蜱体伯氏疏螺旋体分离鉴定及莱姆病ELISA检测方法的建立
|
摘要
莱姆病是经硬蜱传播的,由伯氏疏螺旋体引起的人畜共患病。该病呈世界性分布,目前,全球估计年发病人数约为30万人左右,且有不断增长的趋势。在美国,莱姆病有“第二艾滋病”之称,1992年世界卫生组织将此病列为重点防治对象,现已成为全球性的公共卫生问题之一。近年来,对莱姆病病原生物学、致病机理、流行病学、诊断及综合防治方面的研究已取得了一定的成果,但由于伯氏疏螺旋体抗原结构的复杂性以及莱姆病的临床症状的多样性,使得该病的确诊较困难。另外,莱姆病作为一种新发病,在我国对其传播媒介蜱的种类尚未完全明晰,同时关于其病原体的基因型及分类地位均未阐明,这给诊断和防治该病带来极大的挑战。因此,阐明其传播媒介的种类和病原体的分类地位将有助于我们建立高效、快速诊断手段,合理地开展该病的防治工作。
本文主要在莱姆病病原体的分类学及其诊断学两个方面进行探索。(1)对源于我国部分林区采集的亚洲璃眼蜱(Hyalomma asiaticum)、长角血蜱(Haemaphysalis longicornis)等多种蜱种进行伯氏疏螺旋体的检测;(2)对所分离的螺旋体进行分型研究;(3)对所获得的伯氏疏螺旋体鞭毛基因及鞭毛基因的保守区段进行克隆,并在体外表出达伯氏疏螺旋体鞭毛基因保守区段,用保守区段重组蛋白作为莱姆病的诊断抗原,建立莱姆病ELISA诊断方法。
第一部分,用套式PCR方法扩增蜱体内的伯氏疏螺旋体鞭毛基因,对采自新疆、青岛和延边等地区的亚洲璃眼蜱和长角血蜱及蜱肠培养物进行检测,对PCR所扩增目的基因片段进行测序,测序结果经GenBank中的blast比对分析。采用BSK-H固体培养基分离培养技术,从蜱肠内分离和纯化伯氏疏螺旋体单克隆,并采用分子生物学方法对分离到的单克隆菌落进行分型。首先对新疆地区的亚洲璃眼蜱和镰形扇头蜱在显微镜下进行解剖,取其肠,用BSK-H液体培养基进行培养,收集蜱肠培养物,提取其基因组DNA,采用套式PCR方法扩增鞭毛基因。然后,对经鉴定含有伯氏螺旋体的肠培养物,利用BSK-H固体培养分离技术,对其进行分离纯化,建立了一种快速而方便的分离伯氏疏螺旋体培养方法。最后,应用recA基因和5S-23S间区基因作为分型的靶基因对分离到的伯氏疏螺旋体单克隆进行初步分型,recA基因是与伯氏疏螺旋体进化相关的基因,其GC含量的变化可以通过荧光定量PCR的熔解曲线很容易的反映出来,根据此特性,用定量荧光PCR熔解曲线对分离到的螺旋体单克隆进行初步分型。经初步分型,本次从亚洲璃眼蜱中分离到伽氏疏螺旋体、狭义的伯氏疏螺旋体等菌株。同时为进一步确定螺旋体基因型,对其5S-23S间区进行PCR扩增,并对扩增产物进行测序,根据其测序结果进一步分型。伯氏疏螺旋体单克隆的分离及初步分型的研究,为莱姆病病原生物学研究奠定了基础。
第二部分,对莱姆病血清学诊断技术进行了研究。该研究用重组伯氏疏螺旋体鞭毛基因蛋白作为诊断抗原,建立莱姆病ELISA诊断方法。本研究利用生物工程方法,选取鞭毛基因的氨基酸序列的第131-266区段作为目的片段,对其克隆,连接到PGEX-4T-1表达载体上,构建保守基因片段的重组质粒,然后转化到感受态细胞BL21中,原核表达伯氏疏螺旋体鞭毛基因抗原性和保守性较强的区段的平截性蛋白,用GST.BIND纯化系统纯化伯氏疏螺旋体鞭毛基因重组蛋白,所纯化的蛋白用作诊断抗原;同时将伯氏疏螺旋体分别接种小鼠和兔,两周后收集血清,用上述诊断抗原鉴定,发现经感染的小鼠和兔血清均为阳性血清,建立了敏感性和特异性较高的ELISA诊断方法。此方法为解决莱姆病诊断困难、难以确诊提供了一种有益的借鉴,特别为莱姆病早期的诊断提供了一种有力的手段,且有一定的临床应用价值。综上所述,本研究用简便快捷的固体培养方法对伯氏疏螺旋体单克隆分离株进行分离,并筛选出一种对我国莱姆病病原体分型新方法;建立了检测特异性强、敏感性高的莱姆病血清抗体的ELISA方法。这为有效、快速诊断以及合理防治莱姆病提供理论依据,同时也为该病的流行病学的调查工作开展提供了有力的手段。
Lyme disease is a zoonosis which is caused by Borrelia burgdorferi and transmitted via tick, and it can cause many organs and systems injured. Lyme disease is widely distributed in the word. Nowadays, there are about 0.3 million cases of lyme disease were reported every year, but this tendency is increasing. It is called“the second AIDS”in American. In 1992, it is listed as the main prevent disease by the World Health Organization, nowadays, it become one of the global health problem. Recently, great progreses have been made in many parts of Lyme disease, including pathogen, pathogenesis, epidemiolog, diagnosis and integrated control, but Lyme disease in diagnosis is difficult because of the complex antigenic structure and multiplicity clinical symptoms. Otherwise, as a new disease, the sort and distribution of tick, which can tansmitted it, still clearly got elucidation in China, meanwhile the genetypes of its pathogen are uninterrptly recorded, which will take challege for preventing and controling it. Therefore, to elucidate the sorts of intermediary ticks and classifying of the pathogen, it will be helpful for its rapid diagnosis and controling.
In the thesis, it composed of two parts involving in the etiological agent characterization and its diagnosis. Firstly, we detect Borrelia burgdorferi from Hyalomma asiaticum ,Haemaphysalis longicornis and its’eggs originated from Xinjiang, Jilin, and Shandong forest. Secondly, the typing study of isolated helicoid was made. Thirdly, flagellin gene of Borrelia burgdorferi and its conservative segment were cloned respectively, and the conservative segment was expressed in vitro to establish the ELISA for its diagnosis.
