基于ITS序列鉴别3种感染白牦牛线虫的PCR-RFLP方法的建立

中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (2): 164-168.

基于ITS序列鉴别3种感染白牦牛线虫的PCR-RFLP方法的建立

韩亮^1,², 周东辉², 刘卿², 郑文斌², 朱兴全², 孙晓林^1,^*()

1 甘肃农业大学动物医学院,兰州 730070
2 中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,甘肃省动物寄生虫病重点实验室,兰州 730046

收稿日期:2016-08-03 出版日期:2017-04-20 发布日期:2017-05-02
通讯作者: 孙晓林
基金资助:
国家自然科学基金 (No. 31460659);国家公益性行业（农业）科研专项经费（No. 201303037）;甘肃省国际科技合作专项（No. 144WCGA169）

Establishment of ITS-based PCR-RFLP for identifying three species of nematodes in a white yak

Liang HAN^1,², Dong-hui ZHOU², Qing LIU², Wen-bin ZHEN², Xing-quan ZHU², Xiao-lin SUN^1,^*()

1 College of Veterinary Medicine, Gansu Agricultural University, Gansu 730070, China
2 State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

Received:2016-08-03 Online:2017-04-20 Published:2017-05-02
Contact: Xiao-lin SUN
Supported by:
Supported by the National Natural Science Foundation of China (No. 31460659), the Special Fund for Agro-scientific Research in the Public Interest (No. 201303037), and the Special Fund for International Scientific and Technological Cooperation Projects of Gansu Province (No. 144WCGA169)

摘要/Abstract

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.,GenBank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,EcoR Ⅴ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。

关键词: 肿孔古柏线虫, 环纹背带线虫, 奥斯特线虫, ITS序列, PCR-RFLP

Abstract:

Objective To develop the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method based on internal transcribed spacers (ITS) of ribosomal DNA to identity three nematode species that infected a white yak. Methods Twelve nematodes were collected from the small intestine of a white yak in Tianzhu Tibetan Autonomous County, Gansu Province, and DNA was extracted. Then the ITS region was amplified by PCR and sequenced. The sequences were analysed by DNAstar7.1 software and the phylogenetic tree was constructed by the neighbor-joining (NJ) method. Specific sites for restriction endonucleases were identified, based on which the PCR products were digested, and the PCR-RFLP method was established. Results PCR resulted in specific ITS bands of 900 bp for all the 12 nematodes. Sequencing results showed that each was approximately 900 bp in length. The sequences of 4 of the 12 nematodes had sequence similarity of > 99% with Cooperia oncophora (GenBank accession number AB534601), Teladorsagia circumcincta (GenBank accession number JF680984), and Ostertagia sp. (GenBank accession number HQ844228). However, the interspecies difference in ITS sequence among the three species was 4.3%-13.9%, while the intraspecific difference was < 1%. The interspecies difference in ITS sequence was most significant between C. Oncophora and T. circumcincta (12.9%-13.1%), medium between C. oncophora and Ostertagia sp. (13.6%-13.9%), and the least between T. circumcincta and Ostertagia sp. (4.3%-4.8%). The phylogenetic tree grouped the three nematode species into three branches, and revealed a closer ralationship between T. circumcincta and Ostertagia sp. PCR-RFLP analyses showed that the ITS sequences of C. Oncophora and T. circumcinctais were cut into fragments of 650 bp and 250 bp with endonuclease NdeⅠ, while only the ITS sequence of C. oncophora was cut into fragments of 700 bp and 200 bp with EcoR Ⅴ. The two endonucleases were ineffective for the ITS sequences of Ostertagia sp. Conclusions The three species of nematodes are successfully identified with ITS-based PCR-RFLP.

Key words: Cooperia oncophora, Teladorsagia circumcincta, Ostertagia sp., ITS, PCR-RFLP

中图分类号:

R383.19

韩亮, 周东辉, 刘卿, 郑文斌, 朱兴全, 孙晓林. 基于ITS序列鉴别3种感染白牦牛线虫的PCR-RFLP方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(2): 164-168.

Liang HAN, Dong-hui ZHOU, Qing LIU, Wen-bin ZHEN, Xing-quan ZHU, Xiao-lin SUN. Establishment of ITS-based PCR-RFLP for identifying three species of nematodes in a white yak[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(2): 164-168.

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编号Code	TZN1	TZN2	TZN3	TZN4	AB534601	TZN5	TZN6	TZN7	TZN8	JF680984	TZN9	TZN10	TZN11	TZN12	HQ844228
TZN1	-
TZN2	100	-
TZN3	99.9	99.9	-
TZN4	99.9	99.9	100	-
AB534601	99.5	99.5	99.6	99.6	-
TZN5	87.0	87.0	87.1	87.1	86.9	-
TZN6	87.0	87.0	87.1	87.1	86.9	99.7	-
TZN7	86.9	86.9	87.0	87.0	86.8	99.9	99.6	-
TZN8	87.0	87.0	87.1	87.1	86.9	99.7	100	99.6	-
JF680984	86.9	86.9	87.1	87.0	86.8	99.9	99.9	99.7	99.9	-
TZN9	86.3	86.3	86.4	86.4	86.4	95.7	95.7	95.6	95.7	95.6	-
TZN10	86.3	86.3	86.4	86.4	86.4	95.7	95.7	95.6	95.7	95.6	100	-
TZN11	86.1	86.1	86.3	86.3	86.3	95.6	95.6	95.4	95.6	95.4	99.9	99.9	-
TZN12	86.3	86.3	86.4	86.4	86.4	95.7	95.7	95.6	95.7	95.6	100	100	99.9	-
HQ844228	85.8	85.8	85.9	85.9	85.9	95.2	95.2	95.0	95.2	95.0	99.5	99.5	99.6	99.5	-