基于荧光重组酶介导等温扩增技术的利什曼原虫核酸检测方法的建立

目的　建立一种基于荧光重组酶介导等温扩增（recombinase⁃aided isothermal amplification, RAA）技术的检测利什曼原虫核酸的方法。方法　针对利什曼原虫内转录间隔基因序列1（ITS1）基因设计用于RAA检测的特异性引物和探针,通过引物配对筛选、引物和探针浓度优化,建立检测利什曼原虫核酸的荧光RAA法。分别以构建的含利什曼原虫ITS1基因序列的不同拷贝数重组质粒和不同浓度利什曼原虫基因组DNA为模板进行RAA扩增,评估其检测灵敏度;以田鼠巴贝西虫、刚地弓形虫、间日疟原虫、卵形疟原虫、恶性疟原虫、三日疟原虫、杜氏利什曼原虫、婴儿利什曼原虫等其他经输血传播的寄生虫基因组DNA为模板进行RAA扩增,评价其检测特异度。结果　从9对引物组合中筛选出1对最佳引物对后,引物和探针终浓度经优化分别确定为0.3 μmol/L和0.08 μmol/L。所建立的荧光RAA法在39 ℃等温条件下20 min内可完成样本核酸检测。采用建立的RAA法对田鼠巴贝西虫、刚地弓形虫、间日疟原虫、卵形疟原虫、恶性疟原虫、三日疟原虫、杜氏利什曼原虫、婴儿利什曼原虫等8种寄生虫基因组DNA进行检测,杜氏利什曼原虫、婴儿利什曼原虫基因组DNA在5 min内即出现明显荧光信号,与田鼠巴贝西虫、刚地弓形虫、间日疟原虫、卵形疟原虫、恶性疟原虫、三日疟原虫无交叉反应。以含利什曼原虫ITS1基因序列的重组质粒为模板,荧光RAA法最低检测限为10 拷贝/μL;以利什曼原虫基因组DNA为模板,荧光RAA法最低检测限为1 fg/μL。结论　成功建立了一种基于荧光RAA法的利什曼原虫核酸检测技术,该方法反应快速、操作简便、灵敏度和特异度均较高,可用于利什曼原虫病流行区现场筛查。     

杜氏利什曼原虫蛋白鳞酸酶—2C的基因克隆化与序列分析

目的 克隆杜氏利什曼原虫（Leishmania donovani,Ld)1S株蛋白磷酸酶2C（PP2C）的编码基因，为应用这种编码T细胞抗原的基因进行基因疫苗研究奠定基础。方法 体外培养杜氏利什曼原虫1S株无鞭毛体，常规方法从虫体提取制备基因组DNA。以夏科氏利什曼原虫（Leishmania chagasi,Lc)的PP2C基因序列为参照，设计并合成利什曼原虫PP2C基因序列特异性的引物。结果 以杜氏利什曼原虫的基因组为模板，利用多聚酶链反应（PCR）技术，扩增获得了杜氏利什曼原虫PP2C的全长编码基因。基因序列测定结果表明，杜氏利什曼原虫1S株PP2C基因序列长度为1317bp，开放读码框架由1221bp组成，编码产物为406个氨基酸残基。获得的杜氏利什曼原虫1S株的PP2C基因与来源于夏科氏利什曼原虫的PP2C氨基酸残基序列的同源性为95％（387／406）。结论 本研究克隆了杜氏利什曼虫的PP2C基因，为应用诱导T细胞免疫应答抗原的编码基因进行杜氏利什曼虫的基因疫苗研究奠定了基础。     

