片形吸虫第一内转录间隔区DNA多态性的研究

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。     

酶切图谱及序列分析虫体ITS2鉴别中国的片形吸虫

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。     

酶切图谱及序列分析虫体ITS2鉴别中国的片形吸虫

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,Rca I进行酶切和RFLP技术分析.并对ITS-2 PCR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550 bp的ITS-2目标片段.Hsp92Ⅱ,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362 bp,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.hepattca;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.     

我国片形吸虫(Fasciola)线粒体细胞色素c氧化酶亚基I基因(cox1)部分序列的多态性

用γ^33P对引物进行标记，对来自国内不同地区不同宿主的片形吸虫(Fasciola)的线粒体基因组细胞色素氧化酶亚基I基因(pcox1)部分序列进行PCR扩增及DNA单链构象多态性(SSCP)分析，结果76个虫体均成功地扩增出约450bp的基因片段；SSCP分析显示，不同地区或同一地区的不同样品在pcox1的SSCP带型上存在多态性；代表性样品的测序结果表明，其碱基序列存在差异。试验结果显示，我国片形吸虫pcox1序列种内变异不明显，种间变异显著．表明cox1序列可以作为片形吸虫分类鉴定中一个可靠的遗传标记。     

分子分类结合形态学观察鉴定重庆地区片形吸虫种类

目的明确重庆地区片形吸虫的种类,并为重庆地区片形吸虫的分类研究提供科学的参考依据。方法在对重庆地区黄牛、水牛肝脏上寄生的片形吸虫形态结构进行观察后,根据片形吸虫第一内转录间隔区(ITS-1)和第二内转录间隔区(ITS-2)基因设计特异性引物,运用多聚酶链式反应(PCR)技术,以法国肝片吸虫gDNA为对照,对所采样品gDNA用大片吸虫和肝片吸虫的ITS-1和ITS-2特异性引物进行扩增。结果形态学鉴定均为“不规则”片形吸虫,电泳结果显示均分别扩增出特异性ITS-1和ITS-2条带。结论综合形态学和PCR鉴定结果,初步认为重庆地区存在着大片吸虫和肝片吸虫的“中间型”。     

青海省藏羊片形吸虫18S rRNA基因的扩增及虫种鉴定

为了进一步确定青海地区藏羊体内片形吸虫,并为青海省藏羊体内片形吸虫的分类研究提供科学的参考依据,取片形吸虫基因组DNA,利用保守引物,PCR扩增18S r RNA片段并测序。应用DNAMAN软件用对所测得的序列与Gen Bank中已经发布的大片吸虫(Fasciola gigantica)和肝片吸虫(Fasciola hepatica)的18S r RNA序列进行比对分析。结果发现测得目的片段长度为1 737 bp,测得序列与大片吸虫18S r RNA序列相似度为92.98%,与肝片吸虫的18S r RNA序列相似度为99.77%,从而进一步确定所采虫体为肝片吸虫。     

吸虫线粒体基因组及其应用研究进展

吸虫病(Trematodiasis)如血吸虫病、肝片形吸虫病等是一类重要的人兽共患寄生虫病,在我国和世界各地普遍流行,危害严重。本文将对吸虫线粒体基因组全序列分析的研究进展、应用和今后的发展方向作一综述。目前,已完成包括复殖亚纲10个种和单殖亚纲4个种总计14种吸虫线粒体基因组全序列测定。吸虫线粒体基因组碱基组成、基因结构与排列、基因变异等分析结果为线粒体功能基因组学研究、比较基因组学研究、分子分类学研究、物种起源、分子系统发育与进化分析及其疾病诊断等提供了重要依据和指导作用。线粒体基因组序列分析有助于解决一些新近发现的种如三平并殖吸虫、中华血吸虫等独立种的分类地位;而怡乐村并殖吸虫和佐渡并殖吸虫是大平并殖吸虫的同物异名吸虫。日本片形吸虫与大片形吸虫的cox1基因序列完全一致,这些日本片形吸虫应为大片形吸虫;大片形吸虫不同株虽然在cox1基因序列上无差异,但nad1基因却有8.3%变异性,这表明大片形吸虫不同株中可能存在隐蔽种。总之,线粒体基因组序列可为吸虫虫种与虫株的分类学地位的确定提供重要依据。同时,人们可根据线粒体基因组序列,通过PCR方法有效地对临床上易混淆的吸虫的种、株或群等作出确切的鉴别与诊断,从...     

