棘球绦虫的DNA条形码可行性分析及高原鼠兔棘球绦虫感染率初步研究
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摘要
包虫病(Echinococcosis),又名棘球蚴病(Echinococcosis)是由棘球绦虫(Echinococcus)引起的一种人兽共患病。囊型包虫病(Cysitc Echinococcosis, CE)由细粒棘球绦虫(E. granulosus)引起,泡型包虫病(Alveolar Echinococcosis, AE)由多房棘球绦虫(E. multilocularis)引起。四川省石渠县,位于青藏高原东部,是世界上包虫病感染最严重的地区之一,当地分布着全球常见的细粒棘球绦虫、多房棘球绦虫和本地仅有的石渠棘球绦虫(E. shiquicus)。棘球绦虫属内各种的分类界定一直以来饱受争议：最早对棘球绦虫的分类主要采用形态学及生态学的方法,但此方法多用于成年虫体,且对样本质量的要求较高；后随着分子生物学的发展,大量线粒体基因和核基因也被用来鉴别棘球绦虫。但至今并没有一种受到广泛认可且简单易行的鉴别方法。随着近些年DNA条形码(DNA barcoding)技术的不断发展,越来越多的生物都被证实了可以用此技术进行鉴别：一个适用于全球生物的鉴别手段和数据库也日益形成。本研究的第一部分,即首次以我们开展工作的石渠县已发现的三种棘球绦虫为基础,结合GenBank中棘球绦虫属各个种的序列,探索DNA barcoding技术在棘球绦虫属中的适用性。同时,因本实验选用了在整个带科(Taeniidae)都适用的通用引物,所以也结合以前的研究,探索了DNA barcoding技术在带科中的适用性。
主要研究结果如下：
1、本实验成功扩增测序了13个细粒棘球绦虫,15个多房棘球绦虫和17个石渠棘球绦虫个体的序列,并结合了GenBank中已发表的带科中,棘球绦虫属和带绦虫属26个种的396条可用序列进行遗传距离的分析和基因结构的分析。在棘球绦虫属内,种内的平均遗传距离为0.62%,(标准差SD,0.81%；范围0-8.9%)；种间的平均遗传距离为9.86%,(标准差SD,1.15%；范围,4%-15.28%).在带绦虫属内,种内的平均遗传距离为1.30%,(标准差SD,2.65%；范围0-11.71%)；种间的平均遗传距离为16.06%,(标准差SD,3.28%；范围,3.39%-24.59%).在带科中,种内的平均遗传距离为1.06%,(标准差SD,2.17%；范围0-11.71%)；所有种种间的平均遗传距离为16.95%,(标准差SD,4.12%；范围,3.39%-30.69%)；不同属种问的平均遗传距离为19.29%,(标准差SD,2.46%；范围,11.99%-30.69%)。证明了DNA barcoding是一种鉴别带科各种的行之有效的方法和技术。
2、本研究利用限制性片段长度多态性聚合酶链反应技术(PCR-RFLP),以扩增测序成功的13个细粒棘球绦虫,15个多房棘球绦虫和17个石渠棘球绦虫的444-bp的DNA片段为基础,发现内切酶AluI可将细粒棘球绦虫的444-bp片段切为210-bp和234-bp两段；内切酶BfaI可将多房棘球绦虫444-bp的片段切为281-bp和163-bp两段；以上两种限制性内切酶均不能对石渠棘球绦虫相同位点的序列产生作用。因此可结合琼脂糖凝胶电泳技术,在电泳图上将三种虫区分开。此方法可用于青藏高原地区棘球绦虫的鉴别工作。
本论文中的另一部分,为初步对高原鼠兔感染棘球绦虫的种类和感染率进行研究。
高原鼠兔(Ochotona curzoniae)是广泛分布于青藏高原地区的一种小型哺乳类动物,可以作为棘球绦虫的中间宿主。此前的研究主要通过剖检和病理学的方法对其棘球绦虫感染率进行调查,研究结果发现高原鼠兔主要感染多房棘球绦虫和石渠棘球绦虫：几乎未发现细粒棘球绦虫。本实验采用剖检和分子生物学检验的方法,选取三种棘球绦虫各自特异的引物,对在四川省石渠县捕获的高原鼠兔感染棘球绦虫的种类和感染率进行研究。
主要研究结果如下：
1、筛选了10对已在国内外论文中发表的细粒棘球绦虫、多房棘球绦虫和石渠棘球绦虫各自的特异引物,通过多重对比实验分别选取了对三种棘球绦虫的鉴别效果最好的特异引物。
2、共捕获和剖检105只高原鼠兔,疑似感染量为9只,分子鉴定后,细粒棘球绦虫感染数量0(感染率0%),多房棘球绦虫感染数量2(感染率1.9%),石渠棘球绦虫感染数量2(感染率1.9%)。还存在着多房棘球绦虫和石渠棘球绦虫两种虫同时感染于同一个个体的情况,数量2(感染率1.9%)。
Echinococcosis is a zoonosis caused by Echinococcus. Cystic echinococcosis (CE) caused by E. granulosus and alveolar echinococcosis (AE) caused by E. multilocularis are distributed all around the world. Shiqu County, on the East Qing-hai Tibet Plateau, is one of the most serious Echinococcus infection districts. Besides the two Echinococcus mentioned above, there is another special species, called E. shiquicus, which is only found existing on the Qing-hai Tibet Plateau. The taxonomy of