微小隐孢子虫的基因检测实验研究

目的　探讨聚合酶链反应 (PCR)检测微小隐孢子虫 (Cryptosporidium parvum)感染的特异性和敏感性。　方法用 Sheather's蔗糖梯度离心法从微小隐孢子虫感染者粪便标本中分离纯化卵囊。用酚 -氯仿法抽提卵囊总 DNA。针对本虫 18S r RNA基因片段设计 1对引物 ,对模板 DNA进行 PCR扩增 ,同时以蓝氏贾第鞭毛虫 (Giardia lamblia)、溶组织内阿米巴 (Entamoeba histolytica)、阴道毛滴虫 (Trichomonas vaginalis)、刚地弓形虫 (Toxoplasma gondii)、日本血吸虫(Schistosoma japonicum)和旋毛虫 (Trichinella spiralis) ,以及人体血细胞等 DNA样本为对照。用琼脂糖电泳和溴乙腚染色对 PCR产物进行检测。　结果　仅微小隐孢子虫 DNA被扩增出一 5 0 0 bp的 DNA片段。其它对照 DNA样本均未出现扩增反应。　结论　PCR对隐孢子虫检测的特异性可高达 10 0 % ,敏感性也很强 ,可检测到 2 0 pg微小隐孢子虫卵囊的DNA。表明本实验建立的检测微小隐孢子虫的 PCR方法有效     

微小隐孢子虫的基因检测实验研究

目的 探讨聚合酶链反应（PCR）检测微小隐孢子虫（Cryptosporidium parvum)感染的特异性和敏感性。方法 用Sheather's蔗糖梯度离心法从微小隐孢子虫感染者粪便标本中分离纯化卵囊，用酚-氯仿法抽提卵囊总DNA，针对本虫18SrRNA基因片段设计1对引物，对模板DNA进行PCR扩增，同时以蓝氏贾第鞭毛虫（Giardia lambia)，溶组织内阿米巴（Entamoeba histolytica)。阴道毛滴虫（Trichomonas vaginalis)。刚地弓形虫（Toxoplasma gondii)。日本血吸虫（Schis-tosoma japonicum)和旋毛虫（Trichinella spiralis)，以及人体血细胞等DNA样本为对照，用琼脂糖电泳和溴乙腚染色对PCR产物进行检测。结果 仅微小隐孢子虫DNA被扩增出-500bp的DNA片段，其它对照DNA样本均未出现扩增反应。结论 PCR对隐孢子虫检测的特异性可高达100%，敏感性也很强，可检测到20pg微小隐孢子虫卵囊的DNA，表明本实验建立的检测微小隐孢子虫的PCR方法有效。     

杜氏利什曼原虫特异性kDNA片段扩增及用于内脏利什曼病诊断的研究

本文报道了用杜氏利什曼原虫特异的一对寡核苷酸引物Ⅰ和Ⅱ进行PCR,扩增微环kDNA分子上一种特异性kDNA片段,进行杜氏利什曼原虫虫种鉴定和病原体检测。敏感性分析表明用此法能够检测到的最低模板DNA需要量为1fg,检测健康犬全血稀释的利什曼原虫前鞭毛体,最低可达2个/ml。扩增六种不同的利什曼原虫kDNA样品,在L.donovani四川人株、四川犬株和L.infantum出现阳性产物,其长度和设计扩增长度一致,扩增产物和地高辛标记的重组质粒探针pLK2斑点杂交结果表明为利什曼原虫kDNA序列。应用此对引物,检测8份内脏利什曼病患者骨髓样品和4份血清样品,经琼脂糖凝胶电泳,Southern杂交证实,分别有7例和2例阳性,显示出可喜的应用前景。     

聚合酶链反应结合非放射性标记探针检测粪便中微小隐孢子虫

目的：利用非放射性标记探针技术检测经聚合酶链反应扩增后的人粪便中微小隐孢子虫DNA，观察该方法的特异性和敏感性。方法：用裂解法从粪便标本直接制备微小隐孢子虫模板DNA，用一对人工合成的寡核苷酸作PCR引物，扩增长度为452bp的微小隐孢子虫目的DNA片段。将扩增产物直接点在或经Southern印迹法转移到硝酸纤维素膜上，再和生物素标记的寡核苷酸探针杂交，经底物（BCIP）呈色观察。结果：阳性杂交信号只见于微小隐孢子虫DNA扩增产物，而约氏疟原虫、杜氏利什曼原虫、贾第虫、白色念珠菌、大肠杆菌和人白细胞DNA均无杂交信号。PCR结合斑点杂交或Southern印迹法检测隐孢子虫DNA 的敏感性均为011 pg。结论: 非放射性探针检测人粪便隐孢子虫DNA 的PCR 扩增产物, 具有敏感性高、特异性强、操作较简便及无放射性污染等优点。     

