3种虾肝肠胞虫PCR检测方法的应用比较分析
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摘要
虾肝肠胞虫被认为是凡纳滨对虾"长不大"现象的主要病原之一,国内外学者已建立其PCR检测技术,包括18S-PCR、SSU-PCR及SWP-PCR。采集36个凡纳滨对虾苗种样本、5个卤虫样本和12个水样,应用上述3种PCR方法进行检测,并对阳性产物进行克隆测序和序列同源性分析,以比较检测灵敏度和准确性。试验结果显示,18S-PCR在第一步扩增的灵敏度最好,SSU-PCR在第二步扩增的灵敏度显著高于SWP-PCR,但存在着一定的假阳性现象。建议在对凡纳滨对虾苗种进行虾肝肠胞虫的检测时,宜采用灵敏度更高的套式SSU-PCR方法。
Microsporidian Enterocytozoon hepatopenaei has emerged as a serious pathogen associated with retarded growth in cultured shrimp, and several PCR detection methods were established including 18 S-PCR, SSU-PCR and SWP-PCR which were applied to detect the microsporidian in 36 samples of postlarval Pacific white shrimp Litopenaeus vannamei, 5 samples of Artemia and 12 water samples, the positive bands were cloned and sequenced, and then subjected to homology analysis in order to evaluate the efficiency and accuracy of these 3 methods. The results showed that 18 S-PCR had the best sensitivity in one-step amplification, and that despite the existing of false positive phenomenon, SSU-PCR was significantly more sensitive than SWP-PCR in the second step amplification. It is suggested that nested SSU-PCR method should be used for detection of Enterocytozoon hepatopenaei in Pacific white leg shrimp.
Microsporidian Enterocytozoon hepatopenaei has emerged as a serious pathogen associated with retarded growth in cultured shrimp, and several PCR detection methods were established including 18 S-PCR, SSU-PCR and SWP-PCR which were applied to detect the microsporidian in 36 samples of postlarval Pacific white shrimp Litopenaeus vannamei, 5 samples of Artemia and 12 water samples, the positive bands were cloned and sequenced, and then subjected to homology analysis in order to evaluate the efficiency and accuracy of these 3 methods. The results showed that 18 S-PCR had the best sensitivity in one-step amplification, and that despite the existing of false positive phenomenon, SSU-PCR was significantly more sensitive than SWP-PCR in the second step amplification. It is suggested that nested SSU-PCR method should be used for detection of Enterocytozoon hepatopenaei in Pacific white leg shrimp.
引文
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[10] 骆云慧,石坚,方磊,等.虾肝肠胞虫TaqMan实时荧光定量PCR检测方法的建立及应用[J].中国兽医科学,2016(7):847-852.
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[14] 王博雅,王力,刘美如,等.凡纳滨对虾3种主要病毒和虾肝肠胞虫在辽宁地区的流行情况分析[J].大连海洋大学学报,2017,32(2):150-154.
[15] 刘珍,张庆利,万晓媛,等.虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样本的检测[J].渔业科学进展,2016,37(2):119-126.
[16] 刘宝彬,杨冰,吕秀旺,等.凡纳滨对虾(Litopenaeus vannamei)传染性皮下及造血组织坏死病毒(IHHNV)及虾肝肠胞虫(肠胞虫)的荧光定量PCR检测[J].渔业科学进展,2017,38(2):158-166.
[17] Salachan P V,Jaroenlak P,Thitamadee S,et al.Laboratory cohabitation challenge model for shrimp hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei(EHP)[J].BMC Veterinary Research,2016(13):9.
[18] Suebsing R,Prombun P,Srisala J,et al.Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp[J].Journal of Applied Microbiology,2013,114(5):1254-1263.
[19] Jaroenlak P,Sanguanrut P,Williams B A,et al.A nested PCR assay to avoid false positive detection of the microsporidian Enterocytozoon hepatopenaei (EHP) in environmental samples in shrimp farms[J].PloS One,2016,11(11):e0166320.
[20] Tokarev Y S,Voronin V N,Seliverstova E V,et al.Ultrastructure and molecular phylogeny of Anisofilariata chironomi g.n.sp.n.(Microsporidia:Terresporidia) from Chironomus plumosus L.(Diptera:Chironomidae)[J].Parasitology Research,2010,107(1):39-46.