In the part one, nested PCR was applied to determine the flagellin gene of Lyme burgdorferi to find the carrier with borrelia burgdorferi of tick. Borrelia burgdorferi were isolated from Hyalomma asiaticum and Haemaphysalis longicornis, originated from Xinjiang, Jilin, and Shandong forest, genemic DNA was extrated, the aim gene was amplified, sequenced, and analysed by blast. Then, Borrelia burgdorferi from ticks was isolated and cloned by the BSK solid culture technology, and its typing was made by molecular biology. Firstly, the ticks were dissected under microscope to obtain their guts, and the guts were cultured in BSK-H liquid medium, then the cultures were collected and their genemic DNA was extracted, the nested PCR was applied to amplify the flagellin gene of Borrelia burgdorferi. BSK-H solid medium was used to isolate the clone of borrelia burgdorferi from the positive cultures which were identified by PCR.The reslut indicated that a rapid and convenient separation of Borrelia burgdorferi was established. Finally, initial typing of Borrelia burgdorferi was made by application of recA genes and the 5S-23S inter-region genes. recA gene is related with Borrelia burgdorferi in evolutionthe, and its GC content changes can be determined by fluorescence quantitative PCR curves easily. Based on this feature, we took quantitative fluorescent PCR to type it initially. After initial classification, Borrelia garinii, Borrelia burgdorferi sensu stricto and others from Hyalomma asiaticum. In order to make further determination of the type of spirochete, PCR was amplified 5S-23S inter-region, the amplified product was sequenced and analysed. The study of isolation of Borrelia burgdorferi and typing could be play foundation for pathogenic biology of Lyme disease.
In the part three, the study of diagnostic technique of serology in Lyme disease was made, and the ELISA was established with the recombinant protein. The aim fragment of 131-266 region of flagellar gene was cloned into vector of PGEX-4T-1 by bio-engineering, transformed into E.ciol BL21 competent cells, and expressed. The expressed protein was puried with the purification system of GST.BIND and used for diagnosis. Meanwhile, mice and rabbit were injected with Borrelia burgdorferi to prepare for positve serum, the ELISA with high sensitivity and specificity was set up. It settles for the difficulty in diagnosis to Lyme disease, and provide for the potent measure to diagnose it in clinic.
To sum up, Monoclone of Lyme disease spirochetes was isolated with convenient and shortcut solid culture, and the new type mean of isolation was ensured; the ELISA with sensitivity and specifity for detecteting the disease was established. The conclusion could be made that it provides the reference for its diagnosis and controling and the mean for the investigation of its epidemiology.
本文主要在莱姆病病原体的分类学及其诊断学两个方面进行探索。(1)对源于我国部分林区采集的亚洲璃眼蜱(Hyalomma asiaticum)、长角血蜱(Haemaphysalis longicornis)等多种蜱种进行伯氏疏螺旋体的检测;(2)对所分离的螺旋体进行分型研究;(3)对所获得的伯氏疏螺旋体鞭毛基因及鞭毛基因的保守区段进行克隆,并在体外表出达伯氏疏螺旋体鞭毛基因保守区段,用保守区段重组蛋白作为莱姆病的诊断抗原,建立莱姆病ELISA诊断方法。
第一部分,用套式PCR方法扩增蜱体内的伯氏疏螺旋体鞭毛基因,对采自新疆、青岛和延边等地区的亚洲璃眼蜱和长角血蜱及蜱肠培养物进行检测,对PCR所扩增目的基因片段进行测序,测序结果经GenBank中的blast比对分析。采用BSK-H固体培养基分离培养技术,从蜱肠内分离和纯化伯氏疏螺旋体单克隆,并采用分子生物学方法对分离到的单克隆菌落进行分型。首先对新疆地区的亚洲璃眼蜱和镰形扇头蜱在显微镜下进行解剖,取其肠,用BSK-H液体培养基进行培养,收集蜱肠培养物,提取其基因组DNA,采用套式PCR方法扩增鞭毛基因。然后,对经鉴定含有伯氏螺旋体的肠培养物,利用BSK-H固体培养分离技术,对其进行分离纯化,建立了一种快速而方便的分离伯氏疏螺旋体培养方法。最后,应用recA基因和5S-23S间区基因作为分型的靶基因对分离到的伯氏疏螺旋体单克隆进行初步分型,recA基因是与伯氏疏螺旋体进化相关的基因,其GC含量的变化可以通过荧光定量PCR的熔解曲线很容易的反映出来,根据此特性,用定量荧光PCR熔解曲线对分离到的螺旋体单克隆进行初步分型。经初步分型,本次从亚洲璃眼蜱中分离到伽氏疏螺旋体、狭义的伯氏疏螺旋体等菌株。同时为进一步确定螺旋体基因型,对其5S-23S间区进行PCR扩增,并对扩增产物进行测序,根据其测序结果进一步分型。伯氏疏螺旋体单克隆的分离及初步分型的研究,为莱姆病病原生物学研究奠定了基础。
第二部分,对莱姆病血清学诊断技术进行了研究。该研究用重组伯氏疏螺旋体鞭毛基因蛋白作为诊断抗原,建立莱姆病ELISA诊断方法。本研究利用生物工程方法,选取鞭毛基因的氨基酸序列的第131-266区段作为目的片段,对其克隆,连接到PGEX-4T-1表达载体上,构建保守基因片段的重组质粒,然后转化到感受态细胞BL21中,原核表达伯氏疏螺旋体鞭毛基因抗原性和保守性较强的区段的平截性蛋白,用GST.BIND纯化系统纯化伯氏疏螺旋体鞭毛基因重组蛋白,所纯化的蛋白用作诊断抗原;同时将伯氏疏螺旋体分别接种小鼠和兔,两周后收集血清,用上述诊断抗原鉴定,发现经感染的小鼠和兔血清均为阳性血清,建立了敏感性和特异性较高的ELISA诊断方法。此方法为解决莱姆病诊断困难、难以确诊提供了一种有益的借鉴,特别为莱姆病早期的诊断提供了一种有力的手段,且有一定的临床应用价值。综上所述,本研究用简便快捷的固体培养方法对伯氏疏螺旋体单克隆分离株进行分离,并筛选出一种对我国莱姆病病原体分型新方法;建立了检测特异性强、敏感性高的莱姆病血清抗体的ELISA方法。这为有效、快速诊断以及合理防治莱姆病提供理论依据,同时也为该病的流行病学的调查工作开展提供了有力的手段。
Lyme disease is a zoonosis which is caused by Borrelia burgdorferi and transmitted via tick, and it can cause many organs and systems injured. Lyme disease is widely distributed in the word. Nowadays, there are about 0.3 million cases of lyme disease were reported every year, but this tendency is increasing. It is called“the second AIDS”in American. In 1992, it is listed as the main prevent disease by the World Health Organization, nowadays, it become one of the global health problem. Recently, great progreses have been made in many parts of Lyme disease, including pathogen, pathogenesis, epidemiolog, diagnosis and integrated control, but Lyme disease in diagnosis is difficult because of the complex antigenic structure and multiplicity clinical symptoms. Otherwise, as a new disease, the sort and distribution of tick, which can tansmitted it, still clearly got elucidation in China, meanwhile the genetypes of its pathogen are uninterrptly recorded, which will take challege for preventing and controling it. Therefore, to elucidate the sorts of intermediary ticks and classifying of the pathogen, it will be helpful for its rapid diagnosis and controling.
