微小隐孢子虫种特异性基因片段的克隆及诊断引物的确定

目的　寻找用于诊断微小隐孢子虫病的特异性引物。　方法　在RAPD分析过程中获得了 1条微小隐孢子虫 [Cryptosporidium parvum (C .parvum ) ]种特有 712bp的基因片段 ,将该片段分离、纯化、克隆和测序 ,据此序列合成一对特异性引物FF。用引物FF扩增美国 2株C .parvum和中国 4株C .parvum ,并用该引物与Morgan( 1996 )发表的C .parvum种特异性诊断引物 0 2 1作对照 ,检测镜检C .parvum卵囊阴性的兔粪样 35份和人粪样 5 5份。　 结果　引物FF扩增 6株C .parvum均能产生预计 6 0 3bp的片段 ,引物FF和引物 0 2 1粪样检测结果完全一致 ,其敏感性是可检出 1个卵囊的DNA。　结论　获得的引物FF特异性强 ,敏感性高 ,可用于微小隐孢子虫病的诊断。     

基于凝集素基因对部分隐孢子虫种类的分子鉴定

目的 为了解隐孢子虫凝集素基因在不同分离株的序列差异及其遗传进化关系,以SSU rRNA基因作参照,评价Lectin基因位点作为隐孢子虫基因分型和进化关系分析的可行性。方法 采用巢式PCR,分别在Lectin和SSU rRNA位点处对本实验室分离保存的多个隐孢子虫分离株进行PCR的扩增。用Clustalx对扩增序列与参考序列进行比对,用MEGA5中的邻近法(Neighbor joining method NJ),进行进化树的构建。结果 基于Lectin基因位点,在C. parvum、C. hominis、C. cuniculus及其在SSU rRNA 处与人源C. hominis极为相近的horse genotype、驴源C. hominis均成功扩增出了大小在450 bp左右的目的 条带,并进行了进化树的分析,不同种类和同种不同动物来源的隐孢子虫分布在不同的分支上。结论 Lectin基因位点可很好的区分SSU rRNA序列极为相似的几种隐孢子虫,有望为人兽共患隐孢子虫分类和遗传研究提供新的基因靶标。     

血吸虫种株随机扩增多态性DNA差异研究

目的 对曼氏血吸虫、日本血吸虫的种株基因组DNA进行多态性分析。方法 随机扩增多态性DNA技术（RAPD）。结果 日本血吸虫与曼氏血吸虫种间差异明显,两种间的遗传距离大于0.90;日本血吸虫中国大陆株与台湾株之间存在较大的差异,两者的遗传距离大于0.20;日本血吸虫中国大陆株内,以四川天全为代表的西部山区型地域株与湘、鄂、赣代表的湖沼型地域株之间有一定差异,其遗传距离在0.060-0.067之间,湘、鄂、赣湖区之间并非完全一样,也存在较小的差异,遗传距离在0.000－0.029之间;位于湖北省境内的4个地域株的日本血吸虫存在一定的基因差异;RAPD技术能鉴别血吸虫雌雄性别差异,718bp为雌虫特异扩增条带。结论 日本血吸虫中国大际株已发生不同程度的遗传分化,中国大陆株并非单一的地域株,是由若干不同株构成的复合株;湖北省血吸虫虫株至少存在3个基因类型;RAPD技术能从DNA基因水平区分血吸虫的种株及性别差异,是分析血吸虫基因遗传变异的好方法。     

人源隐孢子虫分离株种类鉴定与种系发育关系分析

目的确定郑州某医院分离到1株隐孢子虫(PRHN)所属种/基因型。方法用PCR对该分离株18S rRNA、HSP70、Cpn60和AOX进行PCR扩增,并对扩增产物进行测序,扩增序列经用DNAstar和ClustalX与GenBank参考序列比对,用PAUP4.0构建种系发育进化树,以确定该分离株与其他隐孢子虫虫种之间的亲缘关系。结果通过18S rRNA、HSP70基因分析该分离株与Cryptosporidium parvum(C.parvum)鼠基因型同源性最高,分别为99.9%和99.2%,在种系发育树上,PRHN均与C.parvum鼠基因型亲缘关系最近;对Chaperponin 60(Cpn60)和Alternative oxidase(AOX)进行序列分析时,该分离株与本实验室分离到的猪隐孢子虫(C.suis)亲缘关系最近。结论该分离株为C.parvum鼠基因型。     

