為58%；將E9的ompA基因與Genebank中其它已知菌株基因進(jìn)行比對也未能對其進(jìn)行確定。 結(jié)論： （1）成功分離Cps禽鳥株7株，并完善了Cps陽性禽鳥株分離鑒定培養(yǎng)技術(shù)； （2）衡陽地區(qū)Cps可能主要以鸚鵡為其宿主，以基因型A為主要型別，，且存在一定的變異。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R374.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 唐國芳;陳麗麗;劉良專;李忠玉;王紹勝;徐磊;吳移謀;;鸚鵡熱嗜衣原體禽鳥株的分離鑒定及小鼠呼吸道感染模型的建立[J];微生物學(xué)報;2010年12期

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