In the thesis, it composed of two parts involving in the etiological agent characterization and its diagnosis. Firstly, we detect Borrelia burgdorferi from Hyalomma asiaticum ,Haemaphysalis longicornis and its’eggs originated from Xinjiang, Jilin, and Shandong forest. Secondly, the typing study of isolated helicoid was made. Thirdly, flagellin gene of Borrelia burgdorferi and its conservative segment were cloned respectively, and the conservative segment was expressed in vitro to establish the ELISA for its diagnosis.
In the part one, nested PCR was applied to determine the flagellin gene of Lyme burgdorferi to find the carrier with borrelia burgdorferi of tick. Borrelia burgdorferi were isolated from Hyalomma asiaticum and Haemaphysalis longicornis, originated from Xinjiang, Jilin, and Shandong forest, genemic DNA was extrated, the aim gene was amplified, sequenced, and analysed by blast. Then, Borrelia burgdorferi from ticks was isolated and cloned by the BSK solid culture technology, and its typing was made by molecular biology. Firstly, the ticks were dissected under microscope to obtain their guts, and the guts were cultured in BSK-H liquid medium, then the cultures were collected and their genemic DNA was extracted, the nested PCR was applied to amplify the flagellin gene of Borrelia burgdorferi. BSK-H solid medium was used to isolate the clone of borrelia burgdorferi from the positive cultures which were identified by PCR.The reslut indicated that a rapid and convenient separation of Borrelia burgdorferi was established. Finally, initial typing of Borrelia burgdorferi was made by application of recA genes and the 5S-23S inter-region genes. recA gene is related with Borrelia burgdorferi in evolutionthe, and its GC content changes can be determined by fluorescence quantitative PCR curves easily. Based on this feature, we took quantitative fluorescent PCR to type it initially. After initial classification, Borrelia garinii, Borrelia burgdorferi sensu stricto and others from Hyalomma asiaticum. In order to make further determination of the type of spirochete, PCR was amplified 5S-23S inter-region, the amplified product was sequenced and analysed. The study of isolation of Borrelia burgdorferi and typing could be play foundation for pathogenic biology of Lyme disease.
In the part three, the study of diagnostic technique of serology in Lyme disease was made, and the ELISA was established with the recombinant protein. The aim fragment of 131-266 region of flagellar gene was cloned into vector of PGEX-4T-1 by bio-engineering, transformed into E.ciol BL21 competent cells, and expressed. The expressed protein was puried with the purification system of GST.BIND and used for diagnosis. Meanwhile, mice and rabbit were injected with Borrelia burgdorferi to prepare for positve serum, the ELISA with high sensitivity and specificity was set up. It settles for the difficulty in diagnosis to Lyme disease, and provide for the potent measure to diagnose it in clinic.
To sum up, Monoclone of Lyme disease spirochetes was isolated with convenient and shortcut solid culture, and the new type mean of isolation was ensured; the ELISA with sensitivity and specifity for detecteting the disease was established. The conclusion could be made that it provides the reference for its diagnosis and controling and the mean for the investigation of its epidemiology.
引文
[1]Obechan, F.D.And Galun, R.Physiology of ticks [M].1982.Pergamon Press, Oxford.
[2]Alberto A. Guglielmone,Richard G.Robbins,Dmitry A. Apanaskevich.et al.Comments on controversial tick (Acari: Ixodida) species names and species described or resurrected from 2003 to 2008[J].Exp appl Acarol.2009.48:311–327
[3]陆支英,陆宝麟.医学昆虫学[M].1990.科学出版社.397-415.
[4]邓国藩,姜在阶.中国经济昆虫志[M].第三十九册.1991.科学出版社.11-13.
[5]Sauer JR, McSwain JL, Bowman AS, Essenberg RC.Tick salivary gland physiology [J]. Annu Rev Entomol.1995.40, 245-267
[6]Fogaca AC,da Sliva PI Jr,Miranda MT,et al.Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus[J].J Biol.Chem.1999.274(36):25330-255340
[7]龚海燕,周金林,周勇志等.镰形扇头蜱半饱血雌蜱cDNA文库的构建[J].中国人兽共患病杂志,2004, 20(6): 458-488
[8]柳支英,陆宝麟.医学昆虫学[M].北京:科学出版社.1990, 397-415
[9]Sauer J R, M cswain J L , Bowman A S, et al. Tick salivary gland physiology[J ]. Annu Rev Entomol, 1995, 40: 245-267
[10]周金林.蜱功能分子研究进展[J].动物医学进展, 2004, 25(1): 53-56.
[11]Shanahan GJ , Hart R J . Change in response of Lucilia cuprina to organophosphorus insecticides in Australia [J].Nature , 1966 , 212(5069) :1466-1467.
[12]仝彩玲,李培英,周金林.莱姆病诊断技术研究进展[J].中国动物传染病学报,2009;3(17):76.
[13]Henry M.Feder,Jr.,M.D.,Barbara J.B.Johnson,Ph.D.,et al.A Critical Appraisal of“Chronic Lyme Disease”.[J]. The new england journal of medicine.2007, 357:1422-1423
[14]廖德丰,陈季武,谢萍,等.蜱传性疾病——莱姆病螺旋体研究进展[J].中国兽医寄生虫病, 2004,12(2):36-38.
[15]Burgdorferi W,Barbour AG,Hayes SF,et al.Lyme disease—A tick borne spirochetosis , Science,1982,216;1317-1319.
[16]C.M.Fraser,S.Casjens,W.M.Huang,et al.Genomic sequence of a Lyme disease spirochaete.Borrelia burgdorferi.Nature,1997,390:580–586
[17]Vander FK.Everything you need to know about Lyme disease and other tick-borne disorders.NewYork.NY John Wiley&Sons Inc,1997,20-47
[18]孙毅,许荣满,张泮河,等.莱姆病人工宿主动物模型的建立[J].寄生虫与医学昆虫学报,2002,9(3):160.
