基于线粒体ND6基因检测犬感染细粒棘球绦虫的粪便PCR方法
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摘要
目的旨在了解动物包虫病流行区内犬细粒棘球绦虫的感染情况。方法本研究利用Qiagen DNeasy Powersoil试剂盒对犬粪提取DNA,并从细粒棘球绦虫线粒体全基因组中筛选出完整线粒体ND6基因作为靶基因,建立一种可对犬粪中细粒棘球绦虫同时进行检测及基因分型的PCR方法。结果该法特异性很高,仅能扩增出细粒棘球绦虫的目的条带而对照组均无条带扩增。其灵敏度达到4 pg的DNA含量。当犬人工感染约50 000只原头蚴时,该法最早可以扩增出感染第13 d粪便中的ND6目的基因。同时,对40份随机采自包虫病流行区的待检犬粪进行PCR检测,共有6份样品可扩增出目的条带且其基因型均属G1型,其检测及分型结果均与经典基因分型依据(COX1基因片段)的结果一致。结论本研究建立的粪便PCR方法具有很高的特异性和灵敏度,并可用于检测犬的细粒棘球绦虫早期感染情况。对于PCR检测阳性者,其产物经测序分析后也可用于细粒棘球绦虫的基因分型及种群遗传结构的分析研究。
In order to evaluate the infection of Echinococcus granulosus in dogs in the echinococcosis epidemic areas, we extracted DNA from canine faeces using the Qiagen DNeasy Powersoil kit and screened the whole mitochondrial ND6 gene as a target gene from the complete mitochondrial genomes of E.granulosus for detection and genotyping of E.granulosus. The results showed that the specificity of the PCR assay was very high, only the E.granulosus could be successfully amplified. And the analytical sensitivity was 4 pg of DNA. Moreover, the assay could amplify the target gene in faeces of the 13 th day as early as possible when the dogs were infected with about 50 000 protoscoleces. In addition, in 40 faeces which were from echinococcosis epidemic areas, 6 samples provided specific DNA fragments using the Copro-PCR assay. The sequence data showed that 6 samples were identified as G1 genotype, which were the same as the sequencing results and the nucleotide sequence analysis of the part of the mitochondrial cytochrome C oxidase subunit 1(COX1) gene-a good candidate in the genotyping of E.granulosus. The Copro-PCR assay developed in this study has high specificity and sensitivity and could be used to detect E.granulosus latent infection in dogs. In addition, the PCR-positive production also could be used for genotyping and population genetic structure analysis of E.granulosus via the nucleotide sequence analysis.
In order to evaluate the infection of Echinococcus granulosus in dogs in the echinococcosis epidemic areas, we extracted DNA from canine faeces using the Qiagen DNeasy Powersoil kit and screened the whole mitochondrial ND6 gene as a target gene from the complete mitochondrial genomes of E.granulosus for detection and genotyping of E.granulosus. The results showed that the specificity of the PCR assay was very high, only the E.granulosus could be successfully amplified. And the analytical sensitivity was 4 pg of DNA. Moreover, the assay could amplify the target gene in faeces of the 13 th day as early as possible when the dogs were infected with about 50 000 protoscoleces. In addition, in 40 faeces which were from echinococcosis epidemic areas, 6 samples provided specific DNA fragments using the Copro-PCR assay. The sequence data showed that 6 samples were identified as G1 genotype, which were the same as the sequencing results and the nucleotide sequence analysis of the part of the mitochondrial cytochrome C oxidase subunit 1(COX1) gene-a good candidate in the genotyping of E.granulosus. The Copro-PCR assay developed in this study has high specificity and sensitivity and could be used to detect E.granulosus latent infection in dogs. In addition, the PCR-positive production also could be used for genotyping and population genetic structure analysis of E.granulosus via the nucleotide sequence analysis.