Echinococcus has been a controversial issue for years:early identification has been based on morphological and ecological features, which were mainly used on adult worms and required high quality samples; as the development of molecular biology, lots of mtDNA and nuclear genes have been used to identify Echinococcus spp., but there has been no standard identification method so far. As a growing technology, DNA barcoding has been proved useful on150,250species (until Feb.2012). In this study, based on the three Echinococcus species, we originally tried to test the utility of DNA barcoding in the genus Echinococcus, with sequences of other Echinococcus spp. in the GenBank. Meanwhile, for the primers we used are family Taeniidae universal primers, we extended the range of our research to the whole family Taeniidae.
The major results and conclusions are as follows:
1.13E. granulosus isolates,15E. multilocularis isolates and17E. shiquicus isolates were successfully amplified and sequenced. These45Echinococcus sequences,136Echinococcus sequences and260Taenia sequences from GenBank were cut and calculated separately and integrally. In the genus Echinococcus, the mean K2P distance within species is0.62%,(SD,0.81%; range,0-8.9%). Mean K2P distance between species is9.86%,(SD,1.15%; range,4%-15.28%). In the genus Taenia, the mean K2P distance within species is1.30%,(SD,2.65%; range,0-11.71%). Mean K2P distance between species is16.06%,(SD,3.28%; range,3.39%-24.59%). In the family Taeniidae, the mean K2P distance within species is1.06%,(SD,2.17%; range,0-11.71%). Mean K2P distance between species is16.95%,(SD,4.12%; range,3.39%-30.69%). Mean K2P distance between genera is19.29%,(SD,2.46%; range,11.99%-30.69%). The COI DNA barcodes were successfully proved to be a powerful and reliable tool for species identification in the family Taeniidae.
2. A polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) based on the444-bp COI gene was developed to give a rapid way to differentiate among E. granulosus, E. multilocularis, and E. shiquicus, for practical application in the Eastern Tibetan Plateau. The E. granulosus samples were yielded to two clear bands of210-bp and234-bp using AluI, and the samples from E. multilocularis were yielded to two clear bands of281-bp and163-bp using BfaI. E. shiquicus could be yielded by neither restriction endonuclease above. These results could be detected directly by agarose gel electrophoresis.