检测利什曼原虫的实时荧光定量PCR的建立及应用

建立一种快速准确检测利什曼原虫的实时荧光定量PCR (qPCR)方法。以利什曼原虫动基体小环保守序列为靶基因,设计1对特异性引物和Taq-Man探针,以利什曼原虫阳性患者骨髓样品DNA为模板进行PCR扩增,扩增产物长83 bp。扩增产物连接至pESI-T载体进行克隆、测序,测序结果在GenBank上进行BLAST比对,结果显示,其序列与婴儿利什曼原虫和杜氏利什曼原虫的序列一致性为100%。取测序正确的质粒梯度稀释至102～107拷贝/μl作为标准品进行qPCR,绘制获得标准曲线方程y=39.23-2.956 x,扩增效率为117.93%,R2为0.994;当阈值循环数为35时,最低检测限为26.98拷贝/μl,理论上可检出小于1个利什曼原虫。用所建立的qPCR检测内脏利什曼病患者骨髓样品14份、血样26份,利什曼病病犬的骨髓、血液、脾、肺、淋巴结、肝、肾组织样品各1份,利什曼原虫阳性白蛉2份,患者和病犬骨髓培养物各1份,结果均为阳性;检测利什曼原虫血清抗体阳性犬骨髓9份,结果 7份阳性;检测利什曼原虫血清抗体阴性人群血样56份,沙门菌、志贺菌、蜡样芽胞杆菌DNA各1份,疟疾患者血样4份,刚地弓形虫病患者血样、卫氏并殖吸虫病患者血样各1份,结果均为阴性。所建立的qPCR具有较高灵敏度及特异度,可用于利什曼病患者、宿主动物、传播媒介利什曼原虫感染与带虫状态的快速检测,可用于患者的疗效判定。     

山丘疫区杜氏利什曼原虫核糖体基因内转录间隔区的克隆及序列分析

目的　构建我国山丘疫区杜氏利什曼原虫分离株前鞭体核糖体DNA(rDNA)内转录间隔区(ITS)片段克隆,并进行测序及同源性分析。　方法　提取杜氏利什曼原虫前鞭毛体DNA进行PCR扩增,将扩增出rDNAITS片段克隆入pMD18 Tvector上,双脱氧链末端终止法测序。　结果　扩增出约 1000bp的rDNAITS片段。测序结果表明山丘疫区的2株利什曼原虫L.d.SC10和L.d.6分别为1027bp和1028bp。序列分析结果表明,L.d.SC10和L.d .6有一定差异。　结论　获得了我国山丘疫区杜氏利什曼原虫分离株L.d.SC10和L.d.6的前鞭体rDNAITS序列。     

FTA-PCR检测水源性隐孢子虫研究

目的 建立FTA-PCR方法,并应用于水源中隐孢子虫污染的基因检测.方法 用免疫磁珠(IMS)分离纯化隐孢子卵囊,采用FTA卡提取隐孢子卵囊DNA,根据隐孢子虫18 S rRNA的基因序列设计引物,对模板DNA进行PCR扩增,同时以刚地弓形虫、猪人肉孢子虫、细粒棘球蚴和华支睾吸虫等DNA样本作对照.用琼脂糖电泳和溴乙腚...     

杜氏利什曼原虫激活蛋白激酶C受体的基因克隆化与序列分析

目的　克隆杜氏利什曼原虫 (LeishmaniadonovaniLd) 1S株激活蛋白激酶C受体 (RACK ,receptorofactivatedpro teinCkinase)的编码基因 ,为应用这种编码T细胞抗原的基因进行基因疫苗的研究奠定基础。方法　体外培养杜氏利什曼原虫 1S株无鞭毛体 ,常规方法提取制备基因组DNA。以硕大利什曼原虫 (Leishmaniaamjor)的RACK基因的核苷酸序列为参照 ,设计并合成利什曼原虫RACK基因序列特异性的引物。以杜氏利什曼原虫的基因组DNA为模板 ,利用多聚酶链反应(PCR)技术 ,扩增获得了杜氏利什曼原虫RACK的全长编码基因。结果　基因序列测定结果表明 ,杜氏利什曼原虫 1S株RACK基因序列长度为 981bp ,开放读码框架由 831bp组成 ,编码产物为 2 76个氨基酸残基。获得的杜氏利什曼原虫 1S株的RACK基因与来源于硕大利什曼原虫的RACK基因序列同源性达 98% (2 6 4 / 2 6 7)。结论　本研究克隆了杜氏利什曼原虫的RACK基因 ,为应用诱导T细胞免疫应答抗原的编码基因进行杜氏利什曼原虫的基因疫苗研究奠定了基础     

