【摘要】： 立克次體(Rickettsias)為原核細(xì)胞型、革蘭染色陰性的專性細(xì)胞內(nèi)寄生菌。分類學(xué)屬于變形綱立克次體目(Rickettsiales),下屬的立克次體科包括立克次體屬( Rickettsia )、恙蟲病東方體屬( Orientia tsutsugamushi)、埃立克體屬(Ehrlichiea),其中立克次體屬又分為斑疹傷寒群、斑點(diǎn)熱群,自然界已發(fā)現(xiàn)有20余種對(duì)人類致病。近年新病種和新疫區(qū)不斷被發(fā)現(xiàn),立克次體病已成為世界性關(guān)注的疾病。由于非特異的臨床表現(xiàn),實(shí)驗(yàn)室診斷成為主要依據(jù)。目前PCR方法已被廣泛用于立克次體的實(shí)驗(yàn)室診斷,但多數(shù)方法只針對(duì)單個(gè)屬或種特異,靈敏度往往也達(dá)不到實(shí)際檢測(cè)要求。為提供更快速、靈敏的檢測(cè)方法,本研究以PCR技術(shù)為基礎(chǔ),應(yīng)用克隆、測(cè)序及序列分析等分子生物學(xué)、生物信息學(xué)手段建立靈敏、特異地立克次體屬和恙蟲病東方體屬多種屬立克次體的檢測(cè)方法,并應(yīng)用此方法對(duì)自然界鼠宿主、媒介生物、臨床病人標(biāo)本進(jìn)行檢測(cè)、產(chǎn)物測(cè)序、序列分析等研究。獲得了以下結(jié)果: 1以PCR為基礎(chǔ)的多種屬立克次體檢測(cè)方法建立 根據(jù)立克次體屬和恙蟲病東方體屬熱休克蛋白編碼基因(groEL)的保守區(qū)設(shè)計(jì)外引物,以文獻(xiàn)屬特異引物對(duì)為內(nèi)引物組成巢式PCR。通過產(chǎn)物片段大小、測(cè)序或PCR-RFLP鑒定不同屬、種甚至不同型別立克次體。特異性地?cái)U(kuò)增立克次體屬和恙蟲病東方體屬立克次體DNA,每個(gè)反應(yīng)可檢測(cè)到1×101拷貝數(shù)標(biāo)準(zhǔn)模板,比普通PCR靈敏度提高10倍。適于地方性斑疹傷寒、流行性斑疹傷寒、斑點(diǎn)熱和恙蟲病地同時(shí)檢測(cè)。 2方法的應(yīng)用 對(duì)從云南省玉溪市紅塔區(qū)收集的各鼠肝臟、脾臟及部分鼠全血,鼠蚤、螨及病人標(biāo)本進(jìn)行g(shù)roEL基因巢式PCR檢測(cè)。34.78%的鼠有陽性擴(kuò)增,斑疹傷寒、恙蟲病和斑點(diǎn)熱擴(kuò)增陽性率分別為28.12%、19.13%、0.29%。從蚤提取的DNA中擴(kuò)增到斑疹傷寒片段,從螨提取的DNA中擴(kuò)增到恙蟲病片段。8例可疑立克次體病病人血標(biāo)本中,4例擴(kuò)增出斑疹傷寒片段,其中1例同時(shí)擴(kuò)增出恙蟲病片段。部分產(chǎn)物序列比較分析發(fā)現(xiàn)斑疹傷寒、恙蟲病和班點(diǎn)熱片段序列分別與GeneBank中已知的莫氏立克次體(R.typhi) W、87-100、Ethiopian AZ322株,恙蟲病(O.t)Karp型和西伯利亞立克次體(R.sibirica)核苷酸和推定氨基酸同源性最高,多數(shù)100%同源,21株斑疹傷寒和恙蟲病產(chǎn)物核苷酸序列有堿基替換,呈99%同源,其中7株推定的氨基酸序列有替換突變,呈99%,98%同源。間接免疫熒光實(shí)驗(yàn)(Indirect fluorescent antibody test,IFA)鼠血清R.typhi W株、康氏立克次體(R.conorii)、O.t Karp型、Kato型、Gilliam型抗體陽性率分別為41.36%、31.82%、35.45%、31.36%、25.91%。從分子生物學(xué)和血清學(xué)方面證實(shí)了紅塔區(qū)有地方性斑疹傷寒、恙蟲病及班點(diǎn)熱的流行,有地方性斑疹傷寒和恙蟲病的核苷酸和氨基酸突變株存在。 各鎮(zhèn)鼠標(biāo)本地方性斑疹傷寒和恙蟲病擴(kuò)增陽性率均有顯著性差異(P㩳0.05),血清R.typhi、O.t Karp型、Kato型IgG抗體陽性率也均有顯著性差異(P㩳0.05),各鎮(zhèn)鼠標(biāo)本斑點(diǎn)熱擴(kuò)增陽性率及血清R.conori和O.t Gilliam IgG抗體陽性率均無顯著性差異(P㧐0.05)。斑點(diǎn)熱在洛河鄉(xiāng)以外的其它鄉(xiāng)鎮(zhèn)有流行,地方性斑疹傷寒和恙蟲病疫源地范圍已擴(kuò)大到全區(qū),地方性斑疹傷寒在研和鎮(zhèn)、春和鎮(zhèn)、北城鎮(zhèn)、州城有高度流行,恙蟲病在全區(qū)各鄉(xiāng)鎮(zhèn)均有高度流行。 PCR檢測(cè)發(fā)現(xiàn)1位病人和13.33%的鼠標(biāo)本有地方性斑疹傷寒、恙蟲病雙重?cái)U(kuò)增,14.09%的鼠血清中同時(shí)存在R.typhi W和O.t Karp型抗體,懷疑自然鼠宿主和病人有地方性斑疹傷寒和恙蟲病的雙重感染,紅塔區(qū)可能為生態(tài)結(jié)構(gòu)復(fù)雜的自然疫源地。 應(yīng)用新建的巢式PCR方法同時(shí)完成了對(duì)紅塔區(qū)全區(qū)各類型標(biāo)本的立克次體屬和恙蟲病東方體屬立克次體的檢測(cè)及分群、分型鑒定,進(jìn)一步證實(shí)了此新建方法的實(shí)用性,為流行病學(xué)調(diào)查提供科學(xué)依據(jù)。
[Abstract]:The rickettsias is a specific intracellular parasitic strain of the prokaryotic cell type and the leather blue staining negative. Taxonomy, belonging to the genus Ricketsiales, of the genus Rickettsiales, includes the genus Rickettsia, the Orientia tsoutsugusi, and Ehrlichia, in which the rickettsia is divided into a group of typhus and a group of spots, More than 20 species have been found to be pathogenic to the human nature. In recent years, the new disease and the new epidemic area have been continuously found, and the disease has become a worldwide concern. Laboratory diagnosis is the main basis for non-specific clinical manifestations. The present PCR method has been widely used in the laboratory diagnosis of the rickettsia, but most of the methods are only for a single genus or species, and the sensitivity is often less than the actual detection requirement. In order to provide a more rapid and sensitive detection method, this study is based on the PCR technology, and uses the molecular biology and bioinformatics methods such as cloning, sequencing and sequence analysis to establish a sensitive, specific and specific species of the genus and the Botrypanosoma. and