CN114214457A - Method for comprehensively evaluating titer of pAd-M3C adenovirus by combining TCID50, MOI, GTU and PFU - Google Patents
Method for comprehensively evaluating titer of pAd-M3C adenovirus by combining TCID50, MOI, GTU and PFU Download PDFInfo
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Abstract
The invention provides a method for comprehensively evaluating titer of adenovirus pAd-M3C by combining TCID50, MOI, GTU and PFU. By integrating three detection methods of TCID50, MOI and GTU, after the operation steps of the improved and refined experiment are improved, the titer of the adenovirus pAd-M3C is detected, and then conversion and comparison are carried out through PFU, namely the TMGP method for short, the original single method can be optimized and improved in repeatability, stability, sensitivity, usability and time consumption, the effectiveness of the adenovirus titer detection is improved, and a foundation is laid for the standard operation of the subsequent experiment. The TMGP method can predict the virus titer MOI value in 24-48 hours, verify GTU data in 36-48 hours and compare the accurate titer of TCID50 on the most critical day 10, so that repeated experiments are reduced, the experiment cost is reduced, and the effectiveness is improved.
Description
Technical Field
The invention belongs to the technical field of cellular immunity in molecular biology. In particular to a method for comprehensively evaluating titer of pAd-M3C adenovirus by combining TCID50, MOI, GTU and PFU.
Background
The current virus titer determination methods mainly comprise 2 types: physical methods (VP) and biological methods (GTU, PFU, TCID50 and MOI).
The method for determining VP (viral Particles) or OPU (Optical Particle Unit) is to determine the absorbance of the viral Particles at 260nm (the total absorbance of viral DNA and proteins is mainly DNA), 1 OD value corresponds to 1.1X 1012And (c) viral particles. Assays performed in this manner are stable in each laboratory, but do not distinguish between infectious and defective viral particles. This method therefore only provides the amount of virus, and does not take into account the nature, e.g.whether defective particles are present. GTU measures the number of cells expressing the reporter gene after infection. If the recombinant adenovirus does not contain a reporter gene, it cannot be assayed in this way, and viral stocks are not typically assayed in this way.
GTU (Gene Transfer Unit) or transduction particles (BFU, Blue dot Forming Unit) determine the number of cells that can express the reporter Gene after infection. In this process, the virus transfers DNA into cells and cells expressing the reporter gene are assayed immediately before the end of an infection cycle. This method can be used to determine if the recombinant adenovirus contains a reporter gene such as GFP or LacZ, etc.
PFU (Plaque Forming Unit) is the earliest standard method for determining adenovirus titer, mainly for determining the formation of Cytopathic Effect (CPE) virus lysis Plaque in monolayer cell culture, generally expressed as PFU/ml, wherein Plaque assay is a commonly used method for detecting titer of virus infectious particles. However, three weeks are typically required to obtain the final result. Generally, the results obtained by this method are rarely repeated in other laboratories, and even in the same laboratory, the same results are rarely obtained by different technician operations.
TCID50 (50% Tissue Culture Infectious Dose, Tissue Culture Infectious Dose 50), i.e., 50% Tissue Culture Infectious Dose, virus dilutions were cultured with cells in 96-well plates, and each well was then monitored for CPE. The TCID50 process has several advantages over the PFU process, being 2 times faster than PFU, more predictable and more stable across different operating units. But have not previously been used for adenovirus. Because PFUs are the earliest and most widely used, TCID50 typically needs to be scaled with the PFU.
MOI (Multiplicity of Infection) this experiment was used to roughly estimate the number of virus particles in the virus supernatant to determine the optimal conditions for Infection. The MOI is generally considered to be a ratio, in units of units, of the number of virus particles to the number of infected cells. Specific viral titers need to be scaled to PFU.
All biological methods the results obtained from the determination of viral titres often vary between different laboratories and are mainly related to the method of viral infection. Many factors such as the amount of virus stock added, the incubation time, the amount of cells and culture medium, etc., affect the results. To date, no method has been recognized as a standard method for determining viral titer. However, in scientific research exchanges, literature references and subject researches, comparison references are often required among the methods, and interconversion among a plurality of main methods has no clear standard, so that various evaluation methods can be comprehensively utilized to form a standard experiment operation, conversion can be rapidly and simply carried out, the effectiveness of titer evaluation is improved, and a foundation is laid for quantification and standardization of subsequent experiments.
