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酶切图谱及序列分析虫体ITS2鉴别中国的片形吸虫 总被引:6,自引:0,他引:6
提取我国广西、四川、黑龙江、甘肃等地及法国的片形吸虫的总DNA,用PCR扩增完整的ITS-2。对得到的PCR产物用限制性内切酶Hsp 92Ⅱ,RcaI进行酶切和RFLP技术分析。并对ITS-2PCR产物进行双向测序。以便确定国内片形吸虫种内及种间ITS-2的特点和序列变异的水平。结果显示:广西、四川、黑龙江、法国等地的样品通过PCR均扩增到约550bp的ITS-2目标片段。Hsp92 Ⅱ,Rcal可区别不同种的片形吸虫。对PCR产物的序列分析结果表明片形吸虫ITS-2全长均为362bp,同种内ITS-2序列无变异,不同种间ITS-2序列存在5-6个碱基差异,变异率约为2.8%。对各地区片形吸虫ITS-2的PCR-RFLP分析与对PCR产物的DNA序列分析一致证明,来自四川的样品和法国样品同属肝片形吸虫F.hepatica;广西样品属大片形吸虫F.gigantica;黑龙江的样品为中间型片形吸虫。本研究首次从分子水平上证实我国除有肝片形吸虫、大片形吸虫外,还存在中间型的片形吸虫。 相似文献
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同工酶与基因和遗传有着密切的关系,生物种类的不同可表现为某些同工防的数量和种类的变化。因此,它可作为一项生化指标,研究生物的分化情况。本文对人畜共患的两种片吸虫的同工配进行研究,发现二者的某些同工酶存在明显差异。1.材料与方法1.1材料来源成虫采自福州市屠宰场宰杀的黄牛和山羊的胆囊和胆管中,分别选出典型的肝片形吸虫和巨片形吸虫的成虫。本研究所使用的试剂均系分析纯。1.2匀浆制各:将典型的肝片形吸虫和巨片形吸虫的成虫分别用生理盐水洗3次,成虫用pH74的缓冲液在冰水浴中研磨5分钟,高速离心(1200转/分)10分… 相似文献
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片形吸虫分泌排泄抗原和虫体抗原的分析 总被引:1,自引:0,他引:1
用牛源肝片吸虫(南京)、牛源大片吸虫(广西)和羊源肝片吸虫(实验感染)制备可溶性虫体抗原(BA)和和分泌排泄抗原(ES),以SDS-PAGE电泳分析比较,结果显示,3株虫体可溶性虫体抗原至少有20条以上的主要蛋白条带,分子量集中于10-90KD之间,它们之间无显著差异,而3株分泌排泄抗原蛋白成分较简单,明显可分的条带不超过5条,分子量集中于10-30KD之间,且均拥有26-28KD的蛋白成分,提示该蛋白应为片形吸虫ES抗原的主要免疫成分。 相似文献
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本文对肝片形吸虫和巨片形吸虫进行了组织化学探讨。结果表明:肝片形吸虫和巨片形吸虫的DNA含量不同,二者比值为2:3,且所含17种氨基酸种类相同,含量却不相同,某些氨基酸含量差别很大。 相似文献
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片形吸虫第一内转录间隔区DNA多态性的研究 总被引:9,自引:0,他引:9
以不同地区的片形吸虫虫株为研究对象,经PCR扩增出了ITS-1部分基因片段,采用单链构象多态性(SSCP)方法,并结合序列分析研究了不同地区片形吸虫ITS-1 DNA的多态性。不同地区的样品经SSCP分析,显示3种带型,第1种为大片形吸虫的带型,第2种为肝片形吸虫的带型,第3种为2种带形的混合带型。广西区样品和大部分贵州省样品属于大片形吸虫带型;四川省、黑龙江省和部分贵州省样品为混合带型;南京市和甘肃省样品为肝片形吸虫带型或混合带型。测序结果表明,根据ITS-1基因的序列变异位点可区分2种片形吸虫;表现为混合带型的样品在变异位点具有多态性。本研究结果显示,ITS-1片段可作为遗传标记用以区分大片形吸虫和肝片形吸虫,同时也证实,在我国除了这2种片形吸虫外,还可能存在着“中间型”的片形吸虫。 相似文献
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用SDS-PAGE和酶联免疫印迹试验(ELIB)分析胰吸虫成虫(EP)及其与肝片形吸虫成虫(Fh)交叉/共同抗原的分子量和免疫活性。SDS-PAGE结果显示,胰吸虫和肝片形吸虫成虫抗原的多肽均达30余条,EP抗原分子量在123kd以下,主带4条;Fb抗原分子量在83kd以下,主带4条;两者具相同分子量的多肽6条。ELIB结果显示,抗原与同源抗血清呈现颜色较深、数量较多的区带,而与异源抗血清则呈现数量不等的交叉/共同反应区带,表明两虫之间存在交叉/共同抗原性多肽。 相似文献
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片形吸虫DNA随机扩增多态性分析 总被引:6,自引:1,他引:6
为区别从南京市江宁县采集的片形吸虫非典型形态虫体,应用随机扩增多态性DNA(RAPD)技术,对6株片形吸虫总DNA进行了扩增。结果,10条引物中有8条能产生扩增图谱,电泳图谱经聚类分析,与传统的分类结果一致,并表明来自江宁的片形吸虫既有形态典型的肝片形吸虫,也有形态不典型的大片形吸虫。 相似文献
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本研究扩增肝片吸虫(Fasciola hepatica,Fh)谷胱甘肽硫转移酶(GST)基因,并进行同源性分析。根据GenBank发表的部分Fh GST基因序列设计并合成一对特异性引物,利用RT-PCR方法扩增出Fh GST基因完整的开放阅读框(open reading frame,ORF),测定序列,使用分子生物学软件进行同源性分析。获得Fh GST基因全长682 bp,编码218个氨基酸,与澳大利亚分离的肝片吸虫GST同源性较高,与大片吸虫和卫氏并殖吸虫的GST也有较高的同源性。不同虫株GST基因具有较高的同源性,因此Fh GST蛋白不适合用作诊断抗原,但由于其存在交叉反应,GST基因作为分子疫苗的候选基因具有重要意义。肝片吸虫GST基因的克隆,为进一步研究GST蛋白的功能和作用奠定了基础。 相似文献
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利用皮肤真菌核糖体内转录间隔区(Internal transcribed spacer,ITS)序列通用引物,对采自山东地区主要兔场的皮肤真菌病的16株分离菌进行了PCR扩增,ITS区的克隆、测序、序列变异及遗传进化关系分析。经与Gen-Bank核酸序列数据库数据比对结果表明:16株病菌分别为须癣毛癣菌(12/16,75%)、犬小孢子菌(2/16,12.5%)、石膏样小孢子菌(2/16,12.5%);不同病原菌的5.8SrDNA序列高度保守,而ITS区的变异性则较高;对该区序列的聚类分析表明,不同种菌株ITS1比ITS2在碱基构成和序列长度上有更大变异;而种内各菌株的ITS1和ITS2在长度上均没有变异,碱基构成上存在微小的变异,可基于该区进行兔皮肤真菌的分类鉴定。该研究确定了兔皮肤病原PCR检测特异引物的靶序列,为兔皮肤真菌病病原的特异性分子鉴定提供了可靠的靶标,为兔皮肤真菌的科学分类提供了分子依据。 相似文献
