凡纳滨对虾肠道上皮细胞微胞子虫PCR检测方法的建立与应用
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摘要
根据Gen Bank中对虾肠道上皮细胞微胞子虫(Enterocytozoon hepatopenaei,EHP)的基因保守序列,设计一对特异性引物,通过优化PCR扩增条件,建立快速检测凡纳滨对虾(Litopenaeus vannamei)EHP的PCR方法。用该方法对EHP阳性虾进行PCR扩增,得到与实验设计相符的330 bp特异性扩增条带,而对白斑综合症病毒(WSSV)、桃拉病毒(TSV)、传染性皮下及造血器官坏死病毒(IHHNV)、对虾杆状病毒(BP)、"棉花虾"微孢子虫、黏孢子虫的阳性虾,以及健康虾的扩增结果为阴性。测序比对发现,PCR产物序列与Gen Bank EHP基因序列的同源性为99.8%,表明该PCR方法检测结果准确。敏感性试验表明,该方法最低可检测出约100 fg的EHP质粒DNA。用该PCR方法检测广东、广西、江苏、山东、海南等地的755份临床样品,共检出阳性样品21份。该PCR方法可用于凡纳滨对虾EHP的快速检测。
A pair of specific primers was designed, and a rapid PCR method was established for detection of microsporidium Enterocytozoon hepatopenaei in Pacific white leg shrimp Litopenaeus vannamei according to the published gene sequences in Gen Bank by optimization of the reaction parameters. The specific band of 330 bp were amplified from the positive shrimps, but no specific band was found from shrimps with white spot syndrome virus(WSSV), Taura syndrome virus(TSV), infectious hypodermal and hematopoietic necrosis virus(IHHNV), Baculovirus penaei(BP), and Pleistophora-like microsporidium, Myxosporea and healthy shrimps. According to the Sequencing analysis, the sequence of PCR product had 99.8% homology with that of EHP gene in Gen Bank which indicated the high accuracy of this method. The sensitivity test of this method showed that the lowest detectable limit DNA plasmid of the microsporida was 100 fg. 755 clinical shrimp samples collected from provinces of Guangdong, Guangxi, Jiangsu, Shandong, and Hainan were detected by this method, and then 21 positive samples were found. The findings indicated that the PCR assay could be used in the detection of Pacific white leg shrimp samples.
A pair of specific primers was designed, and a rapid PCR method was established for detection of microsporidium Enterocytozoon hepatopenaei in Pacific white leg shrimp Litopenaeus vannamei according to the published gene sequences in Gen Bank by optimization of the reaction parameters. The specific band of 330 bp were amplified from the positive shrimps, but no specific band was found from shrimps with white spot syndrome virus(WSSV), Taura syndrome virus(TSV), infectious hypodermal and hematopoietic necrosis virus(IHHNV), Baculovirus penaei(BP), and Pleistophora-like microsporidium, Myxosporea and healthy shrimps. According to the Sequencing analysis, the sequence of PCR product had 99.8% homology with that of EHP gene in Gen Bank which indicated the high accuracy of this method. The sensitivity test of this method showed that the lowest detectable limit DNA plasmid of the microsporida was 100 fg. 755 clinical shrimp samples collected from provinces of Guangdong, Guangxi, Jiangsu, Shandong, and Hainan were detected by this method, and then 21 positive samples were found. The findings indicated that the PCR assay could be used in the detection of Pacific white leg shrimp samples.
引文
[1]谢数涛,何建国,杨晓明,等.套式PCR检测斑节对虾白斑症病毒(WSSV)[J].青岛海洋大学学报,2001,31(2):220-224.
[2]徐丽美,吴成林,邱名毅,等.对虾桃拉病毒(TSV)RT-PCR快速诊断技术的研究[J].厦门大学学报(自然科学版),2005,44(2):264-267.
[3]雷燕,肖洋,张会军,等.凡纳滨对虾对虾杆状病毒PCR检测方法的建立及初步应用[J].广东海洋大学学报,2015,35(4):63-66.
[4]任聪,龚艳清,陈信忠,等.双重PCR同时检测对虾白斑综合征病毒(WSSV)和传染性皮下及造血器官坏死病毒(IHHNV)[J].福建水产,2008(4):38-42.
