牛隐孢子虫多重PCR方法的建立及应用

摘要：目的 为寻求一种牛源隐孢子虫的快速检测手段。方法 根据牛隐孢子虫COWP基因和ITS-1基因,设计合成两对特异性引物,用以特异性扩增牛微小隐孢子虫和安氏隐孢子虫,扩增长度分别为1033bp和263bp。通过反应条件的优化,特异性试验及扩增片段的序列测定的验证,建立多重PCR方法。结果 应用该方法对采集自广西不同地区的1613份牛粪样进行牛隐孢子虫检测,安氏隐孢子虫阳性粪样为189份,微小隐孢子虫阳性粪样1份,与常规隐孢子虫分离鉴定结果一致。结论 表明该方法可用于隐孢子虫的流行病学调查及临床诊断。     

水源性隐孢子虫的PCR检测

水源性隐孢子虫病暴发是一大公共卫生问题 ,且水中的隐孢子虫抵抗力强 ,因其含量少 ,不易检测。PCR技术快速、简便、准确、高度敏感与高度特异。该文就当前在水源性隐孢子虫的PCR检测文献资料进行综述     

牛源隐孢子虫上海分离株的巢式PCR鉴定

目的 用巢式PCR鉴定1株自然感染的上海牛源隐孢子虫。 方法 改良抗酸染色法检查含隐孢子虫卵囊的牛粪,油镜下观察卵囊形态,测量大小。以牛粪中的隐孢子虫卵囊DNA为模板,根据隐孢子虫18S rRNA序列设计2对引物,巢式PCR扩增目的片段,测序,同源性比对,并运用MEGA4.0软件构建系统发育树。 结果 上海牛源隐孢子虫分离株卵囊呈圆形或椭圆形,大小为（5.6±0.49）mm×（5.2±0.51）mm。扩增出的18S rRNA基因片段为812 bp。上海牛源隐孢子虫分离株的18S rRNA与巴西的牛隐孢子虫（Cryptospridium bovis,GenＢank登录号为151935628）序列一致性为100%,两者在种系发育树上为同一分支,亲缘关系最近;与中国青海、蒙古国、美国、突尼斯等地的牛隐孢子虫序列一致性均为99%。 结论 上海牛源隐孢子虫分离株为牛隐孢子虫（C. bovis）。     

新孢子虫病免疫学诊断和免疫预防的研究进展

新孢子虫病是一种对多种动物危害严重的原虫病，给畜牧业带来很大的经济损失，特别是奶牛饲养业。新孢子虫病越来越被人们所关注，特别是诊断和防制成为国内外兽医界研究的热点。该文对新孢子虫病免疫诊断方法的适用范围、优缺点及新孢子虫病免疫预防机制和疫苗研究情况进行了综述。     

应用FTA/PCR检测粪便中隐孢子虫的实验研究

目的 建立用FTA/PCR检测粪便中隐孢子虫的技术,并与常用的商业化的粪便DNA抽提试剂盒进行比较.方法 选择经病原学检测已经确诊的隐孢子虫阳性和阴性粪便,分别采用FTA卡和商业化DNA抽提试剂盒抽提粪便样本中DNA,进行巢式PCR扩增,对PCR阳性产物进行测序并利用BLAST在NCBI上与GenBank数据库进行比对,做同源性分析,确定虫种.结果 应用商业化DNA抽提试剂盒抽提DNA,无论是否反复冻融破壁,2个阳性对照样本扩增出目的 片段,另3个阳性样本未扩增出特异性条带.样本纯化后应用FTA卡抽提DNA进行PCR检测,所有阳性粪样均扩增出830 bp左右的目的 片段,通过测序和比对,其中2个为贝氏隐孢子虫,1个为火鸡隐孢子虫.结论 应用FTA进行粪便样本中隐孢子虫DNA的抽提方法具有简单有效、快速灵敏、准确可靠等特点,可以应用于粪便中隐孢子虫的检测.     

