角蝇体内斯氏副柔线虫的观察及COI基因特异性序列分析
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摘要
斯氏副柔线虫(Parabronema skrjabini)是一种寄生于骆驼皱胃的寄生性线虫,其中间宿主是西方角蝇(Haematobia irritans)和截脉角蝇(Haematobia titillans)。为了明确我国双峰驼养殖地区斯氏副柔线虫对角蝇的感染情况、寄生部位,斯氏副柔线虫所在副柔属的归科问题,以及寻找一种适合于该病生前诊断方法的遗传标记。本文统计了双峰驼生活环境中角蝇体内斯氏副柔线虫的感染率和感染强度,明确了其寄生部位;并利用PCR技术扩增分析了斯氏副柔线虫线粒体DNA的COI基因序列,在此基础上设计出了特异性引物并加以验证。结果如下:
采用生物解剖法对采集于内蒙古巴彦淖尔市乌拉特后旗的176只角蝇(西方角蝇64只,截脉角蝇112只)进行了剖解,共有38只为阳性,感染率为21.59%。其中西方角蝇中13只为阳性,感染率为20.31%,最大感染强度为32条/只,平均感染强度是8条/只;截脉角蝇中25只为阳性,感染率为22.32%,最大感染强度为23条/只,平均感染强度是9条/只。斯氏副柔线虫幼虫主要寄生在角蝇的消化道内。
利用PCR方法,以线虫COI基因通用引物NTF、NTR扩增内蒙古不同地区的斯氏副柔线虫线粒体DNA COI基因。将扩增产物纯化后克隆并测序。经DNAStar5.0和MEGA4.0分析,结果表明:扩增出的COI基因序列均为689bp,不同地区样本间的COI基因无差异;斯氏副柔线虫COI基因与旋尾目(Spirurata)中旋尾科(Spiruridae)、柔线科(Habronematidae)和吸吮科(Thelaziidae),丝虫目(Filariata)盘尾科(Onchocercidae)某些线虫的同源性为79.8%～87.4%,遗传距离为0.137～0.232。基于COI基因构建的系统发育树表明,副柔属归于柔线科更符合生物进化的规律。
以COI基因设计并合成特异性引物STF、STR,利用PCR方法对斯氏副柔线虫基因组进行特异性扩增后可得到约600bp的条带。敏感性试验表明,含有10个虫卵的DNA模板即可通过PCR扩增出来;特异性试验表明,该对引物具有较好的种特异性。
本研究首次报道了斯氏副柔线虫COI基因序列,为斯氏副柔线虫提供了一个可靠的遗传标记,为建立斯氏副柔线虫病分子生物学生前诊断方法奠定了重要基础。
Parabronema skrjabini is a nematode parasite in camels’abmasum and its vectors are Haematobia irritans and Haematobia titillans. In order to ensure the infection, classification and establish an antemortem diagnosis method, the paper do the research about three parts: survey the infection rate, analyse sequences of COI gene within mtDNA, design specific primers. The results as follows:
176 horn flies (64 H.irritans and 112 H.titillans) from Wulatehouqi of Bayannaoer city in Inner Mongolia of China were dissected, then the infection rate and infection density were calculated. The results show: 38 horn flies were infected and the infection rate was 21.59%. Therein, 13 H.irritans were infected which infection rate was 20.13%, the highest infection density was 32; 25 H.titillans were infected which infection rate was 22.32%, the highest infection density was 23. P.skrjabini was parasited in the digestive tract.
The COI gene of P.skrjabini from three different aeras of Inner Mongolia in China was amplified by PCR using universal primers. Then the PCR products were sequenced and analysed. The result showed that the COI gene was 689bp and there were no differences among three samples from different aeras. Comparison with sequences of COI gene from the family in Spirurata and Filariata reported in GenBank, the percent identity was between 79.8% and 87.4%; the nucleotide pairwise distance was between 0.137 and 0.232. Phylogenetic tree based on the sequence of COI gene showed that Parabronema belongs to Habronematidae.
