虾肝肠胞虫(EHP)孢子的纯化和计数
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摘要
采用差速离心和密度梯度离心,从感染虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)对虾的肝胰腺中,尝试纯化出孢子,并应用透射电镜、荧光桃红染色和血球计数板计数的方法对纯化出的孢子进行了观察和计数。结果表明,从1g的EHP载量为(4.7±2.2)×104 copies/ng DNA的肝胰腺组织中经差速离心和密度梯度离心方法分离到EHP的孢子,电镜观察孢子为大小在0.7～1.2μm的椭圆形,纯化孢子悬液用血球计数板计数显示孢子浓度为5.30×103个/μL,蔗糖密度梯度中的纯化孢子区带含有的孢子总数约1.06×106个。
Enterocytozoon hepatopenaei(EHP)spores were purified from total 1g hepatopancreas tissues with an average EHP loading at(4.7±2.2)×10~4copies/ng DNA by differential centrifugation and density gradient centrifugation.The purified spores were observed under transmission electron microscope,and counted with phloxine staining on a blood counting chamber under light microscope.Purified spores of EHP were observed under the electron microscope as ellipsoid at size of 0.7～1.2μm.The concentration of EHP spores in the suspension of purified EHP spores was counted as 5.30×103/μL by blood counting chamber.The total number of purified EHP spores in the band of sucrose gradients was estimated as 1.06×10~6.
Enterocytozoon hepatopenaei(EHP)spores were purified from total 1g hepatopancreas tissues with an average EHP loading at(4.7±2.2)×10~4copies/ng DNA by differential centrifugation and density gradient centrifugation.The purified spores were observed under transmission electron microscope,and counted with phloxine staining on a blood counting chamber under light microscope.Purified spores of EHP were observed under the electron microscope as ellipsoid at size of 0.7～1.2μm.The concentration of EHP spores in the suspension of purified EHP spores was counted as 5.30×103/μL by blood counting chamber.The total number of purified EHP spores in the band of sucrose gradients was estimated as 1.06×10~6.
引文
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[2]Rajendran KV,Shivam S,Praveena PE,et al.Emergence of Enterocytozoon hepatopenaei(EHP),in farmed Penaeus(Litopenaeus)vannamei,in India[J].Aquaculture,2016,454:272-280.
[3]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].J Fish Diseases,2017,40(4):529-539.
[4]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品的检测[J].渔业科学进展,2016,37(2):119-126.
[5]姜义仁,邓真华,王伯阳,等.柞蚕微孢子虫孢子分离纯化方法[J].应用昆虫学报,2011,48(2):452-456.
[6]Liu Y M,Qiu L,Sheng A Z,et al.Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR[J].J Invertebr Pathol,2018,151:191.
[7]Li X P,Wan X Y,Xu T T,et al.Development and validation of a TaqMan RT-qPCR for the detection of convert mortality nodavirus(CMNV)[J].Journal of Virological Methods,2018:65-71.
[8]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.
[9]Yue Z Q,Liu H,Wang W,et al.Development of real-time polymerase chain reaction assay with TaqMan probe for the quantitative detection of infectious hypodermal and hematopoietic necrosis virus from shrimp[J].J AOAC Int.2006,89(1):240-4.