我国片形吸虫线粒体NADH脱氢酶亚单位1基因(nad1)多态性的研究

用γ^33P对引物进行标记，首次对来自国内不同地区、不同宿主的片形吸虫线粒体烟酰胺腺嘌呤二核苷酸(NADH)脱氢酶亚单位Ⅰ基因部分序列(pnad1)进行PCR扩增及DNA单链构象多态性(PCR-SSCP)分析，结果76个虫体均成功地扩增出约200bp的基因片段；76个样品经过PCR-SSCP分析后，筛选出11个代表性样品进行测序。测序结果显示我国片形吸虫pnad1序列种间差异大于种内变异，表明nad1序列可以作为片形吸虫种内和种间遗传多态性研究的标记。     

河南中牟县奶牛片形吸虫感染情况调查及种类鉴定

利用尼龙袋集卵法对奶牛粪便中的虫卵收集、镜检,利用PCR技术对片形吸虫分离株线粒体nad1基因序列进行扩增与分析。结果显示:13份样品检测为阳性,阳性率5.41%(13/240),13个分离株nad1序列长度基本一致,为442~446 bp;通过种系发育分析结果显示,中牟县奶牛片形吸虫种类主要为肝片吸虫和大片吸虫,优势虫种为大片吸虫。说明中牟县奶牛片形吸虫流行情况较为严峻,需要引起当地防疫部门和养殖户的注意。     

黄牛源Fh/Fg杂合型片形吸虫的鉴定

为弄清黄牛感染的片形吸虫种类,首先对来自河南济源市一肉牛屠宰场黄牛肝脏上的片形吸虫进行形态学观察,然后基于核糖体ITS1、ITS2区域和线粒体nad1、cox1基因序列进行分子鉴定。ITS1和ITS2序列分析结果显示,济源黄牛源片形吸虫分离株JY003和JY004为Fh/Fg杂合型片形吸虫。基于nad1和cox1基因序列,JY003和JY004与大片形吸虫的相似性高于肝片形吸虫,其中nad1序列与大片形吸虫中国云南、日本和韩国分离株的相似性高达100%。在基于nad1和cox1基因的进化树上,分离株均与大片形吸虫参考株处于同一分支。结果表明,首次从河南黄牛体内鉴定出Fh/Fg杂合型片形吸虫,为动物和人的片形吸虫病防控提供重要的基础数据。     

猪食道口线虫ITS-1和lTS-2 rDNA的PCR-SSCP分析

以采自我国不同地区猪体的食道口线虫虫株为研究对象,PCR扩增出ITS-1和ITS-2序列片段,然后采用单链构象多态性(SSCP)方法分析PCR产物,对不同地区食道口线虫进行分子鉴定。所有样品经SSCP分析显示两种带型,第一种为有齿食道口线虫带型,另一种为未定种食道口线虫带型。代表性样品的测序结果表明,未定种食道口线虫带型的样品为四棘食道口线虫。本研究在国际上首次报道了中国猪四棘食道口线虫的ITS序列,并建立了区分有齿食道口线虫和四棘食道口线虫的PCR-SSCP方法,从而为食道口线虫的分子生物学的进一步研究奠定了基础。     

Characterization of Bunostomum trigonocephalum and Bunostomum phlebotomum from sheep and cattle by internal transcribed spacers of nuclear ribosomal DNA

