TCID-MOI-PFU意義及其換算

1、 :/bbs.virology /viewthread.php?tid=7423&page=1Multiplicity of infection (MOI)The multiplicity of infection (abbreviated MOI) is the average number of phage per bacterium. The MOI is determined by simply dividing the number of phage added (ml added x PFU/ml) by the number of bacteria added (ml a

2、dded x cells/ml). The average number of phage per bacterium in the population could be 0.1, 1, 2, 10, etc, depending upon how you set up the experiment.Although the MOI tells you the average number of phage per bacterium, the actual number of phage that infect any given bacterial cell is a statistic

3、al function. For example, if the MOI is 1, some cells will get infected with one phage but some cells may be infected with 0 phage and other cells infected with two phage. The proportion of cells in a population infected by a specific number of phage (n) can be calculated from the Poisson distributi

4、on. · 精品文章   金幣 + 100 枚 PFU:plaque forming unit，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(jì)算病毒的感染單位。因此建議也可使用TCID50法。MOI :multiplicity of infection，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(shí)其隱含的單位是pfu number/

5、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)的病毒例如單純皰疹病毒等習(xí)慣上仍用pfu表示病毒數(shù)量，因此其MOI的含義與傳統(tǒng)的概念相同。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(jì)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。其公式為：P = 1- P(0) ,P(0) = e-m 或m = -InP0。其中：P 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%的培養(yǎng)

6、細(xì)胞，那么：P(0) = 1% = 0.01m = -In(0.01)= 4.6 pfu/cell。 TCID50 Protocol：1：制備96孔板單層細(xì)胞2：將病毒做系列稀釋，橫向接種單層細(xì)胞板，每稀釋度重復(fù)3孔3：每日觀察細(xì)胞病變，記錄高于50和低于50病變孔的病毒稀釋度，4：計(jì)算比距，獲得TCID50計(jì)算比距：高于50的病變率50/高于50病變率小于50病變率比距；比距與接近50%病變率的病毒的稀釋度的指數(shù)相加，就獲得了指數(shù)。比方：比色計(jì)算或者顯微鏡觀察病毒的TCID50在10的負(fù)7和8次方之間，那么，指數(shù)8與比距相加，獲得的新的指數(shù)，就是TCID50的指數(shù)，TCID50=10的-7.

7、幾次方TCID50與 PFU換算:PFUs0.7×TCID50的滴度 :/hi.baidu /biovirus/blog/item/86073b01e7b080037aec2cef.htmlMOI感染復(fù)數(shù)TCID50 PFU 病毒的滴度，感染單位2021-06-19 19:40Q: The author infected cells with an M.O.I of 50. The virus I titrated is 5X10(7)p.f.u/ml. So how much virus should I dilute to get an M.O.I of 5?A: An MOI

8、of 50 means 50 active viral particles per cell. So, if you have 10(6) cells on a plate x MOI (50)= 5x10(7) p.f.u for that plate (which equals one ml of your virus stock).The number of p.f.u./ml was then calculated as TCID50 /ml x 0.69. ://cgi/content/abstract/90/10/2364Ian J. Grov

9、es,3 Matthew B. Reeves4 and John H. Sinclair. Lytic infection of permissive cells with human cytomegalovirus is regulated by an intrinsic preimmediate-early repression of viral gene expression mediated by histone post-translational modification. Journal of General Virology (2021), 90, 23642374Conver

10、ting TCID50 to MOIHow do I convert TCID50 to MOI?The easiest way to convert TCID50 to an MOI value is to do the following:Convert the titer by TCID50 to plaque forming units (PFU). To do this, multiply the titer by 0.7.Since plaque forming units represents the estimated number of infectious units pe

11、r volume of virus material, one can estimate the total number of infectous particles.Next, divide the number of infectious particles by the number of cells to be infected to obtain the MOI. Then, dilute the virus accordingly in order to obtain 0.1 to 0.01 MOI.For example,If the TCID50 titer for a gi