The second part of this dissertation is a primary study on the Echinococcus infection of plateau pika (Ochotona curzoniae)
Plateau pika is a kind of small mammal widely distributed on the Qing-hai Tibet Plateau. It can serve as an intermediate host for Echinococcus. Former studies mainly used autopsy and histopathological examinations to survey its infection and found it mainly infected with E. multilocularis and E. shiquicus. This present study used the autopsy method and molecular technology to investigate the Echinococcus infection rates of plateau pika from Shiqu County, Sichuan Province, by choosing specific primers for the three Echinococcus species.
The major results and conclusions are as follows:
1. Three out of ten published and designed specific primers for E. multilocularis, E. shiquicus and E. granulosus were chosen to evaluate the Echinococcus infection rates.
2. In total, nine isolates were suspected infected out of105hunted plateau pikas. The molecular identification result is that zero pikas were infected with E. granulosus (infection rate0); two pikas were infected with E. multilocularis (infection rate1.9%); two pikas were infected with E. shiquicus (infection rate1.9%). Two pikas were mixed infected with E. multilocularis and E. shiquicus (infection rate1.9%)
主要研究结果如下：
1、本实验成功扩增测序了13个细粒棘球绦虫,15个多房棘球绦虫和17个石渠棘球绦虫个体的序列,并结合了GenBank中已发表的带科中,棘球绦虫属和带绦虫属26个种的396条可用序列进行遗传距离的分析和基因结构的分析。在棘球绦虫属内,种内的平均遗传距离为0.62%,(标准差SD,0.81%；范围0-8.9%)；种间的平均遗传距离为9.86%,(标准差SD,1.15%；范围,4%-15.28%).在带绦虫属内,种内的平均遗传距离为1.30%,(标准差SD,2.65%；范围0-11.71%)；种间的平均遗传距离为16.06%,(标准差SD,3.28%；范围,3.39%-24.59%).在带科中,种内的平均遗传距离为1.06%,(标准差SD,2.17%；范围0-11.71%)；所有种种间的平均遗传距离为16.95%,(标准差SD,4.12%；范围,3.39%-30.69%)；不同属种问的平均遗传距离为19.29%,(标准差SD,2.46%；范围,11.99%-30.69%)。证明了DNA barcoding是一种鉴别带科各种的行之有效的方法和技术。
2、本研究利用限制性片段长度多态性聚合酶链反应技术(PCR-RFLP),以扩增测序成功的13个细粒棘球绦虫,15个多房棘球绦虫和17个石渠棘球绦虫的444-bp的DNA片段为基础,发现内切酶AluI可将细粒棘球绦虫的444-bp片段切为210-bp和234-bp两段；内切酶BfaI可将多房棘球绦虫444-bp的片段切为281-bp和163-bp两段；以上两种限制性内切酶均不能对石渠棘球绦虫相同位点的序列产生作用。因此可结合琼脂糖凝胶电泳技术,在电泳图上将三种虫区分开。此方法可用于青藏高原地区棘球绦虫的鉴别工作。
本论文中的另一部分,为初步对高原鼠兔感染棘球绦虫的种类和感染率进行研究。
高原鼠兔(Ochotona curzoniae)是广泛分布于青藏高原地区的一种小型哺乳类动物,可以作为棘球绦虫的中间宿主。此前的研究主要通过剖检和病理学的方法对其棘球绦虫感染率进行调查,研究结果发现高原鼠兔主要感染多房棘球绦虫和石渠棘球绦虫：几乎未发现细粒棘球绦虫。本实验采用剖检和分子生物学检验的方法,选取三种棘球绦虫各自特异的引物,对在四川省石渠县捕获的高原鼠兔感染棘球绦虫的种类和感染率进行研究。
主要研究结果如下：
1、筛选了10对已在国内外论文中发表的细粒棘球绦虫、多房棘球绦虫和石渠棘球绦虫各自的特异引物,通过多重对比实验分别选取了对三种棘球绦虫的鉴别效果最好的特异引物。
2、共捕获和剖检105只高原鼠兔,疑似感染量为9只,分子鉴定后,细粒棘球绦虫感染数量0(感染率0%),多房棘球绦虫感染数量2(感染率1.9%),石渠棘球绦虫感染数量2(感染率1.9%)。还存在着多房棘球绦虫和石渠棘球绦虫两种虫同时感染于同一个个体的情况,数量2(感染率1.9%)。
Echinococcosis is a zoonosis caused by Echinococcus. Cystic echinococcosis (CE) caused by E. granulosus and alveolar echinococcosis (AE) caused by E. multilocularis are distributed all around the world. Shiqu County, on the East Qing-hai Tibet Plateau, is one of the most serious Echinococcus infection districts. Besides the two Echinococcus mentioned above, there is another special species, called E. shiquicus, which is only found existing on the Qing-hai Tibet Plateau. The taxonomy of Echinococcus has been a controversial issue for years:early identification has been based on morphological and ecological features, which were mainly used on adult worms and required high quality samples; as the development of molecular biology, lots of mtDNA and nuclear genes have been used to identify Echinococcus spp., but there has been no standard identification method so far. As a growing technology, DNA barcoding has been proved useful on150,250species (until Feb.2012). In this study, based on the three Echinococcus species, we originally tried to test the utility of DNA barcoding in the genus Echinococcus, with sequences of other Echinococcus spp. in the GenBank. Meanwhile, for the primers we used are family Taeniidae universal primers, we extended the range of our research to the whole family Taeniidae.