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犬隐球菌病病原分离与ITS区序列分析鉴定 《畜牧与饲料科学》2015,36(2):1-1
为确定引起格力犬皮肤病发生的病原菌种属并提供科学的诊治方案,从病犬发病部位真皮层刮取病料,采用沙氏培养基对病料进行分离、培养,根据菌落形态特征与镜检结果对分离到的病原菌进行初步鉴定。采用真菌鉴定通用引物ITS1及ITS4对分离菌的ITS区序列进行PCR扩增、测序,将测序结果与GenBank中的新生隐球菌进行比对分析并构建系统发育树。结果表明,分离到的病原菌经形态学鉴定初步判定为隐球菌属真菌,其ITS区序列与GenBank中的新生隐球菌(JN939462.1和JN939461.1)的ITS序列相似性为99%,且在系统发育树上属同一分支,提示引起该犬隐球菌病的病原菌为隐球菌属新生隐球菌。 相似文献
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Characterisation of Fasciola species from Mainland China by ITS-2 ribosomal DNA sequence 总被引:5,自引:0,他引:5
Isolates of Fasciola (Platyhelminthes: Trematoda: Digenea) from different host species and geographical locations in Mainland China were characterised genetically. The second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) was amplified from individual trematodes by polymerase chain reaction (PCR), and the representative amplicons were cloned and sequenced. The length of the ITS-2 sequences was 361-362bp for all Chinese Fasciola specimens sequenced. While there was no variation in length or composition of the ITS-2 sequences among multiple specimens from France, Sichuan and Guangxi, sequence difference of 1.7% (6/362) was detected between specimens from France and Sichuan, and those from Guangxi. Based on ITS-2 sequence data, it was concluded that the Fasciola from Sichuan represented Fasciola hepatica, the one from Guangxi represented Fasciola gigantica and the one from sheep from Heilongjiang may represent an "intermediate genotype", as its ITS-2 sequences were unique in that two different ITS-2 sequences exist in the rDNA array within a single Fasciola worm. One of the sequences is identical to that of F. hepatica, and the other is almost identical to that of F. gigantica in that nucleotides at five of the six polymorphic positions represent F. gigantica. This microheterogeneity is possibly due to sequence polymorphism among copies of the ITS-2 array within the same worm. Based on the sequence differences, a PCR-linked restriction fragment length polymorphism (PCR-RFLP) assay was established for the unequivocal delineation of the Fasciola spp. from Mainland China using restriction endonuclease Hsp92II or RcaI. This assay should provide a valuable tool for the molecular identification and for studying the ecology and population genetic structures of Fasciola spp. from Mainland China and elsewhere. 相似文献
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采集关中某羊场奶山羊的血液样品,以提取的血液基因组DNA为模板对弓形虫ITS1及529bp基因进行PCR扩增、测序,并对测序结果进行比对,基于529bp重复序列构建系统进化树。序列分析结果表明,待测样品与GenBank中弓形虫RH虫株的ITS1序列(AY259044)完全一致,与529bp重复序列(FJ656209.1)存在差异;基于529bp重复序列构建的系统进化树分析结果显示,待测样品弓形虫与RH虫株遗传距离最近。该结果为奶山羊弓形虫病的分子流行病学、诊断方法的建立等方面的研究奠定了基础。 相似文献
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Ichikawa M Itagaki T 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2012,74(7):899-904