微孢子虫病研究进展

随着艾滋病的流行 ,有关微孢子虫病的报道逐渐增多。本文从检测诊断、免疫学、细胞培养、动物模型、分子生物学及治疗等方面对感染人的微孢子虫进行综述     

卡氏肺孢子虫PCR方法敏感性研究

目的从3种PCR方法中选择出一种检测卡氏肺孢子虫(Pc)敏感性最高的PCR方法,供以后的实验中应用。方法Wistar大鼠30只,皮下注射地塞米松,每周二次,制备PCP动物模型,8周后全部剖杀作大鼠肺印片,六亚甲基四胺银染色。各鼠取肺组织02g,均浆后提取DNA,用三种PCR方法作PCR试验。选取六亚甲基四胺银染色和三种PCR检测均为阳性的大鼠DNA标本20份,对每份标本作对倍稀释1/2、1/4、1/8、1/16……至1/2048,同时用三种PCR方法做扩增试验直到某一稀释度时PCR试验阴性为止,选择出最为敏感的一组PCR方法,并做统计学分析。结果三种方法最低稀释度的均数DHFR-PCT为1/7879±174;TS-PCR为1/4371±207;MT-PCR为1/10975±136。结论三种检测Pc的PCR方法中以MT-PCR方法最为敏感。     

水源性隐孢子虫的PCR检测

水源性隐孢子虫病暴发是一大公共卫生问题，且水中的隐孢子虫抵抗力强，因其含量少，不易检测。PCR技术快速、简便、准确、高度敏感与高度特异，该文就当前在水源性隐孢子虫的PCR检测文献资料进行综述。     

PCR技术检测卡氏肺孢子虫的研究现状

本文简述了近年来多聚酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）在卡氏肺孢子虫（Ｐｎｅｕｍｏｃｙｓｉｔｉｓｃａｒｒｉｎｉ，Ｐ．ｃ．）检测中的研究现状，重点阐述了ＰＣＲ的引物，取材对检测结果的影响，同时也探讨了简化样品制备和改良ＰＣＲ其在临床广泛应用的促进作用。     

FTA/PCR检测加入饮料中的隐孢子虫

用建立的FTA/PCR方法检测饮料样品中隐孢子虫,可检测到隐孢子虫卵囊1.0个/ml,且在6 h内完成。该方法敏感性较高,省时,是检测饮料样品中隐孢子虫的较好方法。     

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。     

Cytokine gene expression at the materno-foetal interface after experimental Neospora caninum infection of heifers at 110 days of gestation

Neospora caninum is a major cause of abortion in cattle, but the reasons why only some animals abort remain unclear. The immunological control of the parasite in the placenta or by the foetus could be the key to determining the mechanism of abortion and/or transplacental transmission to the foetus. In this study, cytokine gene expression, analysed by real-time RT-PCR, at the maternal (caruncle) and foetal placenta (cotyledon) of heifers infected at 110 days of gestation by intravenous inoculation of N. caninum tachyzoites was compared with the responses in uninfected heifers. Animals were euthanized 3 weeks after infection. Upregulated Th1, Th2 and T-regulatory (Treg) cytokine gene expression was observed in both the maternal and the foetal placenta in the infected group. In the caruncle of infected animals, the main changes included upregulation of IFN-γ, IL-12p40, IL-6 and IL-10. In the cotyledon, the main changes included upregulation of IFN-γ and downregulation of TGF-β, being the later the only cytokine downregulated in the infected group. The observed cytokine expression pattern was associated with alive but transplacentally infected foetuses, suggesting that such cytokine pattern is beneficial to foetal survival, but could have a role in the transplacental transmission of the parasite.     

