基于ITS序列鉴别3种感染白牦牛线虫的PCR-RFLP方法的建立

发布时间：2018-06-24 09:05

  本文选题：肿孔古柏线虫 + 环纹背带线虫　； 参考：《中国寄生虫学与寄生虫病杂志》2017年02期

【摘要】：目的基于内转录间隔区(ITS)序列,建立鉴别3种感染白牦牛线虫的PCR-限制性片段长度多态性(PCR-RFLP)方法。方法将从甘肃省天祝藏族自治县1头白牦牛小肠中分离的12条线虫进行消化并提取DNA,PCR扩增ITS序列并送测序,测序结果在GenBank数据库进行同源性分析。用DNAstar7.1软件分析序列之间的差异,用邻接(NJ)法构建系统进化树。根据序列选择特异的酶切位点,对PCR产物分别进行酶切反应,建立PCR-RFLP方法。结果 12条线虫均扩增出长度约900 bp的ITS序列片段,与预期结果一致。测序结果同源性分析显示,其中3种各4条分别与已报道的肿孔古柏线虫(Cooperia oncophora,GenBank登录号为AB534601)、环纹背带线虫(Teladorsagia circumcincta,GenBank登录号为JF680984)、奥斯特线虫(Ostertagia sp.,Gen Bank登录号为HQ844228)的序列相似性高于99%。ITS序列比较发现,3种线虫种间差异为4.3%~13.9%,种内差异均低于1%。其中肿孔古柏线虫与奥斯特线虫的ITS序列差异最大,为13.6%~13.9%;肿孔古柏线虫与环纹背带线虫ITS序列差异为12.9%~13.1%;环纹背带线虫和奥斯特线虫ITS序列的差异最小,为4.3%~4.8%。系统进化树结果显示,3种线虫被分为明显的3支,且环纹背带线虫和奥斯特线虫有更近的亲缘性。ITS序列酶切结果显示,NdeⅠ酶可将环背带线虫和肿孔古柏线虫的ITS序列酶切成约650 bp和250 bp的片段,Eco RⅤ酶仅将肿孔古柏线虫酶切成约700 bp和200 bp的片段,2种内切酶均不能酶切奥斯特线虫的ITS序列。结论建立的基于ITS序列的PCR-RFLP方法可鉴别感染白牦牛的肿孔古柏线虫、环纹背带线虫、奥斯特线虫。
[Abstract]:Objective to establish a PCR- restriction fragment length polymorphism (PCR-RFLP) method for identification of three species of infected yak nematode based on its sequence. Methods 12 nematodes isolated from the small intestine of a white yak in Tianzhu Tibetan Autonomous County of Gansu Province were digested, their DNA sequence was amplified by PCR and sequenced. The sequence was analyzed in GenBank database. DNA star7.1 software was used to analyze the differences between sequences, and NJ method was used to construct phylogenetic tree. The PCR products were digested with specific restriction endonuclease sites according to the sequence and the PCR-RFLP method was established. Results all 12 nematodes amplified its sequences of about 900 BP, which was consistent with the expected results. Homology analysis of sequencing results showed that, The sequence similarity of 3 species (Ostertagia sp. GenBank accession number is AB534601), Teladorsagia circumcinctata GenBank accession number (JF680984) and Ostertagia sp. GenBank accession number (HQ844228) is higher than that of 99.ITS sequence comparison of 3 species of nematodes. The sequence similarity of each species is higher than that of reported Cooperia oncophora GenBank accession number AB534601, Teladorsagia circumcincta-GenBank accession number JF680984, Ostertagia sp. GenBank accession number HQ844228 and Ostertagia sp. GenBank accession number HQ844228. The interspecific difference of nematodes was 4.3 and 13.9.The intraspecific difference was lower than 1. Among them, the difference of its sequence was the biggest (13.6m) between Cymopterus tumefaciens and Orsteria, the difference between them was 12.9% and 13.1, and the difference between them was 4.34.8 (4.34.8), the difference of its sequence was 12.9% (P < 0.05), and the difference of its sequence was 12.9% (P < 0.05), and the difference of its sequence was (4.34.8) (P < 0.05) between A. annularis and C. oryzae (P < 0.05). Phylogenetic tree results showed that the three species of nematodes were divided into three distinct branches. The results of its sequencing showed that Nde 鈪，

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