新孢子虫NcSAG4基因克隆及原核表达

目的构建新孢子虫NcSAG4基因原核表达载体,并在大肠埃希菌BL21(DE3)中表达。方法根据新孢子虫NcSAG4基因序列设计特异性引物,以新孢子虫总核酸为模板PCR扩增目的片段,与pMD-18-T连接,构建克隆载体pMD-NcSAG4,经双酶切后回收目的片段,与表达载体pGEX-T连接,构建原核表达载体pGEX-NcSAG4,用IPTG诱导,通过SDS-PAGE及Western blot进行鉴定。结果成功构建了新孢子虫NcSAG4基因原核表达载体pGEX-Nc-SAG4;SDS-PAGE电泳显示,在IPTG诱导下阳性菌高效表达了分子质量单位为18.79ku的蛋白质;Western blot显示表达产物可被抗新孢子虫的多克隆血清识别。结论成功构建了NcSAG4基因原核表达载体,并在大肠埃希菌BL21(DE3)中高效表达,为建立经济、实用的诊断新孢子虫病的ELISA方法奠定了基础。     

Immunity to experimental neosporosis in pregnant sheep

Neospora caninum is an important cause of fetal loss in cattle but has also infrequently been shown to cause disease in sheep and goats. Experimental infection of pregnant sheep with N. caninum causes clinical and pathological changes very similar to those of neosporosis in cattle. An experiment in sheep was undertaken to examine whether infection with N. caninum before pregnancy conferred immunity to subsequent challenge with the parasite during pregnancy. Primary inoculation of NC1 tachyzoites subcutaneously, either before or during pregnancy, caused a significant temperature response in ewes, while those given a secondary challenge at 90 days gestation (dg) did not show such a response. Primary infection of 12 ewes during pregnancy resulted in the loss of all fetuses while a further 12 ewes inoculated with NC1 tachyzoites before mating and subsequently challenged with the same dose at 90 dg produced nine live and seven dead lambs. There were no fetal deaths in ewes only infected with Neospora before mating although there was serological evidence of vertical transmission in four of their clinically normal offspring while Neospora DNA was detected in the cerebrospinal fluid of a fifth healthy lamb. Thus an experimental primary infection with N. caninum during pregnancy killed all the fetuses while inoculation before pregnancy did not cause any mortality but did provide a degree of protection against subsequent challenge with Neospora during pregnancy.     

Cytokine gene expression in dams and foetuses after experimental Neospora caninum infection of heifers at 110 days of gestation

Neospora caninum is a major cause of abortion in cattle. An essential role for Th1 cytokines, such as IFN-gamma and IL-12 in protective immunity against N. caninum in murine models has been indicated. However, little is known about immunity to Neospora in pregnant cattle where a considerable level of immunomodulation may exist. In this study, the immune response of heifers infected early in the second trimester of pregnancy by intravenous inoculation of N. caninum tachyzoites was compared with immune responses in uninfected pregnant heifers. Animals were killed 3 weeks after infection. No abortion was observed in any infected dam, however, transplacental infection was shown to have already taken place. Infection with N. caninum during pregnancy induced significant immune responses in both dams and their foetuses. Infected dams showed significant changes in lymphocyte subpopulations compared with uninfected pregnant animals and these changes were compartmentalized. Increased levels of T lymphocytes were observed in the infected foetuses. Cytokine gene expression analysed by real time RT-PCR showed increased expression of both Th1 and Th2 cytokines in N. caninum infected animals. This cytokine expression could have a role in the transplacental transmission of the parasite and/or mediate tissue damage.     