Analysis of the immune response to Neospora caninum in a model of intragastric infection in mice

To study experimental Neospora caninum infection initiated at the gastrointestinal tract, Toll-like Receptor 4- and functional IL-12Rbeta2 chain-deficient C57BL/10 ScCr mice were challenged intragastrically with 5 x 10(6) N. caninum tachyzoites. All parasite-inoculated mice eventually died with disseminated infection. In contrast, immunocompetent BALB/c mice challenged with 1 x 10(7) N. caninum tachyzoites by the intragastric (i.g.) or the intraperitoneal (i.p.) route remained alive for at least 6 months. Expansion of splenic B- and T-cells, the latter displaying both activated and regulatory phenotypes, and increased levels of IFN-gamma and IL-10 mRNA were detected in both groups of infected BALB/c mice compared with non-infected controls, whereas in the Peyer's patches only IFN-gamma mRNA levels were found to be increased. Parasite-specific IgG1, IgG2a and IgA antibody levels were elevated in the sera of all infected mice, whereas increased N. caninum-specific IgA levels were detected in intestinal lavage fluids of i.g. challenged mice only. These results show that N. caninum infection can be successfully established in mice by i.g. administration of tachyzoites. They also show that the immune response elicited in i.g. or i.p. infected BALB/c mice, although conferring some degree of protection, was not sufficient for complete parasite clearance.     

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.     

Mechanisms of apoptosis in murine fibroblasts by two intracellular protozoan parasites,Toxoplasma gondii and Neospora caninum

Studies to clarify the mechanisms of apoptosis in host cells, A31 (BALB/3T3 clone A31 fibroblasts), caused by two intracellular protozoan parasites, Toxoplasma gondii and Neospora caninum, were carried out in an in vitro system. The viability of N. caninum-infected cells was significantly reduced following treatment with mouse interferon (IFN)-gamma. By contrast, mouse IFN-gamma treatment had no significant effect on the induction of apoptosis in T. gondii-infected cells. Apoptosis of N. caninum-infected and mouse IFN-gamma-treated cells was shown to be associated with increased DNA fragmentation, and increased caspase-3 and -8 activity, and the administration of caspase-3 and -8 inhibitors inhibited cell death. FasL expression was clearly induced by N. caninum-infection and IFN-gamma treatment compared to the T. gondii-infected cells and the uninfected control with or without IFN-gamma treatment. The reduction in host-cell viability was prevented with the addition of antimouse FasL monoclonal antibody (mAb). Moreover, TUNEL analyses indicated that apoptosis was induced by the treatment with Fas mAb in both T. gondii and N. caninum-infected cells. These results suggest that the Fas/FasL pathway may play a crucial role in the induction of apoptosis in N. caninum- and T. gondii-infected cells mediated by IFN-gamma.     

快速检测血吸虫抗体的乳胶微球层析法(DLIA)的建立和应用

目的建立一种快速、简便、敏感、特异的血吸虫病免疫诊断方法。方法用可溶性血吸虫虫卵抗原作为包被抗原,红色乳胶微球标记鼠抗人单克隆抗体为显色剂,建立一种快速检测血吸虫病患者血清抗体的乳胶微球层析法(DLIA),以该法分别检测血吸虫虫卵阳性者血清69份,健康人血清264份,华支睾吸虫病患者血清15份,广州管圆线虫病患者血清8份,钩虫、蛔虫、鞭虫混合感染者血清11份和卫氏并殖吸虫病患者血清19份,用ELISA平行检测作为对照。结果DLIA和ELISA法检测血吸虫虫卵阳性患者血清的敏感性分别为94.2%(65/69)和95.7%(66/69),检测健康人血清的特异性分别为97.4%(257/264)和94.7%(250/264),差异均无统计学意义(χ2=0.15和2.43,P0.05);两法检测华支睾吸虫病和广州管圆线虫病患者血清,钩虫、蛔虫、鞭虫混合感染者血清,均无交叉反应;两法与卫氏并殖吸虫病患者血清的交叉反应率分别为42.1%(8/19)和47.4%(9/19),差异无统计学意义(χ2=0.11,P0.05)。结论乳胶微球层析法检测血吸虫抗体具有比较敏感、特异、简便、快速和稳定的特点。     