我国利什曼原虫RAPD分析

目的　我国不同疫区利什曼原虫分离株的RAPD分析。　方法　用 7种随机引物 ,扩增来自我国 3个疫区 (得自利什曼病患者、病犬及白蛉 )的利什曼原虫分离株和国际标准株 ,并将扩增产物进行聚类分析。　结果　①来自我国山丘疫区及平原疫区的L d .分离株分别聚为两类 ,两者遗传距离较远 ;②来自新疆荒漠疫区、近荒漠疫区及平原地区的L d .XJ771、L d .XJ90 1和L d .XJ80 1聚在一类 ,表明它们的遗传距离较近 ;③山丘疫区虫株中来自病人和病犬的分离株区别不明显 ,两者同源性高 ,表明犬在传播中起着重要作用 ;④印度平原型标准株L d .DD8与我国平原疫区虫株聚在一类 ;⑤L infantum与我国山丘疫区的L d .分离株分属于两类 ;⑥L dJed与平原疫区L d .分离株亲缘关系较近 ;⑦L infantum与L tropica最早聚合 ,遗传距离最近。　结论　我国不同疫区利什曼原虫分离株在基因水平上存有差异。     

微小隐孢子虫亲环素型肽脯氨酰顺反异构酶基因克隆与分析

摘要：目的 克隆微小隐孢子虫（C.parvm）南京株( NJ) 亲环素型肽脯氨酰顺反异构酶基因,测序并分析C.parvumNJ株与其他隐孢子虫分离株CyP-type PPIase基因序列的差异以及亲缘关系。方法 根据GenBank上C.parvm Iowa II株CyP-type PPIase基因序列,设计2对引物,应用巢式PCR扩增目的基因,并将其克隆入pMD18-T载体,对重组子进行双酶切和菌落PCR鉴定并测序,应用生物信息学方法分析C.parvum NJ株CyP-type PPIase基因与其它隐孢子虫株的核苷酸和氨基酸序列差异。结果 巢式PCR扩增获得特异的目的基因,双酶切和菌落PCR鉴定获得正确的重组子,测序表明,扩增基因组DNA序列为746bp,含有目的基因ORF全长570bp,编码190个氨基酸,该基因已登录GenBank,登录号为JQ284432。序列分析表明,我国C.parvum NJ 株与国外分离的Iowa II 株CyP-type PPIase基因的氨基酸序列具有99%的同源性。进化树分析表明,C.parvm NJ株与Iowa II 株CyP-type PPIase基因之间的亲缘关系最近,与脑膜炎双球菌的亲缘关系最远。结论 成功克隆C.parvum CyP-type PPIase基因,C.parvum CyP-type PPIase基因在氨基酸水平具有高度保守。     

哈尔滨一牛源赖氏隐孢子虫分离株的分子鉴定

目的对来源于哈尔滨某奶牛场一头3月龄奶牛的一个隐孢子虫分离株进行了分子鉴定。选择隐孢子虫基因分型常用的18SrRNA和actin基因位点,设计引物,用巢氏PCR方法分别扩增出约840bp和1066bp目的片段。PCR产物经双向测序后用ClustlX、DNAstar和PHLIP软件进行序列分析。结果表明在18SrRNA基因位点上,此分离株与报道的牛源C.ryanae分离株(DQ871345、AY587166、EU410344)同源性最高,达到100%。在actin位点,此分离株和美国牛源C.ryanae分离株(EU410345)和隐孢子虫鹿样基因型(AY741309)同源性也为100%。种系发育分析显示所获得的分离株在两个基因位点上均和C.ryanae处于同一进化分支。这些分析表明此次分离获得的隐孢子虫分离株为C.ryanae。鉴定结果为研究我国牛源隐孢子虫种类分布以及评价其公共卫生意义提供了重要参考。     