Disclosure of Invention
The invention aims to solve the problems and provide a method for comprehensively evaluating titer of adenovirus pAd-M3C by combining TCID50, MOI, GTU and PFU.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for comprehensively evaluating titer of adenovirus pAd-M3C by combining TCID50, MOI, GTU and PFU, comprises the following steps:
the adenovirus pAd-M3C is amplified in a large quantity by using a tool cell 293A;
(II) purifying the adenovirus harvested after amplification by using a concentration kit;
and (III) performing titer detection on the purified virus solution: diluting the purified adenovirus, infecting cells in vitro by using adenovirus solutions with different dilutions, culturing the cells, and calculating MOI, GTU and TCID50 values;
and (IV) uniformly converting the PFU.
Performing titer detection on the purified virus solution in the step (three), wherein the titer detection method comprises the following steps:
(1) 293A cells were plated in 6, 12, 96-well plates at 37 ℃ with 5% CO according to the following counting requirements2Culturing the cells in a cell culture box overnight;
1) preparation of at least 3X 10 with DMEM 10wt% FBS Medium7The cells of (1), prepared with DMEM 2wt% FBS medium 105At least 20 ml of cells per ml;
2) sample adding: 6. 12, 96-well plates 293A cells were added per well as per the following requirements and 1 plate was repeated:
6, a pore plate: 1X 106Cells/3 ml Medium MEM10% FBS
12 orifice plate: 5X 105 Cells/1.5 ml Medium MEM10% FBS
96-well plate: 1X 10 additions per well4Cells/100 μ l;
(2) diluting the purified adenovirus solution:
1) DMEM 2% FBS medium was added: adding 0.09ml into a tube, 0.9ml into a tube, 0.36ml into tubes from the third to the fifth, and 2.7ml into the rest tubes;
2) adding a purified adenovirus solution: adding 0.01ml of virus solution into the tube, sucking up and down for 5 times, and mixing uniformly;
3) dilution by multiple times: changing a new tip, sucking 0.1ml from the first tube, adding into the second tube, sucking up and down, beating for 5 times, and mixing uniformly; and (4) replacing a new tip, sucking 0.04ml from the second tube, adding into the third tube, sucking and beating for 5 times up and down for uniformly mixing, and diluting the tubes for the fourth to the fifth times. Changing a new tip, sucking 0.3ml from a fifth tube, adding into a sixth tube, sucking and beating for 5 times, uniformly mixing, diluting the seventh to the ninth tubes for the same time, and repeatedly diluting to the highest dilution degree; the second 0- ⑬ tubes correspond to 10 tubes respectively-1—10-13Diluting;
(3) in vitro infection was performed by adding the diluted virus solution of table 1 to 293A cells:
1) second tube 10 was taken from the 6-well plate in which 293A cells were cultured-2Adding 0, 2, 5, 10, 25 and 50 mu l of diluted virus solution into 6 holes respectively;
2) adding the second tube 10 to the 12-well plate cultured with 293A cells-2Dilution and third tube 10-3Diluting virus solution, and adding 0, 2, 5, 10, 25 and 50 mu l into 6 wells of each dilution;
3) adding the last 8 dilutions into a 96-well plate cultured with 293A cells, and placing the tube 10 to the tube ⑬-6—10-13Adding the diluted virus solution into a 96-well plate according to 100 mu l of each well, wherein 10, 11 and 12 wells of each dilution are negative controls; cell survival was monitored by adding 100 μ l DMEM 2% FBS to negative control wells;
（4）37℃，5%CO2cell culture box culture and observation:
1) MOI: culturing for 24 hours, observing under a microscope, and recording the minimum virus solution sample adding volume in the hole with all the cells floating;
2) GTU: culturing for 24-48 hours, and observing and counting the number of fluorescent cells under a fluorescent microscope;
3) TCID 50: culturing for 10 days, observing under an inverted microscope, and calculating the number of holes with CPE in each row; calculating the number of positive wells in each row;
and (5) uniformly converting the PFU in the step (IV), wherein the conversion method is as follows:
(1) MOI result conversion: the calculation is calculated with reference to MOI =10, i.e. 1 cell is infected with 10 viruses, and the value of the viral titer PFU/ml is scaled, if 5 μ Ι wells start drifting all over, then the viral titer is: 10X 5X 105/5μl =1×106 PFU/μl；
(2) And (3) converting a GTU result: viral titration = number of fluorescent cells × 109PFU/ml, mean values obtained from replicate infection samples;
(3) TCID50 results calculation and conversion:
1) TCID50 results calculation: TCID50 titer was T =10 for 100ul virus dilution1+d(s-0.5)D = log10 dilution, s = sum of positive ratios, positive ratio being the ratio CPE positive experimental well/10 experimental wells;
2) conversion of TCID50/ml to PFU/ml:
the formula I is as follows: 1X 10N TCID50 = 1×10( N - 0.7) PFU/ml, where N is an index of the TCID50 value T;
or the formula two: TCID50 × 0.7= PFU/ml.