[19]万康林,张金声,张哲夫.清除莱姆病螺旋体培养物中杂菌的方法研究.中华流行病学杂志1991,12;299-301
[20]张哲夫,万康林,王子敏,等.从湖南省靖县野兔体内分离出莱姆病螺旋体,中国媒介生物学及控制杂志,1992,3(特2);97-98
[21]Kurtti T J,Munderloh U G,Johnson R C,et al.Colony Formation and morphology in Borrelia Burgdorferi [J].J Clin Microbiol, 1987, 25(11); 2054-2058
[22]廖德丰,陈季武,谢萍,傅蓓蓓等.蜱传性疾病——莱姆病螺旋体研究进展[J].中国兽医寄生虫病,2004,12 (2):36-38
[23]J.Ohnishi, J.Piesman and A.M.de.Silva.Antigenic and genetic heterogeneity of Borrelia burgdorferipopulations transmitted by ticks.Proc Natl Acad Sci USA, 2001, 98:670-675.
[24]Kelly R.Cultivation of Borrelia hermsii.Science.1971, 173(995):443-444.
[25]Pollack R.J,S.R.TelfordIII,and A.Spielman.Standardization of medium for culturing Lyme disease spirochetes.J.Clin.Microbiol.1993,31(5):1251–1255
[26]Barbour A.G.Isolation and cultivation of Lyme disease spirochetes.Yale J.Biol.Med. 1984,57(4):521–525
[27]刘敏,王树声.莱姆病研究进展.广西预防医学,2001,7:311-314.
[28]Nadelman R.B.and G.P.Wormser.Lyme borreliosis.Lancent.1998, 352(9127):557-565.
[29]Steere,A.C,Coburn,J,Glickstein,L.The emergence of Lyme disease.The Journal of clinical investigation.2004, 113(8):1093-1101.
[30]Sethi,N,Sondey,M,Bai,Y, et al.Interaction of a Neurotropic Strain of Borrelia turicatae with the Cerebral Microcirculation System.Infect Immum.2006,74(11):6408-6418.
[31]Maria E. Aguero-Rosenfeld, Guiqing Wang, Ira Schwartz,et al.Diagnosis of Lyme Borreliosis[J]. American Society for Microbiology, 2005, 18(3):484-506.
[32]Logan H.Burms,Claire A. Adams,Sean P. et al. BpaB, a novel protein encoded by the Lyme disease spirochete’s cp32 prophages,binds to erp Operator 2 DNA[J].Nucleic Acids research,2010,1-13.
[33]Pick RN,et al.Molecular Characterization of Borrelia burgdorferi sensu Lato from Slovenia Revealing Significant Difference between Tick and Human Isolates.Eur J Microbiol Infect Disease.1996; 15(4):313.
[34]Rogern Picken,et al.Identification of Three Species of Borrelia burgdorefri sensu lato(B.burgdorefri sensu strieto,B.garinii and B.azfelii) among Isolates from Acrodermatitis Chronica Atrophicans Lesions. J Investigative Dermatology.1998; 110:211.
[35]Picken,et al. Genotypic and Phenotypic Charaeterization of Borrelia burgdorefri Isolation fromTicks and Small Animals in Illinosis.J Clin Microbiol.1995; 33(9):2304.
[36]Ulriehh buseh,et al.Moleular Charaeterization of Borrelia burgdorgeri Sensu Lato Strains by Pulsed- Field Gel Electrophoresis. Electrophoresis.1995; 16:747.
[37]Will,G,et al.Sequence Analysis of OspA Genes Shows Homogeneity within Borrelia burgdorferi sensu stricto and Borrelia Strains but Reveals Major Subgroups within the Borrelia garinii Species Med Microbiol Immunol,1995;184:73.
[38]Herman kuiper,et al.Isolation of Borrelia burgdorefrir fom Biopsy Speeimens Taken from HealthyLooking Skin of Patients with Lyme Borreliosis.J.Clin Microbiol.1994;323(3):715.
[39]王槐春.蛋白质家族与分子进化.蛋白质与核酸序列分析基础.北京:人民军医出版社.1989;89-91
[40]Barbour AG,et al.Gurr Mierobiol.1983:8:123
[41]Kurtti T J, Munderloh U G, Johnson R C, et al.Colony Formation and Morphology in Borrelia burgdorferit.[J].J Clin Microbiol, 1987,25(11):2054-2058.
[42]Kurtti T.J.,U.G.Munderloh,G.G.Ahlstrand,and R.C.Johnson.Borrelia burgdorferi in tick cell culture:growth and cellular adherence.J.Med.Entomol.1988,25(4):256–261
[43]Speck S.,K.Failing,B.Reiner,and M.M.Wittenbrink.Evaluation of different media and a BGM cell culture assay for isolation of Borrelia burgdorferi sensu lato from ticks and dogs.Vet.Microbiol.2002,89(4):291–302
[44]Berger B.W.,R.C.Johnson,C.Kodner,and L.Coleman.Cultivation of Borrelia burgdorferi from erythema migrans lesions and perilesional skin.J.Clin.Microbiol.1992,30(2):359–361.
[45]Picken R.N., F.Strle, M.M.Picken, et al.Identification of three species of Borrelia burgdorferi sensu lato (B.burgdorferi sensu stricto, B.garinii, and B.afzelii among isolates from acrodermatitis Chronica atrophicans lesions.J.Investig.Dermatol.1998,110(3):211–214.
[46]Maraspin V.,J.Cimperman,S.Lotric-Furlan,et al.Solitary borrelial lymphocytoma in adult patients.Wien.Klin.Wochenschrift.2002,114:515–523.
[47]KarlssonM. K. Hovind-Hougen, B. Svenungsson, and G. Stiernstedt. Cultivation and characterization of spirochetes from cerebrospinal fluid of patients with Lyme borreliosis. J.Clin.Microbiol.1990,28(3):473–479.
[48]Arnez M.,E.Ruzic-Sabljic,J.Ahcan,A.Radsel-Medvescek,et al.Isolation of Borrelia burgdorferi Sensu lato from blood of children with solitary erythema igrans.Pediatr.Infect.Dis[J] 2001,20(3):251–255
[49]Schmidli J.,T.Hunziker,P.Moesli,and U.B.Schaad.Cultivation of Borrelia burgdorferi from joint fluid three months after treatment of facial palsy due to Lyme borreliosis.J.Infect.Dis.1988,158(4):905–906
[50]Stanek G.,J.Klein,R.Bittner,and D.Glogar.Isolation of Borrelia burgdorferi from the myocardium of a patient with longstanding cardiomyopathy.N.Engl.J.Med. 1990,322(4):249–252
[51]Preac-Mursic V.,H.W.Pfister,H.Spiegel,et al.First isolation of Borrelia burgdorferi from an iris biopsy.J.Clin.Neuroophthalmol.1993,13(3):155–161.