引文
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[6] 郭秀霞,程鹏,刘丽娟,等.基于mtDNA-COⅠ基因的山东省蚊种群遗传多样性分析[J].中国血吸虫病防治杂志,2018(1):37-41.DOI:10.16250/j.32.1374.2017091
[7] Makkonen J,Jussila J,Panteleit J,et al.MtDNA allows the sensitive detection and haplotyping of the crayfish plague disease agent Aphanomyces astaci showing clues about its origin and migration [J].Parasitology,2018,145(9):1-9.DOI:10.1017/S0031182018000227
[8] Senoo T,Yamanaka M,Nakamura A,et al.Quantitative PCR for detection of DNA damage in mitochondrial DNA of the fission yeast Schizosaccharomyces pombe[J].J Microbiol Meth,2016,127:77-81.DOI:10.1016/j.mimet.2016.05.023
[9] 徐丹丹,黄燕,曾庆,等.基于mtDNACytb基因序列的我国不同水系野生鲇种群遗传多样性与种群历史分析[J].水产学报,2017,41(10):1489-1499.DOI:10.11964/jfc.20161010572
[10] Bai J,Xu S,Nie Z,et al.The complete mitochondrial genome of Huananpotamon lichuanense (Decapoda:Brachyura) with phylogenetic implications for freshwater crabs [J].Gene,2018,646:217-226.DOI:10.1016/j.gene.2018.01.015
[11] 沈莹,左成学,吴敏,等.赤眼鳟线粒体NADH脱氢酶6基因克隆及序列特征分析[J].安徽农业科学,2010,38(1):85-87.DOI:10.3969/j.issn.0517-6611.2010.01.032
[12] Silva-Brand?o KL,Lyra ML,Santos TV,et al.Exploitation of mitochondrial nad6 as a complementary marker for studying population variability in Lepidoptera[J].Genet Mol Biol,2011,34(4):719-725.DOI:10.1590/S1415-47572011000400028
[13] 海汀,柴志欣,张成福,等.西藏牦牛mtDNA ND6遗传多样性及系统进化分析[J].家畜生态学报,2014,35(11):11-17.DOI:10.3969/j.issn.1673-1182.2014.11.003
[14] Bowles J,Blair D,Mcmanus DP.Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing[J].Mol Biochem Parasitol,1992,54(2):165-173.DOI:10.1016/0166-6851(92)90109-W
[15] 陈洁,马海梅,古力帕丽,等.用EgAgB8/3重组蛋白ELISA-双抗夹心法建立棘球绦虫感染犬粪抗原检测系统的研究[J].新疆医科大学学报,2011,34(3):240-245.DOI:10.3969/j.issn.1009-5551.2011.03.003
[16] Dinkel A,Nickischrosenegk MV,Bilger B,et al.Detection of Echinococcus multilocularis in the Definitive Host:Coprodiagnosis by PCR as an Alternative to Necropsy[J].J Clin Microbiol,1998,36(7):1871.DOI:mdl-9650927
[17] Deplazes P,Gottstein B,Eckert J,et al.Detection of Echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs,dingoes and foxes[J].Parasitol Res,1992,78(4):303.DOI:10.1007/BF00937088
[18] 张旭,古努尔,吐尔逊,等.犬细粒棘球绦虫粪抗原夹心ELISA检测方法的建立[J].动物医学进展,2012,33(3):19-23.DOI:10.3969/j.issn.1007-5038.2012.03.005
[19] 焦伟,柴君杰,伊斯拉音,等.粪抗原检测法诊断犬细粒棘球绦虫感染的研究Ⅱ.粪抗原检测的敏感性、特异性及实验感染犬排出粪抗原的动态观察[J].中国人兽共患病学报,1999,27(4):41-44.DOI:10.3969/j.issn.1002-2694.1999.04.012
[20] Deplazes P,Alther P,Tanner I,et al.Echinococcus multilocularis coproantigen detection by enzyme-linked immunosorbent assay in fox,dog,and cat populations[J].J Parasitol,1999,85(1):115-121.DOI:10.2307/3285713
[21] 陈家旭,常正山.ELISA检测粪抗原诊断寄生虫感染的研究进展[J].国际医学寄生虫病杂志,2006,33(6):296-300.DOI:10.3760/cma.j.issn.1673-4122.2006.06.004
[22] Nkouawa A,Sako Y,Li T,et al.A loop-mediated isothermal amplification method for a differential identification of Taenia tapeworms from human:application to a field survey[J].Parasitol Int,2012,61(4):723-725.DOI:10.1016/j.parint.2012.06.001
[23] Ni X,Mcmanus DP,Yan H,et al.Loop-Mediated Isothermal Amplification (LAMP) assay for the identification of Echinococcus multilocularis infections in canine definitive hosts[J].Parasite Vector,2014,7(1):254.DOI:10.1186/1756-3305-7-254
[24] Li Y,Fan P,Zhou S,et al.Loop-mediated isothermal amplification (LAMP):A novel rapid detection platform for pathogens[J].Microbial Pathogenesis,2017,107:54.DOI:10.1016/j.micpath.2017.03.016
[25] 吴禹熹,李璞君,王娟,等.LAMP方法及其在病原微生物检测中的应用[J].中国畜牧兽医,2016,43(2):389-393.DOI:10.16431/j.cnki.1671-7236.2016.02.015
[26] Ni XW,Mcmanus DP,Lou ZZ,et al.A Comparison of Loop-Mediated Isothermal Amplification (LAMP) with other surveillance tools for Echinococcus granulosus diagnosis in canine definitive hosts[J].PLoS One,2014,9(7):e100877.DOI:10.1371/journal.pone.0100877
[27] 何琳,徐海圣,王美珍,等.白斑综合征病毒环介导等温扩增快速检测方法的建立[J].水产学报,2010,34(4):598-603.DOI:10.3724/SP.J.1231.2010.06741
[28] 黄火清,郁昂.环介导等温扩增技术的研究进展[J].生物技术,2012,22(3):90-94.DOI:10.3969/j.issn.1004-311X.2012.03.76
[29] Thekisoe OM,Bazie RS,Coronel-Servian AM,et al.Stability of Loop-Mediated Isothermal Amplification (LAMP) reagents and its amplification efficiency on crude trypanosome DNA templates[J].J Vet Med Sci,2009,71(4):471.DOI:10.1292/jvms.71.471
[30] Mcmanus DP,Zhang W,Li J,et al.Echinococcosis[J].Lancet,2003,362(9392):1295-1304.DOI:10.1016/S0140-6736(03)14573-4
[31] 张亚楼,温浩,马旭东,等.家犬粪便中的细粒棘球绦虫DNA PCR检测方法的建立[J].中国病原生物学杂志,2006,1(5):363-365.DOI:10.3969/j.issn.1673-5234.2006.05.013
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