《致病三大弧菌》PPT课件

StudiesonvibrioparahaemolyticusbyPCRduringthefourseasonsinthesouthcoastoftaiwan,指導老師:張福林副教授研究生:陳念群,Introduction,致病三大弧菌,V.vulnificusV.choleraeV.parahaemolyticus霍亂弧菌與腸炎弧菌是食品界及防疫單位最重視的食品中毒菌(資料來源:行政院衛生署),TCBSV.parahaemolyticus:直徑12mm、有光澤、綠色V.cholerae:直徑24mm、平滑、黃色、中心不透明、周圍透明嗜鹽性試驗(0%、1%、6%、8%、10%)V.parahaemolyticus:6%、8%生長良好V.cholerae:0%、1%生長良好,Vibrioparahaemolyticusisoneofthemostimportantfood-bornepathogensinTaiwan,Japanandothercoastalcountries.Vibrioparahaemolyticusisagram-negative,halophilicbacteriumthatoccursnaturallyinestuarineenvironmentsworld-wide.(EnvironmentalMicrobiology5(8),706-710,2003),Thermostabledirecthemolysin(TDH)TDH/TDH-relatedhaemolysin(TRH)UreasepositiveLethaltoxin附著因子,致病因子,PathogenicV.parahaemolyticusgenerallyproducesathermostabledirecthemolysin(TDH).Morethan90%ofclinicalV.parahaemolyticusisolates,butfewerthan1%offoodorenvironmentalstrainsproduceTDH.(APPLIEDANDENVIRONMENTALMICROBIOLOGY,Mar.2003),橫跨四季有系統地蒐集保存與研究台灣南部海域中的腸炎弧菌，並且進行一些基本特性分析，進而由分析結果來評估腸炎弧菌在台灣南部海域的分佈情形，而最後再來加以更深入的探討。,Aim,研究架構,海水、有機樣本收集(水質測量),傳統方法培養腸炎弧菌,利用腸炎弧菌ToxR基因專一性引子進行最確數-聚合酵素連鎖反應(MPN-PCR),偵測樣本中全部的腸炎弧菌密度,利用PCR偵測樣本中致病性腸炎弧菌(tdh/trh)及分析KP和血清型,數據整理與分析,採樣地點,安平漁港興達漁港前鎮漁港,採樣方法,水樣:利用採樣筒丟入海水中採得水樣後放入無菌塑膠試管中有機物質:刮取岸邊岩石上的藻類、貝類或水中魚類後放入無菌塑膠試管中保存於室溫3小時內帶回實驗室分析,水質測量,PH(METTLERTOLEDO)比重鹽度溫度(VWR1551-004)透視度濁度(HACH46500-00)綜合水質DO、TDS、EC(DR/2500ProtHACH5900)COD(HACHCODReactor)、(HACHODYSSEY),Samplecollection(3水樣3有機樣本)APW每管APW各取1loop接種於TCBS抽取DNA挑選典型綠色菌落分別各接種於TSB-2及TSA-2PCR(toxR)由TSB-2抽取DNAPCR(toxR)toxR(+):toxR(-):PCR(tdh)PCR(16SrRNA)toxR(+)PCR(trh)致病性分析生化鑑定菌體抗原PCR(tdh)KP試驗PCR(trh),toxR,對腸炎弧菌的專一性非常強L-GTCTTCTGACGCAATCGTTGR-ATACGAGTGGTTGCTGTCATG,(JOURNALOFCLINICALMICROBIOLOGY,Apr.1999,p.11731177),TDH&TRH,通常致病性腸炎弧菌都會具有的致病因子TDH:L-GGTACTAAATGGCTGACATCR-CCACTACCACTCTCATATGCTRH:L-GGCTCAAAATGGTTAAGCGR-CATTTCCGCTCTCATATGC(AppliedandEnvironmentalMicrobiology,Nov.2004,P.6401-6406),16SrRNA,所有的菌都具有16srRNA,於是選擇保守的序列來做為primer,這樣就可知道樣本中的DNA是否有毀損.L-ACGGCGCAGACTCCTACGGGAGGCR-GGGTTGCGCTCGTTGCGGCACTTA(JournalofClinicalMicrobiology,Aug.1994,P.1911-1917),生化鑑定,KP,使用Wagatsumaagar加以培養，若有溶血(在培養基上形成透明圈)現象則稱為Kanagawaphenomenon(KP陽性)，很可能就是致病菌株.,（一）方式：西元年/月/地點/樣本編號/菌株編號（二）說明：西元年2005取後二碼05月份：01、02、03、04、05、06、07、08、09、10、11、12地點：安平港A興達港B前鎮港C樣本編號:菌株編號：自01開始編號例如:0508A1302,命名系統,RESULTS,Table1.安平港水質結果,Table2.興達港水質結果,Table3.前鎮港水質結果,Fig1.AmplificationofthetoxRgenebyPCR,123456,368bp,Theprimersamplifieda368bpfragmentLane1.100bpmarkerLane2.CCRC10806Lane3.negativecontrolLane4.(+)sampleLane5.(-)sampleLane6.100bpmarker,原液,10倍,100倍,1000倍,90ml緩衝液,90ml緩衝液,90ml緩衝液,10ml原液,每稀釋檢液各接種3支(稱三階三支），並於3537，培養1618小時,Table4.判定為腸炎弧菌陽性者，利用最確數表（如附表），推算出腸炎弧菌之最確數(MPN/g或MPN/ml)。