[2] 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(1):21-29.
[3] Tangprasittipap A,Srisala J,Chouwdee S,et al.The microsporidian Enterocytozoon hepatopenaei,is not the cause of white feces syndrome in whiteleg shrimp Penaeus,(Litopenaeus) vannamei[J].BMC Veterinary Research,2013,9(1):139.
[4] Tang K F,Han J E,Aranguren L F,et al.Dense populations of the microsporidian Enterocytozoon hepatopenaei(EHP) in feces of Penaeus vannamei exhibiting white feces syndrome and pathways of their transmission to healthy shrimp[J].Journal of Invertebrate Pathology,2016(140):1-7.
[5] Tang K F,Pantoja C R,Redman R M,et al.Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei (EHP),a microsporidian parasite infecting penaeid shrimp[J].Journal of Invertebrate Pathology,2015(130):37-41.
[6] Rajendran K V,Shivam S,Praveena P E,et al.Emergence of Enterocytozoon hepatopenaei (EHP) in farmed Penaeus (Litopenaeus) vannamei,in India[J].Aquaculture,2016(454):272-280.
[7] Aranguren L F,Han J E,Tang K F J.Enterocytozoon hepatopenaei (EHP) is a risk factor for acute hepatopancreatic necrosis disease (AHPND) and septic hepatopancreatic necrosis (SHPN) in the Pacific white shrimp Penaeus vannamei[J].Aquaculture,2017(471):37-42.
[8] Kesavan K,Mani R,Toshiaki I,et al.Quick report on prevalence of shrimp microsporidian parasite Enterocytozoon hepatopenaei in India[J].Aquaculture Research,2017,48(7):3980-3984.
[9] 陈禄芝,余霞艳,胡一丞,等.粤西地区凡纳滨对虾虾肝肠胞虫、传染性皮下和造血组织坏死病毒感染情况的初步调查[J].渔业研究,2016,38(4):273-280.
[10] 骆云慧,石坚,方磊,等.虾肝肠胞虫TaqMan实时荧光定量PCR检测方法的建立及应用[J].中国兽医科学,2016(7):847-852.
[11] 浙江省水产技术推广总站.浙江省上半年水产养殖病害形势分析[N].中国渔业报,2015-08-07(02).
[12] 万夕和.江苏省部分淡化场南美白对虾虾苗肠胞虫检出率超两成[J].水产前沿,2016(3):13.
[13] Santhoshkumar S,Sivakumar S,Vimal S,et al.Biochemical changes and tissue distribution of Enterocytozoon hepatopenaei (EHP) in naturally and experimentally EHP-infected whiteleg shrimp,Litopenaeus vannamei (Boone,1931),in India[J].Journal of Fish Diseases,2017,40(4):529-539.
[14] 王博雅,王力,刘美如,等.凡纳滨对虾3种主要病毒和虾肝肠胞虫在辽宁地区的流行情况分析[J].大连海洋大学学报,2017,32(2):150-154.
[15] 刘珍,张庆利,万晓媛,等.虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样本的检测[J].渔业科学进展,2016,37(2):119-126.
[16] 刘宝彬,杨冰,吕秀旺,等.凡纳滨对虾(Litopenaeus vannamei)传染性皮下及造血组织坏死病毒(IHHNV)及虾肝肠胞虫(肠胞虫)的荧光定量PCR检测[J].渔业科学进展,2017,38(2):158-166.
[17] Salachan P V,Jaroenlak P,Thitamadee S,et al.Laboratory cohabitation challenge model for shrimp hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei(EHP)[J].BMC Veterinary Research,2016(13):9.
[18] Suebsing R,Prombun P,Srisala J,et al.Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp[J].Journal of Applied Microbiology,2013,114(5):1254-1263.
[19] Jaroenlak P,Sanguanrut P,Williams B A,et al.A nested PCR assay to avoid false positive detection of the microsporidian Enterocytozoon hepatopenaei (EHP) in environmental samples in shrimp farms[J].PloS One,2016,11(11):e0166320.
[20] Tokarev Y S,Voronin V N,Seliverstova E V,et al.Ultrastructure and molecular phylogeny of Anisofilariata chironomi g.n.sp.n.(Microsporidia:Terresporidia) from Chironomus plumosus L.(Diptera:Chironomidae)[J].Parasitology Research,2010,107(1):39-46.