台灣養殖南美白對蝦感染Enterocytozoon hepatopenaei (EHP ...

本研究針對台灣養殖南美白對蝦(Litopenaeus vannamei)感染Enterocytozoon hepatopenaei (EHP)微孢子蟲之疫情進行調查，並利用分子生物學的技術建立微孢子蟲的偵測方法 ... 資料載入處理中... 跳到主要內容 臺灣博碩士論文加值系統 ::: 網站導覽| 首頁| 關於本站| 聯絡我們| 國圖首頁| 常見問題| 操作說明 English |FB專頁 |Mobile 免費會員 登入| 註冊 功能切換導覽列 (159.65.142.206)您好！臺灣時間：2022/09/3022:48 字體大小：       ::: 詳目顯示 recordfocus 第1筆/ 共1筆  /1頁 論文基本資料 摘要 外文摘要 目次 參考文獻 電子全文 紙本論文 QRCode 本論文永久網址: 複製永久網址Twitter研究生:劉于瑄研究生(外文):LIU,YU-HSUAN論文名稱:台灣養殖南美白對蝦感染Enterocytozoonhepatopenaei(EHP)檢驗方法之建立論文名稱(外文):EstablishmentofDiagnosticMethodstoDetectEnterocytozoonHepatopenaei(EHP)InfectioninCultureLitopenaeusVannameiinTaiwan指導教授:王俊順指導教授(外文):Wang,Chun-Shun口試委員:溫秋明、鄭文騰口試委員(外文):Wen,Chiu-Ming、Winton-Cheng口試日期:2019-06-19學位類別:碩士校院名稱:國立高雄大學系所名稱:生命科學系碩士班學門:生命科學學門學類:生物學類論文種類:學術論文論文出版年:2019畢業學年度:107語文別:中文論文頁數:71中文關鍵詞:南美白對蝦、微孢子蟲、點墨雜交、原位雜交、PCR外文關鍵詞:Litopenaeusvannamei、Enterocytozoonhepatopenaei(EHP)、Dotblothybridization、Insituhybridization、PCR相關次數: 被引用:1點閱:498評分:下載:62書目收藏:0 本研究針對台灣養殖南美白對蝦(Litopenaeusvannamei)感染Enterocytozoonhepatopenaei(EHP)微孢子蟲之疫情進行調查，並利用分子生物學的技術建立微孢子蟲的偵測方法。

實驗中針對台灣南部地區屏東、高雄、台南等養殖場的南美白對蝦進行採樣，以微孢子蟲EHP專一性引子ENF779/ENR779及ENF176/ENR176分別進行PCR及NestedPCR的偵測，確認台灣養殖南美白對蝦確實受到EHP的感染，進而以病理組織切片分析EHP感染的組織趨性及病理變化，實驗中並將PCR反應增幅EHP的SSUrDNA部分基因片段(779bp)進行基因轉殖、定序、序列的比對與親緣關係的分析，同時利用PCR的方式將此核酸片段進行DIG標識製成探針，建立點墨雜交法(Dotblothybridization)及原位雜交反應(Insituhybridization)檢驗EHP的偵測方法。

實驗結果顯示:在台南地區的南美白對蝦在PCR及nestedPCR中，都有偵測到感染EHP的感染，屏東及高雄地區只有在nestedPCR中偵測到EHP微孢子蟲。

在組織病理切片的觀察，顯示南美白對蝦的肝胰臟組織可見上皮細胞有微孢子蟲的感染情況，同時細胞質內呈現嗜酸性的顆粒，除了肝胰臟外的其他器官並無EHP感染的情形。

實驗中EHPSSUrDNA部分基因的基因序列定序、比對與親緣關係分析方面，顯示在台灣所發現的EHP與泰國、越南、印度、中國都有99%以上的核酸相似性，因此應屬同一種微孢子蟲，有關台灣的EHP來源應進一步研究。

在建立南美白對蝦EHP分子生物的檢測方式方面，以點墨雜交法(Dotblothybridization)檢測探針之靈敏度，顯示只要13pg/ul的質體核酸即可呈現陽性反應，進而利用此方法檢測罹患EHP的南美白對蝦所萃取出來的核酸，亦可呈現陽性反應而確認有受到EHP的感染；原位雜交反應(Insituhybridization)的檢驗，亦可成功的偵測到南美白對蝦的肝胰臟被EHP微孢子蟲感染的現象，靈敏度優於傳統的組織切片。

綜合本研究的結果:以PCR、NestedPCR、組織病理切片、點墨雜交法(Dotblothybridization)、原位雜交反應(Insituhybridization)等檢測方法，確認台灣養殖的南美白對蝦確實已受到Enterocytozoonhepatopenaei(EHP)微孢子蟲的感染，有關EHP的防治應進一步進行研究分析。