In the present study, samples representing Bunostomum trigonocephalum and Bunostomum phlebotomum from sheep and cattle in Heilongjiang Province, China, were characterized and grouped genetically by the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). The rDNA region including the ITS-1, 5.8S, ITS-2, and flanking 18S and 28S rDNA sequences was amplified by polymerase chain reaction (PCR), then sequenced and compared with that of other members of the hookworms available in GenBank?, and phylogenetic relationships between them were reconstructed using the Maximum-Parsimony method. The ITS-1, 5.8S, and ITS-2 sequences of the sheep hookworm were 381, 153, and 231 bp in length, respectively, and the corresponding sequences of the cattle hookworm were 392, 153, and 240 bp in length. The identity of ITS sequences of B. trigonocephalum and B. phlebotomum from sheep and cattle was 87.4%. A PCR-linked restriction fragment length polymorphism (PCR-RFLP) assay using restriction endonuclease Nde I was established for the unequivocal differentiation of the two hookworm species. Phylogenetic analyses based on the ITS sequences revealed that B. trigonocephalum and B. phlebotomum were closely related, but they represent two different species.     

Inter- and intra-strain variation and PCR detection of the internal transcribed spacer 1 (ITS-1) sequences of Australian isolates of Eimeria species from chickens

The objective of this study was to confirm the presence of seven species of Eimeria involved in chicken coccidiosis in Australia by comparing internal transcribed spacer 1 (ITS-1) sequences, ITS-1 polymerase chain reaction (PCR) methods and to apply phylogenetic analysis to assess evolutionary relationships of Australian isolates. Twenty-two distinct ITS-1 regions of 15 Australian Eimeria isolates were sequenced, and analysed using maximum parsimony, distance and maximum likelihood methods. Poor bootstrap support, resulting from high ITS-1 sequence heterogeneity between all species groups, resulted in polychotomy of the Eimeria species in all three trees generated by these analyses. Percentage identity analyses revealed two distant ITS-1 lineages in both E. mitis and E. maxima at the same levels that separate the two species E. tenella and E. necatrix. One E. maxima lineage consisted of Australian isolates, the other American isolates, with one European sequence (originating from the same isolate) in each lineage. One Australian E. praecox sequence was only distantly related (33% variation) to three E. praecox sequences from Australian and European isolates. Short and long ITS-1 variants were isolated from both E. tenella (cloned line) and E. necatrix isolates with deletions (106 and 73 bp, respectively) in the short variants within the 3' region of the ITS-1 sequence. ITS-1 sequences of strains of both E. brunetti and E. acervulina species varied the least. Apart from E. maxima, all of the ITS-1 sequences of the six remaining individual species clustered to the exclusion of other species in all phylogenetic trees. Published ITS-1 tests for E. necatrix, E. acervulina, E. brunetti and E. tenella, combined with three new tests for E. mitis, E. praecox and Australian E. maxima amplified all respective Australian isolates specifically in a nested format using conserved ITS-1 PCR products as template to improve the sensitivity. All PCR tests were confirmed against a collection of 24 Australian chicken Eimeria isolates and contaminating species were detected in some instances. In conclusion, once the genetic variation between species and strains is determined, the ITS-1 is a good target for the development of species-specific assays, but the ITS-1 sequences alone do not seem suitable for the confirmation of phylogenetic inferences for these species. This study reports the first attempt at the analysis of the phylogeny and sequence comparison of the Eimeria species involved in chicken coccidiosis in Australia.     

