TCID50與moi值的計算教學(xué)教材

1、TCI D50與moi值的計算實驗四病毒感染力的滴定(TCI®的測定)一、實驗?zāi)康牧私獠《靖腥玖y定的幾種常用方法,掌握半數(shù)細(xì)胞培養(yǎng)物感染量TCID50的操作步驟、計算方法及含義.二、測定病毒感染力的方法半數(shù)致死量LDo( 50% lethal dose):用動物或雞胚來檢測半數(shù)雞胚感染量EID5o(egg 50% infective dose) :用雞胚來檢測半數(shù)細(xì)胞培養(yǎng)物感染量 TCID50 (50% tissue culture infective dose) :用細(xì)胞來檢測蝕斑形成單位(plaque forming unit , PFU :用細(xì)胞來檢測三、材料1、長滿單層的細(xì)

2、胞1瓶2、胰酶、吸球、吸管、生長液3、96孔細(xì)胞培養(yǎng)板4、加樣器、槍頭5、病毒液(PRV四、TCI*的操作步驟1、在青霉素瓶或離心管中將病毒液作連續(xù) 10倍的稀釋,從10-1-10-10.2、將稀釋好的病毒接種到96孔微量培養(yǎng)板中,每一稀釋度接種一縱排共8孔,每孔接種100 U.3、在每孔參加細(xì)胞懸液100d ,使細(xì)胞量到達(dá)23X105個/ml.4、設(shè)正常細(xì)胞對照,正常細(xì)胞對照作兩縱排.100d生長液+100 口細(xì)胞懸液5、逐日觀察并記錄結(jié)果,一般需要觀察 5-7天.6、結(jié)果的計算,按 Reed-Muench兩氏法或Karber法五、TCID50的計算方法1、Reed-Muench兩氏法,內(nèi)毒

3、液稀釋度出現(xiàn)CPE孔數(shù)無CPE孔 數(shù)累 計出現(xiàn)CPEJL所 占的CPEJL 數(shù)無 CPEfL數(shù)10-118 ,0270F 100 (27/27)10-2J8k0190100 (19/19)10-37111191.6 (11/12)10-435 ,4640 (4/10)10-51171130.7 (1/14)10-6080210(0/21)CPE Cytopathic effect距離比例=高于50%|變率的百分?jǐn)?shù)-50% / 高于50%|變率的百分?jǐn)?shù)-低于50%W變率的百分?jǐn)?shù))=(91.6-50 ) / (91.6-40 )=0.8lgTCID50=®離比例X稀釋度對數(shù)之間的差+高于

4、50晚變率的稀釋度的對數(shù)=0.8N-1)+(-3)=-3.8TCID50=10-38/0.1ml含義：將該病毒稀釋10“接種1004可使50%勺細(xì)胞發(fā)生病變2、Karber 法,內(nèi)毒液稀釋度出現(xiàn)CPE的孔數(shù)出現(xiàn)CPEJL的比率10-188/8=110-288/8=110-377/8=0.87510-433/8=0.37510-511/8=0.12510-600/8=0lgTCID50=L-d(s-0.5)L:最高稀釋度的對數(shù)D:稀釋度對數(shù)之間的差S:陽性孔比率總和lgTCID50=-1-1 x(3.375-0.5)=-3.875TCID5o=10-3.875/0.1 ml含義：將該病毒稀釋10

5、“75接種100 d可使50%勺細(xì)胞發(fā)生病變什么是MOPMOI是multiplicity of infection的縮寫,中文譯為感染復(fù)數(shù).傳統(tǒng)的 MOI概念起源于噬菌體感染細(xì)菌的研究.其含義是感染時噬菌體與細(xì)菌的數(shù)量比 值,也就是平均每個細(xì)菌感染噬菌體的數(shù)量.噬菌體的數(shù)量單位為pfu.一般認(rèn)為MOI是一個比值,沒有單位,其實其隱含的單位是pfu number/cell .后來MOI被普遍用于病毒感染細(xì)胞的研究中,含義是感染時病毒與細(xì)胞數(shù)量的比 值.然而,由于病毒的數(shù)量單位有不同的表示方式,從而使MOI產(chǎn)生了不同的含義.能產(chǎn)生細(xì)胞裂解效應(yīng)的病毒例如腺病毒、單純皰疹病毒等習(xí)慣上仍用pfu表示病毒