,DataTemp.pHMPN(MPN/ml或g)MPN-PCR(toxR)(MPN/ml或g)0508Awater1327.6211000508Aorganics2308.383511000508Aorganics3327.5829011000508Bwater1286.893.6930508Bwater2287.4111000508Borganics2287.413.011000508Borganics3297.082811000508Cwater1298.2711000508Corganics3298.2711000509Aorganics2297.9311000509Aorganics3297.9411000509Bwater1307.843.03.00509Bwater2307.913.6430509Bwater3307.891511000509Borganics1307.843.011000509Borganics2307.91152900509Borganics3307.896.111000509Cwater1298.113.63.60509Cwater2298.143.63.60509Cwater3298.163.63.60509Corganics1298.1111000510Aorganics230.17.7611000510Bwater131.18.029.2930510Bwater230.87.836.111000510Bwater330.87.949.2430510Borganics131.18.023.011000510Borganics230.87.8315930510Borganics330.87.946.211000510Cwater130.28.183.63.60510Cwater230.28.233.0750510Cwater330.28.173.63.60510Corganics130.28.173.63.60510Corganics230.28.233.6230510Corganics330.28.173.623,Table7.Vibrioparabaemolyficusdensitiesindifferentsamplesfromthreeareas,V.parahaemolyticusdetectedbySample(n)CulturemethodtoxR-PCRWater(n=27)14(51.9)20(74.1)Organics(n=27)18(66.7)26(96.3)Total(n=54)32(59.3)46(85.2),Table8.DetectionofVibrioparahaemolyticusfromwaterandorganicsamples,123456789101112,Fig2.Amplificationofthe16SrRNAgenebyPCR,Theprimersamplifieda763bpfragmentLane1.markerLane2.0508C32Lane3.0508C33Lane4.0508C34Lane5.0508C35Lane6.0508C36Lane7.0508C45Lane8.0508C47Lane9.0508C48Lane10.negativecontrolLane11nosampleLane12.nosample,763bp,Fig3.tdhFig4.trh,Theprimersamplifieda251bpfragmentLane1markerLane20508A41Lane30508A42Lane40508A43Lane50508A44Lane60508A45Lane70508A46Lane80508A47Lane90508A48Lane10.0508A49Lane11vp1Lane12negativecontrol,123456789101112,123456789101112,Theprimersamplifieda250bpfragmentLane1markerLane20508A41Lane30508A42Lane40508A43Lane50508A44Lane60508A45Lane70508A46Lane80508A47Lane90508A48Lane10.0508A49Lane11vp2Lane12negativecontrol,250bp,251bp,Fig5.AmplificationofthetdhgenebyPCR,300bp,200bp,Theprimersamplifieda251bpfragmentLane1.markerLane2.0508B44Lane3.0508B45Lane4.0508B46Lane5.0508B47Lane6.0508B48Lane7.0508B49Lane8.0508B50Lane9.0508B51Lane10.0508B52Lane11.positivecontrolLane12.negativecontrol,123456789101112,Fig6.AmplificationofthetrhgenebyPCR,300bp,200bp,Theprimersamplifieda250bpfragment.Lane1.markerLane2.0508B53Lane3.0508B54Lane4.0508B55Lane5.0508B56Lane6.0508B57Lane7.0508B58Lane8.0508B59Lane9.0508B60Lane100508B61Lane11.positivecontrolLane12.negativecontrol,123456789101112,tdhdetectedbySample(n)Culturemethodtdh-PCRWater(n=27)0(0)0(0)Organics(n=27)0(0)4(14.8)Total(n=54)0(0)4(7.4),Table9.Detectionofhaemolysingenes(tdh)fromwaterandorganicsamples,trhdetectedbySample(n)Culturemethodtrh-PCRWater(n=27)0(0)0(0)Organics(n=27)1(3.7)5(27.8)Total(n=54)1(1.9)5(13.9),Table10.Detectionofhaemolysingenes(trh)fromwaterandorganicsamples,Table11.Serotypingofthe101strainsisolate