PCR-SSCP对我国鲁道夫对盲囊线虫的分子鉴定

对来自青海湖的鲁道夫对盲囊线虫（Contracaecum rudolphii）核糖体DNA第一、第二内转录间隔区（ITS-1、ITS-2）进行PCR扩增、DNA单链构象多态性（SSCP）分析及序列分析。并与来自欧洲的2个姊妹种鲁道夫对盲囊线虫进行了比较。结果显示，我国青海湖的鲁道夫对盲囊线虫与来自意大利的（.rudolphii B具有一样的SSCP带型及ITS序列，但不同于C．rudolphiiA．因此属于C.rudolphii B。本试验证实了ITS片段可作为遗传标记用于鉴别鲁道夫对盲囊线虫的姊妹种，从而为鲁道夫对盲囊线虫的进一步研究奠定了基础。     

安氏隐孢子虫ITS-1序列的PCR扩增、克隆及分析

通过对国内三株安氏隐孢子虫(Cryptosporidium andersoni)即GD株、HN株和AH株的rDNA的内转录间隔区Ⅰ(ITS_1)序列进行PCR扩增、克隆、测序和序列分析,旨在确定ITS_1是否可作为C.andersoni分子分类的遗传标记。结果表明:GD株、HN株和AH株的ITS_1序列基本一致,仅AH株有三个碱基的差异;但与GenBank注册的C.muris和C.parvum存在种间差异,而且差异显著。说明ITS_1可作为C.andersoni种的遗传标记,从而为隐孢子虫属的种间鉴定以及进一步的分子流行病学调查和分子诊断学研究奠定了基础。     

Characterization of the second internal transcribed spacer of ribosomal DNA of Calicophoron daubneyi from various hosts and locations in southern Italy

Isolates of the rumen fluke Calicophoron daubneyi (Digenea: Paramphistomidae) from various hosts and three locations in southern Italy were characterized genetically. The second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) plus flanking 5.8S and 28S sequence (ITS-2+) was amplified from individual rumen flukes by PCR. PCR-linked restriction fragment length polymorphism (PCR-RFLP) analysis was performed using four different restriction endonucleases, and PCR products were sequenced. The PCR analyses from all the C. daubneyi specimens produced identical fragments, and the PCR-RFLP analyses did not show, with respect to any of the four restriction endonucleases, any differences between the C. daubneyi specimens. The sequence analyses of the ITS-2+ from each of the C. daubneyi specimens showed them all to be 428 bp, and composed of the entire ITS-2 sequence (282 bp) plus the two partial flanking conserved sequences, 5.8S (99 bp) and 28S (47 bp). No intra-specific variation was observed in the nucleotide composition of the ITS-2+ (homology=100%). There was, however, an observable inter-specific variation between the ITS-2+ of C. daubneyi and the ITS-2+ of both Calicophoron calicophorum (homology=97.2 %) and Calicophoronmicrobothrioides (homology=97.4 %), both previously deposited in the GenBank. The finding of the present study shows that, as has already demonstrated for other parasitic helminths, ITS-2 can serve as an effective genetic marker for the molecular identification of paramphistomes, and as a useful tool for developing molecular epidemiological techniques for the study of C. daubneyi transmission patterns and prevalence in definitive and intermediate hosts.     

Is ITS-2 rDNA suitable marker for genetic characterization of Sarcoptes mites from different wild animals in different geographic areas?

The present study examined the relationship among individual Sarcoptes scabiei mites from 13 wild mammalian populations belonging to nine species in four European countries using the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) as genetic marker. The ITS-2 plus primer flanking 5.8S and 28S rDNA (ITS-2+) was amplified from individual mites by polymerase chain reaction (PCR) and the amplicons were sequenced directly. A total of 148 ITS-2+ sequences of 404 bp in length were obtained and 67 variable sites were identified (16.59%). UPGMA analyses did not show any geographical or host-specific clustering, and a similar outcome was obtained using population pairwise Fst statistics. These results demonstrated that ITS-2 rDNA does not appear to be suitable for examining genetic diversity among mite populations.     