6、數(shù)量,因此其 MOI的含義與傳統(tǒng)的概念相同.而對于某些病毒如AAV病毒,無法用pfu表示病毒的數(shù)量,而是采用 TUK IU、病毒顆粒(viralparticles, v.p ) 或基因組數(shù)量(vector genome,v.g.) 來表示病毒數(shù)量,因 此其MOI就有了不同含義.采用 TU或IU, MOI的含義便是TU number/cell或 IU number/cell .采用 v.p. , MOI的含義便是 v.p. number/cell .采用 v.g , MOI的含義便是 v.g. number/cell .可以將上述不同的MOI表示方式分為2種：1)以活性單位表示病毒數(shù)量,如 pf

7、u, TU, IU .這時MOI的含義是指平均每 個細(xì)胞感染病毒的活性單位數(shù).2)以病毒顆?；蚧蚪M數(shù)表示病毒數(shù)量,如 v.p.或v.g.這時MOI的含義 是指平均每個細(xì)胞感染病毒的病毒顆?；蚧蚪M數(shù).值得一提的是,上述2種不同的MOI表示方式在含義和數(shù)值上都有所不同.前 一種是傳統(tǒng)意義上的MOL后一種MOI表示方式的含義更為簡化和直觀,也逐 步被一些研究者采用.傳統(tǒng)意義上的MOI的測定,其原理是基于病毒感染細(xì)胞是一種隨機(jī)事件,遵循 Poisson分布規(guī)律,可計算出感染一定比例的培養(yǎng)細(xì)胞所需的感染復(fù)數(shù)(MOI .具公式為：P(k) = 1- P(0) ,P(0) = e-m 或 m = -In

8、P (0).其中：P(k)為被感染細(xì)胞的百分率P(0)為未被感染細(xì)胞的百分率 m為MOI值例如,如果要感染培養(yǎng)皿中99%勺培養(yǎng)細(xì)胞,那么：P(0) = 1% = 0.01m = -In(0.01)= 4.6 pfu/cell .由此可以看出,由于病毒特性和研究者習(xí)慣的不同,MOI的單位由原來約定俗成的pfu number/cell 變成多種表示單位.在閱讀文獻(xiàn)和寫作論文時,應(yīng)注意 MOI 的具體含義和單位.MOI= (pfu/ml的值)乘(取的病毒液體積)再除以你要轉(zhuǎn)染的細(xì)胞數(shù).TCID50 與 PFU 換算：PFUs =0.7 刈CID50 的滴度PFU:plaque forming uni

9、t ,空斑形成單位.感染性滴度的單位一般表示為PFU/ml.由于測定pfu往往重復(fù)性較差,因此近些年許多研究又開始采用TCID50方法來計算病毒的感染單位.因此建議也可使用TCID50法.Sample problem:You infect a monolayer of 1X107 cells with 0.1 ml. of a sample of virus the titer of which is 1X 109 PFU/ml.What is the MOI of this infection, and how many cells are infected?0.1ml. ( 1 X 109

10、 PFU/ml ) = 1 X 108 PFUMOI = PFU/cell =1 X 108 / 1 X 107 =10The proportion of infected cells = 1- the proportion of uninfected cells = 1-e- m =1-e-10 =0.999952The number of infected cells = the total number of cells multiplied by the proportion of infected cells = (1X107) (0.999952) = 0.999952X107.

11、Thus at an MOI of 10, most cells are infected.Now you calculate the number of cells that are uninfected, infected with one PFU, and multiply infected.The One-step growth curve was devised by Ellis and Delbruck to study the single cell life cycle of bacteriophage. The key element of this experiment i

12、s synchronization of the infected cells so that the population reflects events occurring in the single cells. Synchronization is achieved by infecting the culture of cells at an MOI at which most of the cells are infected (MOI of 510). At various times aliquots of the infected culture are withdrawn

13、and assayed for PFU by plaque assay. Samples can be tittered directly, or lysed prior to analysis to observe intracellular events. Refer to your text for a graphic plot of the curves. Pay special attention to the events in the life cycle of a virus (eg. Adsorption, uncoating, biosynthesis, assemble,