The major results and conclusions are as follows:
1.13E. granulosus isolates,15E. multilocularis isolates and17E. shiquicus isolates were successfully amplified and sequenced. These45Echinococcus sequences,136Echinococcus sequences and260Taenia sequences from GenBank were cut and calculated separately and integrally. In the genus Echinococcus, the mean K2P distance within species is0.62%,(SD,0.81%; range,0-8.9%). Mean K2P distance between species is9.86%,(SD,1.15%; range,4%-15.28%). In the genus Taenia, the mean K2P distance within species is1.30%,(SD,2.65%; range,0-11.71%). Mean K2P distance between species is16.06%,(SD,3.28%; range,3.39%-24.59%). In the family Taeniidae, the mean K2P distance within species is1.06%,(SD,2.17%; range,0-11.71%). Mean K2P distance between species is16.95%,(SD,4.12%; range,3.39%-30.69%). Mean K2P distance between genera is19.29%,(SD,2.46%; range,11.99%-30.69%). The COI DNA barcodes were successfully proved to be a powerful and reliable tool for species identification in the family Taeniidae.
2. A polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) based on the444-bp COI gene was developed to give a rapid way to differentiate among E. granulosus, E. multilocularis, and E. shiquicus, for practical application in the Eastern Tibetan Plateau. The E. granulosus samples were yielded to two clear bands of210-bp and234-bp using AluI, and the samples from E. multilocularis were yielded to two clear bands of281-bp and163-bp using BfaI. E. shiquicus could be yielded by neither restriction endonuclease above. These results could be detected directly by agarose gel electrophoresis.
The second part of this dissertation is a primary study on the Echinococcus infection of plateau pika (Ochotona curzoniae)
Plateau pika is a kind of small mammal widely distributed on the Qing-hai Tibet Plateau. It can serve as an intermediate host for Echinococcus. Former studies mainly used autopsy and histopathological examinations to survey its infection and found it mainly infected with E. multilocularis and E. shiquicus. This present study used the autopsy method and molecular technology to investigate the Echinococcus infection rates of plateau pika from Shiqu County, Sichuan Province, by choosing specific primers for the three Echinococcus species.
The major results and conclusions are as follows:
1. Three out of ten published and designed specific primers for E. multilocularis, E. shiquicus and E. granulosus were chosen to evaluate the Echinococcus infection rates.
2. In total, nine isolates were suspected infected out of105hunted plateau pikas. The molecular identification result is that zero pikas were infected with E. granulosus (infection rate0); two pikas were infected with E. multilocularis (infection rate1.9%); two pikas were infected with E. shiquicus (infection rate1.9%). Two pikas were mixed infected with E. multilocularis and E. shiquicus (infection rate1.9%)
引文
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