ThisstudyinvestigatedtheepizooticofEnterocytozoonhepatopenaei(EHP)infectionfromLitopenaeusvannameiculturedinTaiwan.Inaddition,thediagnosticmethodsforEHPinfectionwereestablishedusingbiotechnology.ThediseasedL.vannameiwerecollectedinsouthernTaiwan,mainlyinPingtung,KaohsiungandTainan.ThePCRandnestedPCRwerecarriedoutwiththespecificprimers(ENF779/ENR779andENF176/ENR176)inordertoconfirmtheEHPinfection.Furthermore,thetissuestropismandhistopathologicalchangesofEHPinfectionwereevaluatedbytraditionalhistologicalmethod.TheampliconofEHPSSUrDNApartialgenefragments(779bp)byPCRreactionwerecloned,sequencing,andsequencealignmentandphylogeneticanalysis.TheDIG-probeofEHPSSUrDNApartialgenefragmentswerepreparedbyPCRmethod.ThedotblothybridizationandinsituhybridizationdetectivemethodsforEHPweredeveloped.TheresultsshowedEHPmicrosporidiaweredetectedinL.vannameiinTainanusingPCRandNestedPCR.InPingtungandKaohsiung,EHPinfectionwereconfirmedbynestedPCR.Histopathologicalstudy,theepithelialcellsofhepatopancreasofL.vannameiweretargettissueinfectedbyEHPusingH&Estaining.Exceptforhepatopancreas,EHPinfectionswerenotobservedinotherorgansinthediseasedshrimp.TheEHPSSUrDNApartialgenefragmentofEHPisolatedfromTaiwanwerefoundmorethan99%nucleotidesequencesimilaritieswithisolatesofThailand,Vietnam,India,andChina.Thephylogenetictreeanalysis,TaiwanisolateshouldbesameasEHPisolatedfromL.vannameiculturedinSoutheastAsia.ThesensitivityofDIG-probeforEHPwas13pg/ulusingdotblothybridization.ByDotblothybridization,theEHPinfectionwasdetectedinthediseasedshrimpusingtheDNAextractedfromEHPinfectedL.vannameiandconfirmeditissufferedfromEHPmicrosporidian.InsituhybridizationtestalsosuccessfullydetectedtheEHPinfectionofthehepatopancreasofL.vannameibyDIG-probe.Thesensitivitywasexcellentthanthatoftraditionalhistopathology.Conclusion,theEnterocytozoonhepatopenaei(EHP)microsporidianinfectionwasconfirmedintheL.vannameiculturedinTaiwanusingPCR,nestedPCR,histopathology,dotblothybridization,insituhybridizationmethods.Infuture,thepreventiveandtreatmentmethodsforEHPshouldbefurtherdevelopedinTaiwan. 目錄I圖目錄III表目錄V摘要：VI第一章前言11.1南美白對蝦的習性介紹11.2南美白對蝦之常見的疾病21.3微孢子蟲的特性41.4Enterocytozoonhepatopenaei(EHP)的特性與歸類51.5Enterocytozoonhepatopenaei(EHP)之地理分布及症狀61.6Enterocytozoonhepatopenaei(EHP)傳播方式及感染途徑71.7Enterocytozoonhepatopenaei(EHP)的檢測方法81.7.1外表病徵觀察81.7.2電子顯微鏡技術91.7.3組織病理切片91.7.4DNA探針101.7.5聚合鏈連鎖反應(Polymerasechainreaction，PCR)101.7.6環介導的等溫擴增(loop-mediatedisothermalamplification，LAMP)101.8實驗目的11第二章材料與方法122.1南美白對蝦微孢子EHP病學之研究122.1.1採樣122.1.2病理組織切片觀察122.2EHP之部分基因片段序列選殖132.2.1去氧核醣核酸的(TotalDNA)萃取132.2.2聚合酶連鎖反應(PCR)142.2.3膠體純析(Gelelution)152.2.4DNA接合(DNALigation)152.2.5質體轉型(Transformation)152.2.6質體抽取(Plasmidextraction)162.2.7限制酶素切割(Restrictionenzymedigestion)162.2.8核酸序列之分析與比對162.3EHP微孢子蟲之檢測方式與分析172.3.1點墨雜交(DotblotHybridization)172.3.1.1探針之製備與純化172.3.1.2點墨雜交反應(DotblotHybridization)182.3.2原位雜交反應(Insituhybridization)18第三章結果203.1南美白對蝦EHP微孢子蟲流行病學之研究203.2組織病理觀察213.3EHP微孢子蟲片段基因的選殖213.3.1SSUrDNA之片段基因PCR轉質和序列分析213.3.2EHP微孢子蟲SSURNA片段基因序列比對223.4EHP微孢子蟲之檢測方式之建立與分析233.4.1點墨雜交試驗(Dotblothybridization)233.4.2原位雜交試驗(Insituhybridization)23第四章討論40第五章結論45第六章參考文獻46附錄55 丁雲源(2005)白蝦養殖發展史。

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水試專訊：42-44。

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