微小隐孢子虫LDH基因的克隆与序列分析

目的克隆微小隐孢子虫(Cryptosporidium parvum,Cp)南京株(NJ)乳酸脱氢酶(lactate dehydrogenase,LDH)基因,测序并分析微小隐孢子虫NJ株CpLDH与其他隐孢子虫分离株LDH基因序列的差异。方法根据微小隐孢子虫已知LDH 基因序列设计合成2对引物, 应用巢式PCR 技术从微小隐孢子虫NJ株基因组DNA 中扩增LDH 基因, 并将其克隆到pMD18 T 载体上,阳性克隆的重组质粒经PCR及双酶切鉴定后, 用双脱氧链终止法对重组质粒中的插入序列进行测序,应用生物信息学方法分析 CpLDH 基因序列和其他物种LDH序列的同源性。结果巢式PCR 扩增得到特异的CpLDH 基因序列,经PCR及双酶切鉴定获得了正确的pMD18 T CpLDH 重组质粒。测序表明, NJ株微小隐孢子虫LDH 基因全长966 bp, 编码322个氨基酸,该基因序列已登录GenBank,登录号为 HM001298。序列分析表明, 我国微小隐孢子虫NJ株与国外分离的Iowa II株 LDH基因编码的氨基酸序列具有98%的同源性。结论成功克隆了微小隐孢子虫NJ株LDH 基因;序列测定及同源性分析表明, 微小隐孢子虫NJ株在LDH酶关键结构位点存在突变。     

用SSR-PCR和RAPD技术研究中国不同地域旋毛虫株的遗传变异

目的　对中国不同地域的旋毛虫株基因组DNA进行多态性分析 ,并为其种及种下分类提供依据。方法　采用微卫星锚定PCR(SSR PCR)及随机扩增多态性DNA技术 (RAPD)对我国 6旋毛虫株基因组DNA进行PCR扩增 ,根据扩增产物电泳条带情况进行SAS分析 ,计算遗传距离并构建进化树。结果　T3、T7与国内各地域株间遗传距离均大于 0 85 ;湖北株与天津株、黑龙江株与云南株之间的遗传距离均小于 0 2。河南株与前 4株的遗传距离在SSR PCR小于 0 3,在RAPD小于0 6。结论　中国 6株旋毛虫在基因水平可分为不同层次的 3类 :a 湖北株、天津株为一类 ;b 黑龙江株、云南株为一类 ;c 不明株与河南株归为一类或者不明株归为a类中。国内 6株归属于T1。此外 ,国际标准株T3、T7分为另两大类     

RAPD技术在宁夏人源细粒棘球蚴基因分型中的应用

目的建立RAPD分子分型技术并将其应用于人源细粒棘球蚴基因分型研究。方法用单因素筛选方法建立PCR-RAPD分析体系,并用建立的RAPD分析方法对分离自宁夏不同地区13例包虫病病人的细粒棘球蚴进行RAPD指纹图谱分析。结果采用20条随机引物对细粒棘球蚴DNA进行扩增,其中11条引物呈现良好的多态性和稳定性,共得到232条片段。根据RAPD指纹图谱将13株人源细粒棘球蚴分为Ⅰ型(53.85%)、Ⅱ型(23.08%)、Ⅲ型(7.69%)和Ⅳ型(15.38%)4个种内基因类群。结论宁夏地区人源细粒棘球蚴之间存在不同的种内遗传差异。     

七种媒介硬蜱基因组随机扩增多态性DNA分析

目的　研究7种硬蜱的基因组随机扩增多态性DNA(RAPD)以及种间的遗传距离。　方法　用5条不同的多聚核苷酸单链引物对草原革蜱、森林革蜱、青海血蜱、台湾血蜱、刻点血蜱、龟形花蜱、卵形硬蜱7种硬蜱基因组DNA进行随机扩增,分析DNA图谱并计算 7种硬蜱间的遗传距离。　结果　 7种硬蜱基因组随机扩增产物均有各自独特的DNA条带,种间的平均遗传距离为071。　结论　RAPD技术可以区分这7种硬蜱。     