Bioinformatics analysis of CO I gene of Simulium quinquestriatum

The genomic DNA was extracted from Simulium quinquestriatum (Sq) and its CO I gene was amplified by PCR. The PCR product was purified and cloned into plasmid pMD18-T vector. The recombinant plasmid was transformed into Escherichia coli DH5alpha and then identified by digestion with restriction enzyme and PCR amplification. The amplified fractions (1 621 bp) included complete CO I gene (1 542 bp, GenBank accession number: DQ534949), 5' tRNA-Tyr and 3' tRNA-Leu partial fraction. The CO I gene sequence had a high identity (99%) with that of S. quinquestriatum (GenBank accession number: AY251520). Bioinformatics analysis showed that the Sq-CO I open reading frame encoded a 513-amino acid protein with M(r) 5565, pI5.84. Structural prediction showed this protein possessed a conservative domain of CO I gene.     

Cellular immune responses in cattle experimentally infected with Neospora caninum

Neospora caninum has recently been identified as an important cause of infectious abortion in cattle. The parasite is closely related to Toxoplasma gondii , but the two species are antigenically distinct. To examine cell proliferative responses and the induction of IFN-γ in experimentally infected cattle, four 2–4 months old calves were subcutaneously inoculated with N. caninum tachyzoites. Peripheral blood mononuclear cells were collected regularly and stimulated in vitro with a crude lysate of N. caninum or T. gondii tachyzoites. Significant proliferative responses to N. caninum antigen were recorded in all calves from days 4–6 postinoculation. This response was accompanied by production of high levels of IFN-γ. Although the calves remained seronegative to T. gondii, while seroconverting to N. caninum , stimulation with T. gondii lysate resulted in cell proliferation of a similar magnitude as that obtained using the N. caninum lysate. However, the T. gondii lysate appeared less effective than the N. caninum lysate to stimulate IFN-γ production. Cells taken from uninfected control animals did not show any significant proliferation to either N. caninum or T. gondii antigen and no IFN-γ was produced. These results suggest that the two parasites may possess cross-reacting T-cell epitopes, but that the T cells specific for N. caninum may have a different functional capacity. This highlights the need to investigate the antigen specificity and cytokine profile of T cells from infected animals to help understand their role in immunity to N. caninum.     

绵羊附红细胞体部分16S rRNA基因序列测定和系统进化分析

从自然感染附红细胞体的石河子和杭州两地的绵羊无菌采集血液,分离附红细胞体并提取基因组,根据已公布的绵羊附红细胞体16S rRNA基因序列设计一对引物,进行PCR扩增。结果扩增出约1 100bp目的片段。测序结果表明:目的片段长1 080bp、1 079bp(GenBank收录号EU916726、FJ440328),同源性分析表明该序列与参考序列(AF338268)同源性达99.7%,证实该病原是绵羊附红细胞体。将该序列与5种支原体、14种血营养菌及立克次氏体等相应序列进行同源性比对,系统进化树表明,绵羊附红细胞体和其他血营养菌在进化关系上组成一个大的分支,与支原体科,支原体属病原最为接近,与立克次氏体科的病原较远。分析结果与Neimark等提出的观点一致,将这类血营养菌划归支原体科、支原体属。     

Recognition patterns of Neospora caninum tachyzoite antigens by bovine IgG at different IFAT titres

This study describes qualitative and quantitative antibody response in cows naturally infected with Neospora caninum. The study was carried out with 269 serum samples obtained from 24 cows over a period of 15 weeks. Prior to sample collection, the cows were tested with ELISA. The 269 samples were screened with IFAT and categorized into seven IFAT titre groups (< 1 : 80, 1 : 80, 1 : 200, 1 : 600, 1 : 1000, 1 : 2000, > 1 : 2000). The samples were finally analysed by Western blotting. Seven immunodominant antigens (approximately 18-, approximately 25-, approximately 33-, approximately 35-36-, approximately 45-46-, approximately 47-, approximately 60-62 kDa) and five minor antigens (approximately 25, approximately 51, approximately 64, approximately 77, approximately 116 kDa) were recognized by cow sera. The recognition of approximately 46 kDa antigen by cow sera was common to samples with IFAT titre 1 : 80 and above. Another common antigen was the approximately 18 kDa antigen, which was recognized by samples with IFAT titre 1 : 200 and above. The most remarkable observation was the presence of the 45-46 kDa, the 77 kDa, and absence of the 18 kDa antigenic bands in samples with IFAT titre 1 : 80. This observation was consistent even in the face of fluctuating antibody titre where serum antibody titres from an animal exceeded then failed to reach 1 : 80. Antibody fluctuation was observed across all cows (pregnant and aborted) with no discernible fluctuation pattern. However, the fluctuation in antibody titre observed appeared to be most remarkable in initially ELISA-negative pregnant cows, and to a lesser extent in ELISA-positive pregnant cows, and ELISA-positive aborted cows. Although there was fluctuation in antibody titre, the banding patterns of N. caninum tachyzoite antigens by cows within the same IFAT titre group remained similar.     