[5]雷燕,张会军,王娟,等.鱼类类立克次氏体PCR检测方法的建立及初步应用[J].广东海洋大学学报,2015,35(6):1-5.
[6]LIGHTNER D V.A handbook of pathology and diagnostic procedures for diseases of penaeid shrimp[M].Baton Rouge,L A:World Aquaculture Society,1996.
[7]TOURTIP S.Histology,ultrastructure and molecular biology of a new microsporidium infecting the black tiger shrimp Penaeus monodon[D].Ph D Thesis,Bangkok:Mahidol University,2000.
[8]TOURTIP S,WONGTRIPOP S,STENTIFORD G D,et al.Enterocytozoon hepatopenaei sp.nov.(Microsporida:Enterocytozoonidae),a parasite of the black tiger shrimp Penaeus monodon(Decapoda:Penaeidae):fine structure and phylogenetic relationships[J].Journal of Invertebrate Pathology,2009,102:21-29.
[9]HA N T H,HA D T,THUY N T,et al.Enterocytozoon hepatopenaei parasitizing on tiger shrimp(Penaeus monodon)infected by white feces culture in Vietnam,has been detected(In Vietnamese with English abstract)[J].Agriculture and Rural Development:Science and Technology(translation from Vietnamese),2010,12:45-50.
[10]AMORNRAT T,JIRAPORN S,SAISUNEE C.The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in white leg shrimp Penaeus(Litopenaeus)vannamei[J].BMC Veterinary Research,2013,9:139.
[11]SRITUNYALUCKSANA K,SANGUANRUT P,SALACHAN P V,et al.Urgent appeal to control spread of the shrimp microsporidium parasite Enterocytozoon hepatopenaei(EHP).Network of Aquaculture Centre in Asia-Pacific[EB/OL].(2014-11-24).http://www.enaca.org/modules/news/article.php?article_id=2039.
[2]徐丽美,吴成林,邱名毅,等.对虾桃拉病毒(TSV)RT-PCR快速诊断技术的研究[J].厦门大学学报(自然科学版),2005,44(2):264-267.
[3]雷燕,肖洋,张会军,等.凡纳滨对虾对虾杆状病毒PCR检测方法的建立及初步应用[J].广东海洋大学学报,2015,35(4):63-66.
[4]任聪,龚艳清,陈信忠,等.双重PCR同时检测对虾白斑综合征病毒(WSSV)和传染性皮下及造血器官坏死病毒(IHHNV)[J].福建水产,2008(4):38-42.
[5]雷燕,张会军,王娟,等.鱼类类立克次氏体PCR检测方法的建立及初步应用[J].广东海洋大学学报,2015,35(6):1-5.
[6]LIGHTNER D V.A handbook of pathology and diagnostic procedures for diseases of penaeid shrimp[M].Baton Rouge,L A:World Aquaculture Society,1996.
[7]TOURTIP S.Histology,ultrastructure and molecular biology of a new microsporidium infecting the black tiger shrimp Penaeus monodon[D].Ph D Thesis,Bangkok:Mahidol University,2000.
[8]TOURTIP S,WONGTRIPOP S,STENTIFORD G D,et al.Enterocytozoon hepatopenaei sp.nov.(Microsporida:Enterocytozoonidae),a parasite of the black tiger shrimp Penaeus monodon(Decapoda:Penaeidae):fine structure and phylogenetic relationships[J].Journal of Invertebrate Pathology,2009,102:21-29.
[9]HA N T H,HA D T,THUY N T,et al.Enterocytozoon hepatopenaei parasitizing on tiger shrimp(Penaeus monodon)infected by white feces culture in Vietnam,has been detected(In Vietnamese with English abstract)[J].Agriculture and Rural Development:Science and Technology(translation from Vietnamese),2010,12:45-50.
[10]AMORNRAT T,JIRAPORN S,SAISUNEE C.The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in white leg shrimp Penaeus(Litopenaeus)vannamei[J].BMC Veterinary Research,2013,9:139.
[11]SRITUNYALUCKSANA K,SANGUANRUT P,SALACHAN P V,et al.Urgent appeal to control spread of the shrimp microsporidium parasite Enterocytozoon hepatopenaei(EHP).Network of Aquaculture Centre in Asia-Pacific[EB/OL].(2014-11-24).http://www.enaca.org/modules/news/article.php?article_id=2039.