14、 release) that occur in the latent, eclipse, exponential rise, and plateau regions of the curve. From the total number of cells in the culture, and the total number of progeny PFU produced at the end of the rise (" burst " ) one can calculate the number ofPFU produced per cell.MOI, pfu, an

15、d TCID 50? Plaque forming units (pfu) is a measure of number of infectious virus particles. It is determined by plaque forming assay.? Multiplicity of infection (moi)is the average number of virusparticles infecting each cell.? TCID50 is the tissue culture infectious dose which will infect 50% if th

16、e cell monolayers challenged with the defined inoculum. If the titer is "103 TCID0/0.2 ml, MK, 2 days," it means that when a 0.2 mlinoculum of a 1:1,000 dilution of the virus is added to each of four tubes containing monkey kidney (MK) cells, two tubes are expected to become infected.MOI i

17、s related to pfu by the following formula:Multiplicity of infection (moi) = Plaque forming units (pfu) of virus used for infection / number of cells.For example, if 2x106 cells is infected by 50 ml of virus with atiter of 108 pfu/ml. The moi will be 0.05*108/2*10 6 = 2.5.The fraction of cells that a

18、re not infected isP(0) = 1 - e -moi, i. e., 8% for moi = 2.5.To ensure 99% of cells are infected requires moi > 4.6.Assume the conditions used for plaque assay and TCID assay don't alter the expression of infectious virus, TCID50/ml and pfu/ml arerelated bypfu/ml = 0.7 * TCID 犯As a working es

19、timate, one can usepfu/ml = 0.5 * TCID50.H5N1流感病毒TCID 50 組織半數(shù)感染量測定一生物材料與試劑1.病毒與細(xì)胞高致病性H5N1禽流感病毒A/Chicken/Yangzhou/35/2022 以下簡稱：YZ35MDCK細(xì)胞O成關(guān)苴2,口仆/10%DMEM、無血無抗 DMEM、1%DMEM3.其他96孔板、血凝板、PBS、1%的雞紅細(xì)胞二操作步驟1 .細(xì)胞準(zhǔn)備取出一塊96孔細(xì)胞培養(yǎng)板,每個孔大約傳 800010000個細(xì)胞一個T25 瓶的細(xì)胞消化后加10mL培養(yǎng)液正好傳一塊96孔板,要傳勻.每個孔的細(xì)胞 鋪成單層大約70-90%豐度即可接種病毒晚

20、上傳好板第二天早上就能用.2 .稀釋待測病毒YZ35尿囊液YZ35尿囊液用無血無抗的DMEM進(jìn)行10倍倍比稀釋,即向每支 EP管中 參加900也無血無抗的DMEM,向1號試管中參加100 pL病毒尿囊液,振蕩混 勻,再從1號管中取100山稀釋液依次10倍系列稀釋至10-1°.注意：1病毒稀釋過程中一定將病毒液與孵育液充分混勻.2此過程中 需要使用加樣器和tip頭.使用前用75%乙醇擦拭加樣器,并用紫外線照射 20min,保證無菌.使用新高壓的tip頭,外包裝一定在超凈臺或平安柜中翻開. 3.病毒稀釋液接種細(xì)胞取細(xì)胞培養(yǎng)板,用多道加樣器即排槍吸去96孔板中的培養(yǎng)液,再吸取無血無抗DMEM加在每孔中再輕輕吹打一次,然后吸出孵育液此步目的是去 除血清,由于血清能干擾病毒的吸附.將稀釋好的病毒液加到96孔板上,每孔100山,每個稀釋度做8個重復(fù)即1豎排.根據(jù)觀察的習(xí)慣,一般從右 到左,從上到下,從高稀釋度到低稀釋度加樣.同時設(shè)置正常的細(xì)胞對照16孔即2豎排.37CCO2培養(yǎng)箱中孵育1h,取出培養(yǎng)板吸去病毒液從低濃度向高濃度吸 取可防止竄孔,參加1%DMEM維持液200山繼續(xù)在37C CO2培養(yǎng)箱中培 養(yǎng).注意：低致病性流感病毒一般在胰酶存在的條件下才能感染MDCK0胞,因此病毒稀釋液中需參加2國/mlTPCK-胰酶.高致病性禽流感病毒在無胰酶存在 條件下即