Schistosoma japonicum strains: differentiation by RAPD and SSR-PCR

Eight geographical isolates of Schistosoma japonicum from Taiwan and mainland China and one isolate of Schistosoma mansoni were studied by RAPD analysis using six arbitrary primers and SSR-PCR analysis using a (CA)8RY primer. The genetic distance was determined by the percentage of unshared bands. The RAPD and SSR-PCR results showed that the genetic distance between S. mansoni and S. japonicum was more than 0.900 and 0.850 respectively; the genetic distance between the eight geographical isolates of S. japonicum was 0.000 to 0.232 and 0.066 to 0.368 respectively. These results demonstrated the usefulness of RAPD and SSR-PCR for showing the differences of inter- and intra-species of Schistosoma. The results also suggest that there is genetic diversity among the different geographical strains of S. japonicum in China.     

安徽省不同血吸虫病流行区湖北钉螺遗传差异性研究

目的研究安徽省不同血吸虫病流行区湖北钉螺的遗传差异性。方法采集宁国市有螺无病地区和芜湖市有螺有病地区的湖北钉螺,提取其头足部基因组DNA,用7条GC含量不同的随机引物进行RAPD扩增,根据扩增产物琼脂糖凝胶电泳的DNA带型,比较两地湖北钉螺基因组DNA的遗传差异性,并计算其遗传距离。结果两地区湖北钉螺PCR产物呈多态性,扩增产物中除具有相同的DNA条带外,又具有各自独特的DNA条带,扩增片段共享度F值为0.603,DNA条带亮度也有不同。两地湖北钉螺遗传距离为0.387。结论安徽省不同血吸虫病流行区湖北钉螺基因组DNA存在较大的遗传差异。     

应用RAPD技术对我国11个地域株并殖吸虫遗传变异的初步探讨

目的对来自中国3省9地11个地域株并殖吸虫进行遗传变异研究,明确它们的亲缘关系,为并殖吸虫的分类提供依据,尤其为湖北省并殖吸虫流行区划分提供分子生物学依据;探讨采用不同虫期标本及其不同保存方法对该研究的影响。方法采用随机扩增多态性DNA(RAPD)技术,对11个地域株并殖吸虫进行PCR扩增,根据扩增产物琼脂糖凝胶电泳的带型,计算各株并殖吸虫的遗传距离,并绘制系统进化树。结果RAPD技术将11个地域株并殖吸虫分为3类:①江西武宁、浙江兰亭、湖北温泉和湖北赤壁;②湖北走马(a)、湖北走马(b)、湖北五峰(a)、湖北五峰(b)、湖北十堰和湖北五里;③湖北神农架。结论11个地域株并殖吸虫在基因水平上存在差异,其中有种间和种内差异。我国湖北、江西和浙江3省存在3种并殖吸虫:卫氏并殖吸虫、斯氏并殖吸虫和一种尚未定种的神农架并殖吸虫;湖北存在除卫氏、斯氏并殖吸虫外不明种的神农架并殖吸虫。采用不同虫期(成虫和囊蚴)标本及其不同保存方法不影响遗传变异研究的结果。     

Virulence profile of ten Paracoccidioides brasiliensis isolates: association with morphologic and genetic patterns

