立克次体分子生物学检测方法建立及云南红塔立克次体感染情况调查
发布时间：2019-01-26 09:23
【摘要】： 立克次体(Rickettsias)为原核细胞型、革兰染色阴性的专性细胞内寄生菌。分类学属于变形纲立克次体目(Rickettsiales),下属的立克次体科包括立克次体属( Rickettsia )、恙虫病东方体属( Orientia tsutsugamushi)、埃立克体属(Ehrlichiea),其中立克次体属又分为斑疹伤寒群、斑点热群,自然界已发现有20余种对人类致病。近年新病种和新疫区不断被发现,立克次体病已成为世界性关注的疾病。由于非特异的临床表现,实验室诊断成为主要依据。目前PCR方法已被广泛用于立克次体的实验室诊断,但多数方法只针对单个属或种特异,灵敏度往往也达不到实际检测要求。为提供更快速、灵敏的检测方法,本研究以PCR技术为基础,应用克隆、测序及序列分析等分子生物学、生物信息学手段建立灵敏、特异地立克次体属和恙虫病东方体属多种属立克次体的检测方法,并应用此方法对自然界鼠宿主、媒介生物、临床病人标本进行检测、产物测序、序列分析等研究。获得了以下结果: 1以PCR为基础的多种属立克次体检测方法建立 根据立克次体属和恙虫病东方体属热休克蛋白编码基因(groEL)的保守区设计外引物,以文献属特异引物对为内引物组成巢式PCR。通过产物片段大小、测序或PCR-RFLP鉴定不同属、种甚至不同型别立克次体。特异性地扩增立克次体属和恙虫病东方体属立克次体DNA,每个反应可检测到1×101拷贝数标准模板,比普通PCR灵敏度提高10倍。适于地方性斑疹伤寒、流行性斑疹伤寒、斑点热和恙虫病地同时检测。 2方法的应用 对从云南省玉溪市红塔区收集的各鼠肝脏、脾脏及部分鼠全血,鼠蚤、螨及病人标本进行groEL基因巢式PCR检测。34.78%的鼠有阳性扩增,斑疹伤寒、恙虫病和斑点热扩增阳性率分别为28.12%、19.13%、0.29%。从蚤提取的DNA中扩增到斑疹伤寒片段,从螨提取的DNA中扩增到恙虫病片段。8例可疑立克次体病病人血标本中,4例扩增出斑疹伤寒片段,其中1例同时扩增出恙虫病片段。部分产物序列比较分析发现斑疹伤寒、恙虫病和班点热片段序列分别与GeneBank中已知的莫氏立克次体(R.typhi) W、87-100、Ethiopian AZ322株,恙虫病(O.t)Karp型和西伯利亚立克次体(R.sibirica)核苷酸和推定氨基酸同源性最高,多数100%同源,21株斑疹伤寒和恙虫病产物核苷酸序列有碱基替换,呈99%同源,其中7株推定的氨基酸序列有替换突变,呈99%,98%同源。间接免疫荧光实验(Indirect fluorescent antibody test,IFA)鼠血清R.typhi W株、康氏立克次体(R.conorii)、O.t Karp型、Kato型、Gilliam型抗体阳性率分别为41.36%、31.82%、35.45%、31.36%、25.91%。从分子生物学和血清学方面证实了红塔区有地方性斑疹伤寒、恙虫病及班点热的流行,有地方性斑疹伤寒和恙虫病的核苷酸和氨基酸突变株存在。 各镇鼠标本地方性斑疹伤寒和恙虫病扩增阳性率均有显著性差异(P㩳0.05),血清R.typhi、O.t Karp型、Kato型IgG抗体阳性率也均有显著性差异(P㩳0.05),各镇鼠标本斑点热扩增阳性率及血清R.conori和O.t Gilliam IgG抗体阳性率均无显著性差异(P㧐0.05)。斑点热在洛河乡以外的其它乡镇有流行,地方性斑疹伤寒和恙虫病疫源地范围已扩大到全区,地方性斑疹伤寒在研和镇、春和镇、北城镇、州城有高度流行,恙虫病在全区各乡镇均有高度流行。 PCR检测发现1位病人和13.33%的鼠标本有地方性斑疹伤寒、恙虫病双重扩增,14.09%的鼠血清中同时存在R.typhi W和O.t Karp型抗体,怀疑自然鼠宿主和病人有地方性斑疹伤寒和恙虫病的双重感染,红塔区可能为生态结构复杂的自然疫源地。 应用新建的巢式PCR方法同时完成了对红塔区全区各类型标本的立克次体属和恙虫病东方体属立克次体的检测及分群、分型鉴定,进一步证实了此新建方法的实用性,为流行病学调查提供科学依据。
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R376;R450;R513
本文编号：2415348
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R376;R450;R513
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