实时荧光定量PCR法检测日本血吸虫

目的 运用实时荧光定量PCR法检测日本血吸虫。 方法 根据日本血吸虫18 S小亚基单位核糖体核酸（18S rRNA）基因设计特异性引物,PCR扩增出1 450 bp序列,经TA克隆后转入大肠埃希菌DH5α,提取重组质粒,鉴定后作为模板制作荧光定量PCR 标准曲线。方法重现性评价,用初始循环数（Ct,拷贝/反应）进行标准差分析,并计算变异系数（CV）。 结果 制作的标准曲线循环阈值与模板浓度具有良好的线性关系,相关系数为0.998 7。方法重现性评价,在1.05×107～1.05×103个拷贝范围内,对应的Ct平均值分别为17.55、20.93、24.32、27.59和30.95;CV值分别为1.31%、1.53%、0.90%、1.85%及0.90%,在重复性试验中试验间数据平均变异系数为1.27%,无非特异性扩增。 在试验检测范围内（Ct≤30.95）,可检测的日本血吸虫基因组浓度为6.15 pg,3 h内完成。 结论 运用荧光定量PCR方法检测日本血吸虫DNA,快速、灵敏、特异性高。     

动态窗口扫描统计量法在急性血吸虫病时间聚集性分析中的应用

使用动态窗口扫描统计量法、集中度法和圆形分布法分析2001-2006年安徽省池州市贵池区急性血吸虫病病例的发病时间,确定其时间聚集性。动态窗口扫描统计量法分析结果显示,贵池区急性血吸虫病病例的发病时间从2001年到2003年越来越集中,之后越来越分散,而2006年的发病时间聚集性消失（对数似然比LLR=4.14,P>0.05）;分析所有病例发现,2002年8～9月是6年中发病的最大聚集期（LLR=18.5,P＜0.01）,与实际情况相符。而集中度和圆形分布法提供的信息较少,且2002年的分析结果与动态窗口扫描统计量法所得结果有差异。本研究结果显示,与其他两种分析方法相比,动态窗口扫描统计量法在急性血吸虫病的时间聚集性分析中提供信息较多,结果可靠。     

套式/多重PCR方法应用于疟疾诊断与监测的初步评价

目的 与镜检法比较评价标签引物-套式/多重PCR（UT-PCR）在疟疾监测中的应用价值。 方法 在海南、云南省恶性疟和间日疟混合流行区和广西疟疾控制区的疟疾监测中,采集初诊为疟疾或疑似疟疾的发热患者的血片与滤纸血样400份,在双盲条件下比较镜检法与UT-PCR的初检结果,对结果不一致的血片再次镜检复查,同时对其滤纸血样重复PCR 2～3次;评估UT-PCR与镜检法的敏感性和特异性。 结果 400例发热患者血样中,镜检法初检检出疟原虫阳性234例,其中恶性疟125例,间日疟109例;UT-PCR检出疟原虫阳性235例,其中恶性疟124例,间日疟109例;恶性疟和间日疟混合感染2例。两法初检结果一致的血样占92.5%（370/400）,其中阴性154例,阳性216例（间日疟117例,恶性疟99例）。复查25份初检结果不一致的血样,包括镜检阴性PCR阳性11例,镜检阳性PCR阴性10例,镜检为恶性疟PCR为间日疟3例,镜检为间日疟而PCR为混合感染1例,其中15份与UT-PCR的初检结果一致,7份“假阳性”原因不明,仅3份为PCR的假阴性。根据复查结果评估PCR的敏感性为99.6%,特异性为98.8%。 结论 采用更敏感的UT?鄄PCR疟疾诊断方法有助于解决疟疾诊断与鉴别诊断中的疑难问题,提高疟疾监测的质量和效率。