[52]Steere A.C.,R.L.Grodzicki,A.N.Kornblatt,et al.The spirochetal etiology of Lyme disease.N.Engl.J.Med.1983,308(13):733–740.
[53]Wallach F.R., A.L.Forni, J.Hariprashad, et al.Circulating Borrelia burgdorferi in patients with acute Lyme disease: results of blood cultures and serum DNA analysis.J. Infect.Dis. 1993, 168(6):1541–1543.
[54]Nadelman R.B.,J.Nowakowski,G.Forseter,et al.Failure to isolate Borrelia burgdorferi after Antimicrobial therapy in culture-documented Lyme borreliosis associated with erythema migrans:report of a prospective study.Am.J.Med.1993,94(6):583–588
[55]Hauser U, Krahl H, Peters H, et al. Impact of strain heterogeneity on Lyme disease serology in Europe: comparison of enzyme-linked immunosorbent assays using different species of Borrelia Burgdorferi sensu lato [J]. J Clin Microbiol,1998,36(2):427–436.
[56]Coleman J. L, J. L. Benach. Isolation of antigenic components from the Lyme disease spirochete: their role in early diagnosis. J. Infect. Dis. 1987, 155(4): 756–765.
[57]Luft B.J,J.Dunn,R.J.Dattwyler, G. Gorgone, P.D.Gorevic,and W.H Schubach.Cross-reactive antigenic domains of the flagellin protein of Borrelia burgdorferi. Res.Microbiol. 1993, 144(4):251–257
[58]Maria E. Aguero-Rosenfeld, Guiqing Wang, Ira Schwartz, and Gary P.Wormser. Diagosis Of Lyme Borreliosis. [J]. Clinical Microbiology Revivews,2005.18(3):488-450
[59]Jauris-Heipke S.,R.Fuchs,M.Motz,et al.Genetic heterogenity of the genes coding for the outer surface protein C(OspC)and the flagellin of Borrelia burgdorferi.Med.Microbiol.Immunol .(Berlin)1993, 182(1):37–50
[60]Theisen M.,B.Frederiksen,A.M.Lebech,et al.Polymorphism in ospC gene of Borrelia burgdorferi and immunoreactivity of OspC protein:implications for taxonomy and for use of OspC protein as a diagnostic antigen.J.Clin.Microbiol.1993,31(10):2570–2576
[61]Liang F. T., A. L. Alvarez, Y. Gu, J. M. Nowling, R. Ramamoorthy, and M. T. Philipp.An Immunodominant conserved region within the variable domain of VlsE, the variable surface Antigen of Borrelia burgdorferi. [J]. Immunol. 1999, 163(10):5566–5573.
[62]Magnarelli L.A, J. M. Meegan, J. F. Anderson, and W. A. Chappell. Comparison of an indirect Fluorescent-antibody test with an enzyme-linked immunosorbent assay for serological studies of Lyme disease. [J].Clin Microbiol. 1984,20(2):181–184.
[63]Brown S.L.,S.L.Hansen,and J.J.Langone.Role of serology in the diagnosis of Lyme disease.JAMA.1999,282(1):62–66
[64]Engstrom S. M, E.Shoop, and R.C.Johnson. Immunoblot interpretation criteria for serodiagnosis of early Lyme disease. [J]. Clin. Microbiol. 1995, 33 (2):419–427.
[65]Dressler F., J. A. Whalen, B. N. Reinhardt, and A.C. Steere. Western blotting in the serodiagnosis of Lyme disease. [J]. Infect. Dis. 1993, 167 (2): 392–400.
[66]Hauser U., G. Lehnert, R. Lobentanzer, and B. Wilske. Interpretation criteria for standardized Western blots for three European species of Borrelia burgdorferi sensu lato.[J]. Clin. Microbiol. 1997,35(6):1433–1444.
[67]蒋毅,万康林,耿震,等.中国莱姆病伽氏疏螺旋体蛋白免疫印迹法诊断标准的研究[J].中华微生物学和免疫学杂志,2005,7 25(7):595-598.
[68]Aguero-Rosenfeld M. E, J. Nowakowski, et al. Evolution of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans.[J]. Clin.Microbiol. 1996,34(1):1–9.
[69]Callister S.M., R.F. Schell, L.C. Lim, D.A. Jobe, et al. Detection of borreliacidal antibodies by flow cytometry. An accurate, highly specific serodiagnostic test for Lyme disease.[J].Arch. Intern.Med. 1994, 154 (14): 1625–1632.
[70]DumLer J.S. Molecular diagnosis of Lyme disease: Review and meta-analysis. [J] Mol.Diagn. 2001, 6(1):1–11.
[71]Wang G, C.Ojaimi, R.Iyer, V. Saksenberg, et al. Impact of genotypic variation of Borrelia Burgdorferi sensu stricto on kinetics of dissemination and severity of disease in C3H/HeJ mice. [J].Infect. Immun. 2001, 69 (7): 4303–4312.
[72]Liebling M.R, M. J. Nishio, A. Rodriguez, et al. The polymerase chain reaction for the detection of Borrelia burgdorferi in human body fluids. [J].Arthritis Rheum.1993, 36(5):665–675.
[73]Liveris D, G. Wang, G. Girao, et al. Quantitative detection of Borrelia burgdorferi in-millimeter Skin samples of erythema migrans lesions: correlation of results with clinical and laboratory Findings. [J]. Clin. Microbiol. 2002, 40(4): 1249–1253.