It has been speculated that populations of aspermic Fasciola flukes in Korea and Japan have a close phylogenetic relationship. To evaluate this, we analyzed 33 Korean aspermic Fasciola flukes on the basis of nuclear ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (nad1) and cytochrome c oxidase 1 (cox1) sequences. Fh, Fg, and Fh/Fg types were detected in the ITS1 region and displayed the fragment patterns of F. hepatica, F. gigantica, and both species, respectively by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Additionally, three concatenated haplotypes of nad1 and cox1(nad1/cox1) were detected, and 2 of these, Kor1/Kor1 (Fsp1/Fsp1) haplotype and Kor2a/Kor2 (Fsp2/Fsp2) haplotype, were shared by Korean and Japanese aspermic flukes. The Fst value (0.019), calculated using the concatenated sequences, indicated that Korean and Japanese aspermic Fasciola populations were genetically undifferentiated. Interestingly, a combination of the Fh/Fg type and Kor1/Kor1 haplotype was found at the highest frequency in Korean aspermic flukes, whereas the Fg type and Fsp2/Fsp2 haplotype combination was found at a conspicuously high frequency in Japanese aspermic flukes. This indicates that a founder effect caused by the introduction of infected hosts may have played a key role in the introduction of aspermic Fasciola flukes from Korea into Japan. 相似文献
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为分析捻转血矛线虫ITS序列的变异与虫株特性之间的关系,对捻转血矛线虫不同药物抗性虫株(苯并咪唑类药物抗性虫株、伊维菌素抗性虫株、莫西菌素抗性虫株、药物敏感虫株)、不同宿主来源虫株(长颈鹿、牛、绵羊、山羊、牦牛)和不同地理位置(中国、美国、意大利、法国、也门、马来西亚、澳大利亚)的分离株,以及毛圆科的其他线虫的ITS序列进行了分析。结果发现捻转血矛线虫与毛圆科其他线虫的ITS-2基因的相似性高,基于该基因建立的系统进化树可以很好地区分毛圆科不同属的线虫;捻转血矛线虫的抗药性特点与其ITS-1基因的变异进化关系不大;不同地理位置的捻转血矛线虫分离株的ITS-2基因变异很小;从野生动物长颈鹿体内分离的捻转血矛线虫的ITS-1基因与家养反刍动物分离株的差异较明显。研究结果表明捻转血矛线虫ITS序列在种内相对保守,不同虫株间变异较小,在毛圆科线虫的分子分类中具有一定意义。 相似文献
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Ai L Li C Elsheikha HM Hong SJ Chen JX Chen SH Li X Cai XQ Chen MX Zhu XQ 《Veterinary parasitology》2010,174(3-4):228-233
The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of Fasciola hepatica and Fasciola gigantica. The LAMP assay is inexpensive, easy to perform and shows rapid reaction, wherein the amplification can be obtained in 45 min under isothermal conditions of 61 °C or 62 °C by employing a set of four species-specific primer mixtures and results can be checked through naked-eye visualization. The optimal assay conditions with no cross-reaction with other closely related trematodes (Clonorchis sinensis, Opisthorchis viverrini, Orientobilharzia turkestanicum and Schistosoma japonicum) as well as within the two Fasciola species were established. The assay was validated by examining F. gigantica DNA in the intermediate host snails and in faecal samples. The results indicated that the LAMP assay is approximately 10(4) times more sensitive than the conventional specific PCR assays. These findings indicate that this Fasciola species-specific LAMP assay may have a potential clinical application for detection and differentiation of Fasciola species, especially in endemic countries. 相似文献