the method is used for detecting the host, the medium organism, the clinical patient specimen, the product sequencing, the sequence analysis and the like of the natural mouse host, the medium organism and the clinical patient. The following results were obtained: 1. Multiple genera based on PCR in that method, an external primer is designed accord to the conserved region of the heat shock protein coding gene (ground EL) of the genus Rickettsia and the oriental body of the cysticercosis, so as to A specific primer pair is used as an inner primer to form a nest type PCR. The product fragment size, sequencing or PCR-RF LP identification of different genera, species, or even different species. The specific amplification of the species of the genus of the genus of the genus of the genus Rickettsia and the Orientina of the genus Euplotes is the Rickettsial DNA, each of which can be detected to have a copy number of 1 to 101. Standard template, 10-fold higher than common PCR sensitivity. Suitable for endemic typhus, flow typhus The method was used to detect the whole blood of the rat liver, spleen and part of the rat's liver, spleen and part of the mice from the Hongta district of Yuxi city of Yunnan province. 4.78% of the mice were positive for amplification, The positive rates of heat amplification of typhus, cysticercosis and spot were 28. 12%, 19. 13%, and 0. 29%, respectively. In the blood samples of the patients with suspected rickettsugamushi disease, 4 cases were amplified with typhus, one of which was amplified out of the disease. The results of partial product sequence analysis showed that the sequences of typhus, cysticercosis and spot heat were respectively similar to that of the known Moritz type (R. typhi) W, 87-100, Ethopian AZ322 and Opt. K in GeneBank, respectively. The homology of R. sibirica and R. sibirica has the highest homology with the putative amino acid, most of which are 100% homologous, 21 strains of typhus and cysticercosis, and the acid sequence is base. The substitution of the base was 99%, of which 7 putative amino acid sequences had the substitution mutation, which was 99% and 98% homologous. The indirect immunofluorescence assay (IFA) rat serum R. typhi W, Concorii, O.t. Karp, Kato type, and Gilliam type antibody were positive. The sex rate was 41. 36%, 31. 82%, 35. 45%, 31. 36%, 25. 91%, respectively. There was a significant difference in the positive rates of the amplification of local typhus and malarial disease (P? 0.05). The positive rates of serum R. typhi, O. t Karp and Kato-type IgG were significantly different (P <0.05). The positive rate of the positive rate and the positive rate of the serum R. conori and the O. t Gilliam IgG antibody had no significant difference (P <0.05). The spot heat was prevalent in other towns other than the Luohe Township, and the range of the local typhus and the plague foci of the endemic typhus was already in the range. The prevalence of endemic typhus in the whole district and local typhus in the areas of research and town, spring and town, north and town, and state city is highly popular. R. typhi was present at the same time in 09% of the rat serum W and O. t Karp-type antibodies, suspected of the double infection of the host and the patient of the natural mouse and the endemic typhus and the parasitic disease of the patient, may be the natural foci of the complex structure of the ecosystem. The new nested PCR method is applied in the same time.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R376;R450;R513

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