12、ven virus is 10(7.25) TCID50 per 0.2 ml, 10(7.25) is approximately 17,782,794 (the inverse log of 7.25), and when multiplied by 0.7 gives 12,447,956 PFU per 0.2 ml. If you were going to infect 10 million cells at an MOI of 0.1, you need 1,000,000 infectious particles. For the above example, you shou

13、ld dilute 0.2 ml of the virus stock at least 1:12.5 to obtain 1,000,000 infectious particles per 0.2 ml, then this volume (0.2 ml x 12.5 = 2.5 ml) should be diluted appropriately to infect the number of vessels. (Usually we infect a 75 cm2 flask with 2 mL of diluted virus or a 150 cm2 flask with 5 m

14、L of diluted virus).為什么病毒載體的感染單位有不同的表示方法？不同的病毒載體，其表示感染單位的方式有所不同。早期的逆轉(zhuǎn)錄病毒載體一般用CFU，是“clone forming unit的簡稱，叫做“克隆形成單位。感染性滴度的單位一般表示為CFU/ml。這是因?yàn)樽钤绲哪孓D(zhuǎn)錄病毒系統(tǒng)是Dr. Dusty Miller建立的LN系列包括LN，LXSN，LNSX，LXCN，LNCX等，包裝細(xì)胞株是PA317, psi2，G&P86等。在這套載體中N代表了neo基因，X代表外源基因，S代表SV40啟動子，C代表CMV啟動子，L代表逆轉(zhuǎn)錄病毒的LTR，具有啟動子功能。因此，早期的

15、LN系列載體是雙表達(dá)載體，重組病毒中有neo基因的表達(dá)單位，滴度測定時用病毒感染NIH3T3細(xì)胞，用G418選擇培養(yǎng)，根據(jù)形成neo基因抗性克隆的多少來計(jì)算病毒滴度，因此滴度的單位就用cfu/ml來表示了。這種表示方法至今還很常用。腺病毒載體的感染單位一般用PFU表示，是“plaque forming unit的簡稱，叫做“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古老的TCID50方法來計(jì)算腺病毒的感染單位。What do MOI and PFU mean?The term MOI stands for Multiplic

16、ity Of Infection. This value describes the ratio of the number of virions per target cell at the time the virus is added to the cuture. The term PFU stands for Plaque Forming Unit. One PFU/ml is defined as 1 lytic event (seen as a plaque on a lawn of 293 cells) per milliliter of media. This is a mea

17、sure of viral titer.    Additional information can be found in the following reference: Nature Medicine, vol. 3, number 7, July 1997. Sensitivity and Reproducibility in Adenoviral Infectious Titer Determination. Once the viral titer is determined, a given MOI can be specified and used for

18、the target cells.什么是MOI？MOI 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(shí)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表示病毒數(shù)量，因此其

19、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TU、IU、病毒顆粒viral particles, 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pfu, TU, IU。這時MOI的含義是指平均每個細(xì)胞

20、感染病毒的活性單位數(shù)。 2 以病毒顆粒或基因組數(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MOI，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(jì)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。其公式為： P(k) = 1- P(0) ,P(0) = e-m 或m = -InP0。 其中： P(k) 為被感染細(xì)胞的百分率 P(0

21、)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%的培養(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。-PFU: plaque forming units    A measure of the biological activity of a virus sample. MOI: multiplicity of in

22、fection         MOI= #PFU/cell    eg. If 1X106 cells are infected with 5X106 PFU, the MOI is calculated as 5X106/1X106=5 CPE: cytopathic effect The Poisson distribution is used to calculate the proportion (P) of cells infected with K (0, 1, 2, 3

23、, etc.) PFU when virus is added to cells at a particular MOI (m).-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 PFU/ml ) = 1 X 108 PFU MOI

24、 = PFU/cell =1 X 108 / 1 X 107 =10 The proportion of infected cells = 1- the proportion of uninfected cells = 1-e-m =1-e-10 =0.999952 The number of infected cells = the total number of cells multiplied by the proportion of infected cells = (1X107) (0.999952) = 0.999952X107. Thus at an MOI of 10, mos

25、t 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 is synchronization of the infected cells s