[74]Emir Hodzic, Sunlian Feng, Kimberly J. Freet, Dori L. Borjesson, and Stephen W. Barthold. Borrelia burgdorferi Population Kinetics and Selected Gene Expression at the Host-Vector Interface.[J].Infection and immunity, 2002,70(7) :3382–3388
[75]Joseph Piesman, Bradley S. Schneider, and Nordin S. Zeidner. Use of Quantitative PCR To Measure Density of Borrelia burgdorferi in the Midgut and Salivary Glands of Feeding Tick Vectors.[J].Journal of clinical microbiology, 2001, 39(11):4145–4148
[76]Joshua W. Courtney, Leah M. Kostelnik, Nordin S. Zeidner, and Robert F. Massung. MultiplexReal- Time PCR for Detection of Anaplasma phagocytophilum and Borrelia burgdorferi.[J].Journal of clinical microbiology,2004, 42(7):3164–3168
[77]Morrison T. B, Y. Ma, J. H. Weis, and J. J. Weis. Rapid and sensitive quantification of Borrelia Burgdorferi-infected mouse tissues by continuous fluorescent monitoring of PCR. [J]. Clin. Microbiol. 1999,37(6):987–992
[78]Rauter C, R.Oehme, I. Diterich, M. Engele, and T. Hartung. Distribution of clinically relevant Borrelia genospecies in ticks assessed by a novel, single-run, real-time PCR. [J].Clin. Microbiol. 2002,40(1):36–43
[79]Du W, Xu ZH, Ma X, et al. Biochip as a potential plat form of serological interfere onα2 antibody assay. [J] Biotech, 2003, 106 (1):87-100.
[80]Pavlickova P , Jensen NM , Paul H , et al. Antibody detection of human serum using a versatile Protein chip platformconst ructed by applying of nano-scale self-assembled archi-tecture on gold. [J] Proteome Res , 2002 ,1 (3) :227-231.
[81]Pavlickova P, Hug H. Ast reptavidin-biotin based mi-croarray platform for immunoassays. [J].Methods Mol Biol, 2004, 264:73283.
[82]于冬冬.莱姆病分子生物学和诊断新技术的研究[D].吉林:吉林大学,2008:2-3.
[83]Nicholas H.Ogden Dphil,L.Robbin Lindsay phD,et al.The emergence of Lyme disease in Canada[J].Canadian Medical Association or its licensors.2009,180(12):1221-1224.
[84]Muddasir Qureshi, MD, Richard S.Bedlack, MD, PhD, et al.Lyme disease serology in amyotrophic lateral sclerosis [J]. MUSCLE & NERVE.2009, 5(12):1-2.
[2]Alberto A. Guglielmone,Richard G.Robbins,Dmitry A. Apanaskevich.et al.Comments on controversial tick (Acari: Ixodida) species names and species described or resurrected from 2003 to 2008[J].Exp appl Acarol.2009.48:311–327
[3]陆支英,陆宝麟.医学昆虫学[M].1990.科学出版社.397-415.
[4]邓国藩,姜在阶.中国经济昆虫志[M].第三十九册.1991.科学出版社.11-13.
[5]Sauer JR, McSwain JL, Bowman AS, Essenberg RC.Tick salivary gland physiology [J]. Annu Rev Entomol.1995.40, 245-267
[6]Fogaca AC,da Sliva PI Jr,Miranda MT,et al.Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus[J].J Biol.Chem.1999.274(36):25330-255340
[7]龚海燕,周金林,周勇志等.镰形扇头蜱半饱血雌蜱cDNA文库的构建[J].中国人兽共患病杂志,2004, 20(6): 458-488
[8]柳支英,陆宝麟.医学昆虫学[M].北京:科学出版社.1990, 397-415
[9]Sauer J R, M cswain J L , Bowman A S, et al. Tick salivary gland physiology[J ]. Annu Rev Entomol, 1995, 40: 245-267
[10]周金林.蜱功能分子研究进展[J].动物医学进展, 2004, 25(1): 53-56.
[11]Shanahan GJ , Hart R J . Change in response of Lucilia cuprina to organophosphorus insecticides in Australia [J].Nature , 1966 , 212(5069) :1466-1467.
[12]仝彩玲,李培英,周金林.莱姆病诊断技术研究进展[J].中国动物传染病学报,2009;3(17):76.
[13]Henry M.Feder,Jr.,M.D.,Barbara J.B.Johnson,Ph.D.,et al.A Critical Appraisal of“Chronic Lyme Disease”.[J]. The new england journal of medicine.2007, 357:1422-1423
[14]廖德丰,陈季武,谢萍,等.蜱传性疾病——莱姆病螺旋体研究进展[J].中国兽医寄生虫病, 2004,12(2):36-38.
[15]Burgdorferi W,Barbour AG,Hayes SF,et al.Lyme disease—A tick borne spirochetosis , Science,1982,216;1317-1319.
[16]C.M.Fraser,S.Casjens,W.M.Huang,et al.Genomic sequence of a Lyme disease spirochaete.Borrelia burgdorferi.Nature,1997,390:580–586
[17]Vander FK.Everything you need to know about Lyme disease and other tick-borne disorders.NewYork.NY John Wiley&Sons Inc,1997,20-47
[18]孙毅,许荣满,张泮河,等.莱姆病人工宿主动物模型的建立[J].寄生虫与医学昆虫学报,2002,9(3):160.
[19]万康林,张金声,张哲夫.清除莱姆病螺旋体培养物中杂菌的方法研究.中华流行病学杂志1991,12;299-301
[20]张哲夫,万康林,王子敏,等.从湖南省靖县野兔体内分离出莱姆病螺旋体,中国媒介生物学及控制杂志,1992,3(特2);97-98
[21]Kurtti T J,Munderloh U G,Johnson R C,et al.Colony Formation and morphology in Borrelia Burgdorferi [J].J Clin Microbiol, 1987, 25(11); 2054-2058
[22]廖德丰,陈季武,谢萍,傅蓓蓓等.蜱传性疾病——莱姆病螺旋体研究进展[J].中国兽医寄生虫病,2004,12 (2):36-38
[23]J.Ohnishi, J.Piesman and A.M.de.Silva.Antigenic and genetic heterogeneity of Borrelia burgdorferipopulations transmitted by ticks.Proc Natl Acad Sci USA, 2001, 98:670-675.
[24]Kelly R.Cultivation of Borrelia hermsii.Science.1971, 173(995):443-444.
[25]Pollack R.J,S.R.TelfordIII,and A.Spielman.Standardization of medium for culturing Lyme disease spirochetes.J.Clin.Microbiol.1993,31(5):1251–1255
[26]Barbour A.G.Isolation and cultivation of Lyme disease spirochetes.Yale J.Biol.Med. 1984,57(4):521–525
[27]刘敏,王树声.莱姆病研究进展.广西预防医学,2001,7:311-314.
[28]Nadelman R.B.and G.P.Wormser.Lyme borreliosis.Lancent.1998, 352(9127):557-565.
[29]Steere,A.C,Coburn,J,Glickstein,L.The emergence of Lyme disease.The Journal of clinical investigation.2004, 113(8):1093-1101.