志贺菌耐多药相关基因水平转移的验证及其耐药性研究

目的对志贺菌耐多药相关基因(T1C4)水平转移进行验证,并对其与细菌耐药性的关系进行初步判定。方法培养已筛选出的包含T1C4基因片段的阳性克隆,提取其质粒,PCR扩增T1C4基因片段、纯化、制备探针,与志贺菌敏感株SS23、志贺菌耐多药转移株(ST11)、大肠埃希菌耐多药株(E667)的基因组DNA和质粒DNA进行斑点杂交。采用药敏纸片法研究含T1C4基因片段大肠埃希菌的耐药性。结果应用PCR方法可以从ST11和E667菌株基因组DNA中扩增出T1C4基因片段,而SS23菌株不含该基因片段;应用T1C4基因探针进行斑点杂交显示,ST11和E667菌株的全基因组和质粒DNA均可显示杂交信号,而SS23菌株的基因组和质粒DNA均不产生杂交信号;14种药物的药敏试验表明,含有T1C4基因的大肠埃希菌(DH5α)对四环素、先锋V、头孢噻吩、氟哌酸、复方新诺明等五种药物的抑菌环直径明显小于(相差3mm以上)不含T1C4基因的DH5α菌株。结论 T1C4基因片段为ST11菌株从E667菌株获得,且存在于两菌株的质粒上;T1C4基因可能介导细菌对多种药物的抗性。     

奶牛乳腺炎无乳链球菌临床分离株fbsA基因的克隆与序列分析

目的克隆并分析牛乳腺炎无乳链球菌内蒙古地区临床分离株表面蛋白fbsA基因核苷酸及编码氨基酸序列,预测其潜在的抗原表位。方法以无乳链球菌内蒙古地区临床分离株为材料,根据GenBank中公布的无乳链球菌fbsA基因序列设计特异性克隆引物,采用同源克隆法,PCR扩增fbsA基因序列,采用DNA Star生物信息学软件预测分析其编码氨基酸的潜在抗原性。结果克隆的fbsA基因序列大小为321bp,编码107个氨基酸残基,与GenBank中公布的B群无乳链球菌fbsA基因核苷酸序列同源性为98.13%,氨基酸序列同源性为99%。预测克隆基因编码氨基酸抗原性指数良好。结论成功克隆出内蒙古地区奶牛乳腺炎无乳链球菌表面蛋白fbsA基因序列,并预测其编码氨基酸具有潜在抗原性为进一步研究其原核表达产物的抗原性及致病机制奠定了基础。     

马来丝虫3-磷酸甘油醛脱氢酶基因的克隆、序列分析及编码产物B细胞表位预测

根据GenBank中马来丝虫3-磷酸甘油醛脱氢酶基因(BmG3PD基因)序列设计引物, 以马来丝虫mRNA为模板, RT-PCR扩增BmG3PD基因, 将其克隆入pGEM-T载体, 转化大肠埃希菌（E. coli）DH5α, 筛选阳性克隆。经EcoRⅠ和XhoⅠ双酶切及PCR鉴定, 获得阳性重组质粒pGEM-BmG3PD, 经序列分析及同源性比较, 以及对其编码产物进行B细胞表位预测, 结果表明PCR扩增的特异性条带为1 020 bp, 与预期相符, 与GenBank已知基因序列同源性为99%。编码产物B细胞表位预测, 氨基酸区域可能在22～36、242～255、303～318和326～336位。