STF and STR primers based on COI gene were designed, and a 600bp amplicon was amplified from the DNA of P.skrjabini. Sensitivity tests showed that specific amplicon could amplified from DNA of ten eggs supplied, and the specific tests showed that the primers have a high specificity to DNA of P.skrjabini.
It was the first time to report the sequence of P.skrjabini COI gene. The study is not only provide a reliable genetic marker, but also offer the basis for the molecular biology antemortem diagnosis of camel’s parabronemosis.
采用生物解剖法对采集于内蒙古巴彦淖尔市乌拉特后旗的176只角蝇(西方角蝇64只,截脉角蝇112只)进行了剖解,共有38只为阳性,感染率为21.59%。其中西方角蝇中13只为阳性,感染率为20.31%,最大感染强度为32条/只,平均感染强度是8条/只;截脉角蝇中25只为阳性,感染率为22.32%,最大感染强度为23条/只,平均感染强度是9条/只。斯氏副柔线虫幼虫主要寄生在角蝇的消化道内。
利用PCR方法,以线虫COI基因通用引物NTF、NTR扩增内蒙古不同地区的斯氏副柔线虫线粒体DNA COI基因。将扩增产物纯化后克隆并测序。经DNAStar5.0和MEGA4.0分析,结果表明:扩增出的COI基因序列均为689bp,不同地区样本间的COI基因无差异;斯氏副柔线虫COI基因与旋尾目(Spirurata)中旋尾科(Spiruridae)、柔线科(Habronematidae)和吸吮科(Thelaziidae),丝虫目(Filariata)盘尾科(Onchocercidae)某些线虫的同源性为79.8%～87.4%,遗传距离为0.137～0.232。基于COI基因构建的系统发育树表明,副柔属归于柔线科更符合生物进化的规律。
以COI基因设计并合成特异性引物STF、STR,利用PCR方法对斯氏副柔线虫基因组进行特异性扩增后可得到约600bp的条带。敏感性试验表明,含有10个虫卵的DNA模板即可通过PCR扩增出来;特异性试验表明,该对引物具有较好的种特异性。
本研究首次报道了斯氏副柔线虫COI基因序列,为斯氏副柔线虫提供了一个可靠的遗传标记,为建立斯氏副柔线虫病分子生物学生前诊断方法奠定了重要基础。
Parabronema skrjabini is a nematode parasite in camels’abmasum and its vectors are Haematobia irritans and Haematobia titillans. In order to ensure the infection, classification and establish an antemortem diagnosis method, the paper do the research about three parts: survey the infection rate, analyse sequences of COI gene within mtDNA, design specific primers. The results as follows:
176 horn flies (64 H.irritans and 112 H.titillans) from Wulatehouqi of Bayannaoer city in Inner Mongolia of China were dissected, then the infection rate and infection density were calculated. The results show: 38 horn flies were infected and the infection rate was 21.59%. Therein, 13 H.irritans were infected which infection rate was 20.13%, the highest infection density was 32; 25 H.titillans were infected which infection rate was 22.32%, the highest infection density was 23. P.skrjabini was parasited in the digestive tract.
The COI gene of P.skrjabini from three different aeras of Inner Mongolia in China was amplified by PCR using universal primers. Then the PCR products were sequenced and analysed. The result showed that the COI gene was 689bp and there were no differences among three samples from different aeras. Comparison with sequences of COI gene from the family in Spirurata and Filariata reported in GenBank, the percent identity was between 79.8% and 87.4%; the nucleotide pairwise distance was between 0.137 and 0.232. Phylogenetic tree based on the sequence of COI gene showed that Parabronema belongs to Habronematidae.
STF and STR primers based on COI gene were designed, and a 600bp amplicon was amplified from the DNA of P.skrjabini. Sensitivity tests showed that specific amplicon could amplified from DNA of ten eggs supplied, and the specific tests showed that the primers have a high specificity to DNA of P.skrjabini.
It was the first time to report the sequence of P.skrjabini COI gene. The study is not only provide a reliable genetic marker, but also offer the basis for the molecular biology antemortem diagnosis of camel’s parabronemosis.
引文
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