[30]Sethi,N,Sondey,M,Bai,Y, et al.Interaction of a Neurotropic Strain of Borrelia turicatae with the Cerebral Microcirculation System.Infect Immum.2006,74(11):6408-6418.
[31]Maria E. Aguero-Rosenfeld, Guiqing Wang, Ira Schwartz,et al.Diagnosis of Lyme Borreliosis[J]. American Society for Microbiology, 2005, 18(3):484-506.
[32]Logan H.Burms,Claire A. Adams,Sean P. et al. BpaB, a novel protein encoded by the Lyme disease spirochete’s cp32 prophages,binds to erp Operator 2 DNA[J].Nucleic Acids research,2010,1-13.
[33]Pick RN,et al.Molecular Characterization of Borrelia burgdorferi sensu Lato from Slovenia Revealing Significant Difference between Tick and Human Isolates.Eur J Microbiol Infect Disease.1996; 15(4):313.
[34]Rogern Picken,et al.Identification of Three Species of Borrelia burgdorefri sensu lato(B.burgdorefri sensu strieto,B.garinii and B.azfelii) among Isolates from Acrodermatitis Chronica Atrophicans Lesions. J Investigative Dermatology.1998; 110:211.
[35]Picken,et al. Genotypic and Phenotypic Charaeterization of Borrelia burgdorefri Isolation fromTicks and Small Animals in Illinosis.J Clin Microbiol.1995; 33(9):2304.
[36]Ulriehh buseh,et al.Moleular Charaeterization of Borrelia burgdorgeri Sensu Lato Strains by Pulsed- Field Gel Electrophoresis. Electrophoresis.1995; 16:747.
[37]Will,G,et al.Sequence Analysis of OspA Genes Shows Homogeneity within Borrelia burgdorferi sensu stricto and Borrelia Strains but Reveals Major Subgroups within the Borrelia garinii Species Med Microbiol Immunol,1995;184:73.
[38]Herman kuiper,et al.Isolation of Borrelia burgdorefrir fom Biopsy Speeimens Taken from HealthyLooking Skin of Patients with Lyme Borreliosis.J.Clin Microbiol.1994;323(3):715.
[39]王槐春.蛋白质家族与分子进化.蛋白质与核酸序列分析基础.北京:人民军医出版社.1989;89-91
[40]Barbour AG,et al.Gurr Mierobiol.1983:8:123
[41]Kurtti T J, Munderloh U G, Johnson R C, et al.Colony Formation and Morphology in Borrelia burgdorferit.[J].J Clin Microbiol, 1987,25(11):2054-2058.
[42]Kurtti T.J.,U.G.Munderloh,G.G.Ahlstrand,and R.C.Johnson.Borrelia burgdorferi in tick cell culture:growth and cellular adherence.J.Med.Entomol.1988,25(4):256–261
[43]Speck S.,K.Failing,B.Reiner,and M.M.Wittenbrink.Evaluation of different media and a BGM cell culture assay for isolation of Borrelia burgdorferi sensu lato from ticks and dogs.Vet.Microbiol.2002,89(4):291–302
[44]Berger B.W.,R.C.Johnson,C.Kodner,and L.Coleman.Cultivation of Borrelia burgdorferi from erythema migrans lesions and perilesional skin.J.Clin.Microbiol.1992,30(2):359–361.
[45]Picken R.N., F.Strle, M.M.Picken, et al.Identification of three species of Borrelia burgdorferi sensu lato (B.burgdorferi sensu stricto, B.garinii, and B.afzelii among isolates from acrodermatitis Chronica atrophicans lesions.J.Investig.Dermatol.1998,110(3):211–214.
[46]Maraspin V.,J.Cimperman,S.Lotric-Furlan,et al.Solitary borrelial lymphocytoma in adult patients.Wien.Klin.Wochenschrift.2002,114:515–523.
[47]KarlssonM. K. Hovind-Hougen, B. Svenungsson, and G. Stiernstedt. Cultivation and characterization of spirochetes from cerebrospinal fluid of patients with Lyme borreliosis. J.Clin.Microbiol.1990,28(3):473–479.
[48]Arnez M.,E.Ruzic-Sabljic,J.Ahcan,A.Radsel-Medvescek,et al.Isolation of Borrelia burgdorferi Sensu lato from blood of children with solitary erythema igrans.Pediatr.Infect.Dis[J] 2001,20(3):251–255
[49]Schmidli J.,T.Hunziker,P.Moesli,and U.B.Schaad.Cultivation of Borrelia burgdorferi from joint fluid three months after treatment of facial palsy due to Lyme borreliosis.J.Infect.Dis.1988,158(4):905–906
[50]Stanek G.,J.Klein,R.Bittner,and D.Glogar.Isolation of Borrelia burgdorferi from the myocardium of a patient with longstanding cardiomyopathy.N.Engl.J.Med. 1990,322(4):249–252
[51]Preac-Mursic V.,H.W.Pfister,H.Spiegel,et al.First isolation of Borrelia burgdorferi from an iris biopsy.J.Clin.Neuroophthalmol.1993,13(3):155–161.
[52]Steere A.C.,R.L.Grodzicki,A.N.Kornblatt,et al.The spirochetal etiology of Lyme disease.N.Engl.J.Med.1983,308(13):733–740.
[53]Wallach F.R., A.L.Forni, J.Hariprashad, et al.Circulating Borrelia burgdorferi in patients with acute Lyme disease: results of blood cultures and serum DNA analysis.J. Infect.Dis. 1993, 168(6):1541–1543.
[54]Nadelman R.B.,J.Nowakowski,G.Forseter,et al.Failure to isolate Borrelia burgdorferi after Antimicrobial therapy in culture-documented Lyme borreliosis associated with erythema migrans:report of a prospective study.Am.J.Med.1993,94(6):583–588
[55]Hauser U, Krahl H, Peters H, et al. Impact of strain heterogeneity on Lyme disease serology in Europe: comparison of enzyme-linked immunosorbent assays using different species of Borrelia Burgdorferi sensu lato [J]. J Clin Microbiol,1998,36(2):427–436.
[56]Coleman J. L, J. L. Benach. Isolation of antigenic components from the Lyme disease spirochete: their role in early diagnosis. J. Infect. Dis. 1987, 155(4): 756–765.