Phylogenetic relationships of three species within the family Heligmonellidae (Nematoda; Heligmosomoidea) from Japanese rodents and a lagomorph based on the sequences of ribosomal DNA internal transcribed spacers, ITS-1 and ITS-2

Nematodes of the family Heligmonellidae (Heligmosomoidea; Trichostrongylina) reside in the digestive tracts of rodents and lagomorphs. Although this family contains large numbers of genera and species, genetic information on the Heligmonellidae is very limited. We collected and isolated adult worms of three species in Japan that belong to the family Heligmonellidae, namely Heligmonoides speciosus (Konno, 1963) Durette-Desset, 1970 (Hs) from Apodemus argenteus, Orientostrongylus ezoensis Tada, 1975 (Oe) from Rattus norvegicus and Lagostrongylus leporis (Schulz, 1931) (Ll) from Pentalagus furnessi, and sequenced the entire internal transcribed spacer regions, ITS-1 and ITS-2 of ribosomal DNA. ITS-1 of Hs, Oe and Ll was 426, 468 and 449 bp in length, and had a G+C content of about 41, 41 and 37 %, respectively. ITS-2 of Hs, Oe and Ll was 297, 319 and 276 bp in length and had a G+C content of about 38, 40 and 28%, respectively. The data of Hs, Oe and Ll were compared with those of two other known species within the family Heligmonellidae, Calorinensis minutus (Dujardin, 1845) (Cm) and Nippostrogylus brasiliensis (Travassos, 1914) (Nb), and with those of two species of Heligmosomidae (Heligmosomoidea), Heligmosomoides polygyrus bakeri and Ohbayashinema erbaevae. Phylogenetic analysis placed Hs, Oe and Ll in the same clade with Cm and Nb, forming a Heligmonellidae branch in both ITS-1 and ITS-2, separate from the Heligmosomoidea branch. These results demonstrated that the ITS-1 and ITS-2 sequences are useful for differentiating the Heligmonellidae nematode species. This study is the first to describe the ITS-1 and ITS-2 sequences of Hs, Oe and Ll.     

A method for extracting genomic DNA from individual elaphostrongyline (Nematoda: Protostrongylidae) larvae and differentiation of Elaphostrongylus spp. from Parelaphostrongylus spp. by PCR assay.

This article reports a rapid and effective method for the extraction and purification of genomic DNA (gDNA) from individual first-stage larvae (L1) of elaphostrongyline nematodes that had been stored frozen or fixed in 95% ethanol for 1 to 5 years. The method was highly effective for L1s of all 6 species of elaphostrongylines, based on polymerase chain reaction (PCR) amplification of a partial fragment of the first internal transcribed spacer (ITS-1) of the ribosomal DNA. Differences were detected in the sizes of partial ITS-1 amplicons between the 2 elaphostrongyline genera, Elaphostrongylus and Parelaphostrongylus. The reliability of the ITS-1 PCR assay was tested by using L1s of unknown identity from Newfoundland and Labrador, Canada. The ability to consistently isolate gDNA from individual L1s, together with a simple PCR-based method to distinguish between Parelaphostrongylus and Elaphostrongylus, have important implications for diagnostic testing and for conducting epizootiological studies on these parasites of veterinary importance.     

长颈鹿血矛线虫ITS的PCR扩增与序列分析

目的利用分子生物学方法对来自长颈鹿皱胃的血矛线虫进行虫种鉴定。方法对样品XM9和XM11的核糖体DNA内转录间隔区(ITS-1、5.8 S、ITS-2)进行PCR扩增及序列分析,并与GenBank公布的血矛线虫(Hae-monchus)相应序列进行比较。结果来自长颈鹿皱胃的2条血矛线虫具有相同的ITS序列,5.8 S与ITS-1分别为153 bp、404 bp,与GenBank分布的捻转血矛线虫序列是一致的。ITS-2序列为231 bp,第753位是一个多态位点,该序列与来自国外的H.contortus,H.placei,H.longistipes存在0-18个碱基差异。结论来自长颈鹿的血矛线虫是捻转血矛线虫。