Ten isolates of Paracoccidioides brasiliensis were examined for differences in virulence in outbred mice intravenously inoculated with the fungus, associated with mycelial morphology, and genetic patterns measured by random amplified polymorphic DNA (RAPD). Virulence was evaluated by viable yeast cell recovery from lungs and demonstration of histopathologic lesions in different organs. The results showed that the isolates presented four virulence degrees: high virulence, intermediate, low and non-virulence. RAPD clustered the isolates studied in two main groups with 56% of genetic similarity. Strains with low virulence, Pb265 or the non-virulent, Pb192, showed glabrous/cerebriform morphology and high genetic similarity (98.7%) when compared to the other isolates studied. The same was observed with Bt79 and Bt83 that shared 96% genetic similarity, cottony colonies and high virulence. The RAPD technique could only discriminate P. brasiliensis isolates according to glabrous/cerebriform or cottony colonies, and also high from low virulence strains. Isolates with intermediate virulence such as Pb18, Pb18B6, Bt32 and Bt56 showed variability in their similarity coefficient suggesting that RAPD was able to detect genetic variability in this fungal species. Virulence profile of P. brasiliensis demonstrated that both mycelial morphologic extreme phenotypes may be associated with fungal virulence and their in vitro subculture time. Thus, RAPD technique analysis employed in association with virulence, morphologic and immunologic aspects might prove adequate to detect differences between P. brasiliensis isolates.     

Comparative study of PCR-based Cryptosporidium discriminating techniques with a review of the literature

We identified the species or genotypes of the six Cryptosporidium isolates from patients and C. parvum strain HNJ-1 using the seven previously described species-differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. In addition, we also discussed about the usefulness of these PCR-based protocols on the basis of the reports previously published. Cryptosporidium diagnostic fragment was amplified by PCR with each primer pair, targeting the 18S ribosomal RNA (18SrRNA), Cryptosporidium oocyst wall protein (COWP). Heat shock protein 70 (HSP70), Polythreonine (Poly-T), Thrombospondin related adhesive protein of Cryptosporidium-1 (TRAP-C1), and unknown gene locus, in all isolates from patients and the strain HNJ-1. The RFLP profiles of 18SrRNA, COWP, HSP70, Poly-T, and TRAP-C1 PCR products in all isolates from patients were found to be the same among isolates, and were correspondent to those of C. parvum human genotype. While the RFLP profiles of HNJ-1 were strictly different from those of isolates from patients, and were correspondent to C. parvum cattle genotype. In addition, nucleotide sequences in 18 SrRNA gene of all isolates from patients and HNJ-1 were found to be identical to that of C. parvum, human or cattle genotype, respectively. Therefore, the isolates from patients and HNJ-1 were identified as C. parvum human and cattle genotype, respectively. According to the reports related to the PCR-based protocols applied in the present study, RFLP profiles targeting the HSP70, Poly-T, TRAP-C1 genes had been revealed in only a few species or genotypes, but those of 18SrRNA and COWP genes were in all species and genotypes. However, we supposed that it was difficult to distinguish between human or cattle genotype and other species or genotypes by RFLP profiles of 18SrRNA or COWP because the RFLP profiles of human or cattle genotype were identical or similar to those of other species or genotypes. On the other hand, it has been known that the nucleotide sequences in 18SrRNA or COWP gene are different among Cryptosporidium species and/or genotypes. Therefore, the direct sequencing method targeting the variable regions which can be used to distinguish among Cryptosporidium species, as well as the genotypes within C. parvum in either 18SrRNA or COWP gene is the most useful tool for accurate identification of Cryptosporidium isolates.     

安徽省三种常见蜚蠊基因组多态性DNA的研究

目的；研究随机扩增多态性DNA（RAPD）技术，用于蜚蠊分子分类。方法：提取3种蜚蠊的基因组DNA，鉴定及定量分析。确定RAPD反应总体积，反应条件。选取3条不同的随机排列碱基顺序的多聚核苷酸单链为引物，进行RAPD－PCR扩增反应。将扩增产物制备成DNA图谱，通过对DNA图谱和遗传距离的分析，研究这三种蜚蠊基因组DNA的多态性。结果：分别扩增出不同数量和分子大小的DNA片段。3种蜚蠊基因组DNA扩增产物中具有相同的DNA条带，这反映了各蜚蠊基因组DNA具有同源性；3种蜚蠊基因组DNA扩增产物又具有各自独特的DNA条带，DNA条带亮度也有差别。结论：RAPD技术可以精确地区别这三种蜚蠊。