[57]Luft B.J,J.Dunn,R.J.Dattwyler, G. Gorgone, P.D.Gorevic,and W.H Schubach.Cross-reactive antigenic domains of the flagellin protein of Borrelia burgdorferi. Res.Microbiol. 1993, 144(4):251–257
[58]Maria E. Aguero-Rosenfeld, Guiqing Wang, Ira Schwartz, and Gary P.Wormser. Diagosis Of Lyme Borreliosis. [J]. Clinical Microbiology Revivews,2005.18(3):488-450
[59]Jauris-Heipke S.,R.Fuchs,M.Motz,et al.Genetic heterogenity of the genes coding for the outer surface protein C(OspC)and the flagellin of Borrelia burgdorferi.Med.Microbiol.Immunol .(Berlin)1993, 182(1):37–50
[60]Theisen M.,B.Frederiksen,A.M.Lebech,et al.Polymorphism in ospC gene of Borrelia burgdorferi and immunoreactivity of OspC protein:implications for taxonomy and for use of OspC protein as a diagnostic antigen.J.Clin.Microbiol.1993,31(10):2570–2576
[61]Liang F. T., A. L. Alvarez, Y. Gu, J. M. Nowling, R. Ramamoorthy, and M. T. Philipp.An Immunodominant conserved region within the variable domain of VlsE, the variable surface Antigen of Borrelia burgdorferi. [J]. Immunol. 1999, 163(10):5566–5573.
[62]Magnarelli L.A, J. M. Meegan, J. F. Anderson, and W. A. Chappell. Comparison of an indirect Fluorescent-antibody test with an enzyme-linked immunosorbent assay for serological studies of Lyme disease. [J].Clin Microbiol. 1984,20(2):181–184.
[63]Brown S.L.,S.L.Hansen,and J.J.Langone.Role of serology in the diagnosis of Lyme disease.JAMA.1999,282(1):62–66
[64]Engstrom S. M, E.Shoop, and R.C.Johnson. Immunoblot interpretation criteria for serodiagnosis of early Lyme disease. [J]. Clin. Microbiol. 1995, 33 (2):419–427.
[65]Dressler F., J. A. Whalen, B. N. Reinhardt, and A.C. Steere. Western blotting in the serodiagnosis of Lyme disease. [J]. Infect. Dis. 1993, 167 (2): 392–400.
[66]Hauser U., G. Lehnert, R. Lobentanzer, and B. Wilske. Interpretation criteria for standardized Western blots for three European species of Borrelia burgdorferi sensu lato.[J]. Clin. Microbiol. 1997,35(6):1433–1444.
[67]蒋毅,万康林,耿震,等.中国莱姆病伽氏疏螺旋体蛋白免疫印迹法诊断标准的研究[J].中华微生物学和免疫学杂志,2005,7 25(7):595-598.
[68]Aguero-Rosenfeld M. E, J. Nowakowski, et al. Evolution of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans.[J]. Clin.Microbiol. 1996,34(1):1–9.
[69]Callister S.M., R.F. Schell, L.C. Lim, D.A. Jobe, et al. Detection of borreliacidal antibodies by flow cytometry. An accurate, highly specific serodiagnostic test for Lyme disease.[J].Arch. Intern.Med. 1994, 154 (14): 1625–1632.
[70]DumLer J.S. Molecular diagnosis of Lyme disease: Review and meta-analysis. [J] Mol.Diagn. 2001, 6(1):1–11.
[71]Wang G, C.Ojaimi, R.Iyer, V. Saksenberg, et al. Impact of genotypic variation of Borrelia Burgdorferi sensu stricto on kinetics of dissemination and severity of disease in C3H/HeJ mice. [J].Infect. Immun. 2001, 69 (7): 4303–4312.
[72]Liebling M.R, M. J. Nishio, A. Rodriguez, et al. The polymerase chain reaction for the detection of Borrelia burgdorferi in human body fluids. [J].Arthritis Rheum.1993, 36(5):665–675.
[73]Liveris D, G. Wang, G. Girao, et al. Quantitative detection of Borrelia burgdorferi in-millimeter Skin samples of erythema migrans lesions: correlation of results with clinical and laboratory Findings. [J]. Clin. Microbiol. 2002, 40(4): 1249–1253.
[74]Emir Hodzic, Sunlian Feng, Kimberly J. Freet, Dori L. Borjesson, and Stephen W. Barthold. Borrelia burgdorferi Population Kinetics and Selected Gene Expression at the Host-Vector Interface.[J].Infection and immunity, 2002,70(7) :3382–3388
[75]Joseph Piesman, Bradley S. Schneider, and Nordin S. Zeidner. Use of Quantitative PCR To Measure Density of Borrelia burgdorferi in the Midgut and Salivary Glands of Feeding Tick Vectors.[J].Journal of clinical microbiology, 2001, 39(11):4145–4148
[76]Joshua W. Courtney, Leah M. Kostelnik, Nordin S. Zeidner, and Robert F. Massung. MultiplexReal- Time PCR for Detection of Anaplasma phagocytophilum and Borrelia burgdorferi.[J].Journal of clinical microbiology,2004, 42(7):3164–3168
[77]Morrison T. B, Y. Ma, J. H. Weis, and J. J. Weis. Rapid and sensitive quantification of Borrelia Burgdorferi-infected mouse tissues by continuous fluorescent monitoring of PCR. [J]. Clin. Microbiol. 1999,37(6):987–992
[78]Rauter C, R.Oehme, I. Diterich, M. Engele, and T. Hartung. Distribution of clinically relevant Borrelia genospecies in ticks assessed by a novel, single-run, real-time PCR. [J].Clin. Microbiol. 2002,40(1):36–43
[79]Du W, Xu ZH, Ma X, et al. Biochip as a potential plat form of serological interfere onα2 antibody assay. [J] Biotech, 2003, 106 (1):87-100.
[80]Pavlickova P , Jensen NM , Paul H , et al. Antibody detection of human serum using a versatile Protein chip platformconst ructed by applying of nano-scale self-assembled archi-tecture on gold. [J] Proteome Res , 2002 ,1 (3) :227-231.
[81]Pavlickova P, Hug H. Ast reptavidin-biotin based mi-croarray platform for immunoassays. [J].Methods Mol Biol, 2004, 264:73283.
[82]于冬冬.莱姆病分子生物学和诊断新技术的研究[D].吉林:吉林大学,2008:2-3.
[83]Nicholas H.Ogden Dphil,L.Robbin Lindsay phD,et al.The emergence of Lyme disease in Canada[J].Canadian Medical Association or its licensors.2009,180(12):1221-1224.
[84]Muddasir Qureshi, MD, Richard S.Bedlack, MD, PhD, et al.Lyme disease serology in amyotrophic lateral sclerosis [J]. MUSCLE & NERVE.2009, 5(12):1-2.