The method is applied to adenovirus titer detection.
The invention has the beneficial effects that:
three detection methods of TCID50, MOI and GTU are integrated, after the operation steps of the improved and refined experiment are improved, the titer of the adenovirus pAd-M3C is detected, and then conversion and comparison are carried out through PFU, namely the TMGP method for short, so that the repeatability, stability, sensitivity, usability and time consumption can be optimized and improved compared with the original single method, the effectiveness of the adenovirus titer detection is improved, and a foundation is laid for the standard operation of the subsequent experiment.
The TMGP method integrates three detection methods of TCID50, MOI and GTU, improves and refines the operation steps of the experiment, is beneficial to the comparison of subsequent results and the standardized operation in the experiment, reduces errors and improves the reliability and stability of the results. The TMGP method is used for detecting the titer of the pAd-M3C adenovirus for three times, so that the time consumption is greatly shortened, the MOI value of the titer of the virus can be estimated within 24-48 hours, the GTU data can be verified within 36-48 hours, and the accurate titer of the TCID50 on the most critical day 10 can be compared before and after, repeated experiments are reduced, the experiment cost is reduced, and the effectiveness is improved.
The three detection methods of TCID50, MOI and GTU are converted and compared through PFU, and are unified by conversion of a formula, so that the reliability of horizontal and vertical comparison of virus titer detection is accurately and quickly improved.
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FIG. 1 is a technical scheme of the present application.
Detailed Description
The present invention will be further described with reference to examples, which will help to better understand the present invention, but the present invention is not limited to only the following examples.
Example 1 method for comprehensive assessment of adenovirus pAd-M3C Titers by TCID50, MOI, GTU in combination with PFU
One large scale amplification of adenovirus pAd-M3C (purchased from Addgene) was performed using tool cell 293A.
(II) adenovirus harvested after amplification was purified using concentration kit (Vivapure AdenoPACK ™ SARTORIUS (Sadoris, cat # VS-AVPQ 022)).
And (III) performing titer detection on the purified virus solution:
(1) 293A cells were plated in 6, 12, 96-well plates at 37 ℃ with 5% CO according to the following counting requirements2The cell culture box was cultured overnight.
1) Preparation of at least 3X 10 with DMEM 10wt% FBS Medium7The cells of (1), prepared with DMEM 2wt% FBS medium 105At least 20 ml of cells per ml.
2) Sample adding: 6. 12, 96-well plates 293A cells were added per well as per the following requirements and 1 plate was repeated:
6, a pore plate: 1X 106Cells/3 ml medium MEM10% FBS;
12 orifice plate: 5X 105Cells/1.5 ml medium MEM10% FBS;
96-well plate: 1X 10 additions per well4Cells/100. mu.l.
(2) The purified adenovirus solution was diluted according to the following ratio in table 1:
1) DMEM 2% FBS medium was added: adding 0.09ml into the first tube, 0.9ml into the second tube, 0.36ml into the third to fifth tubes, and 2.7ml into the rest tubes.
2) Adding a purified adenovirus solution: 0.01ml of virus solution is added into the first tube, and the mixture is sucked up and down for 5 times and mixed evenly.
3) Dilution by multiple times: changing a new tip, sucking 0.1ml from the first tube, adding into the second tube, sucking up and down, beating for 5 times, and mixing uniformly; and (4) replacing a new tip, sucking 0.04ml from the second tube, adding into the third tube, sucking and beating for 5 times up and down for uniformly mixing, and diluting the tubes for the fourth to the fifth times. And (3) replacing a new tip, sucking 0.3ml of the new tip from the fifth tube, adding the new tip into the sixth tube, sucking and beating the new tip for 5 times up and down, uniformly mixing, diluting the tubes from the seventh tube to the sixth tube by the seventh tube to the seventh tube by the seventh tube to the ⑬ tubes, and repeatedly diluting the tubes to the highest dilution degree.
4) The same tube adenovirus solution was used for 2 nd dilution and the 96-well plate was repeated.
TABLE 1
(3) In vitro infection was performed by adding the diluted virus solution of Table 1 to 293A cells.
6, a pore plate: take the second tube 10-2Each well was added in the following volume (μ l)
12 orifice plate: take the second tube 10-2Third pipe 10-3Each well was added in the following volume (μ l)
96-well plate: and adding the final 8 dilutions and tubes of sixth to ⑬ into a 96-well plate according to 100 mu l of each well, wherein 10 wells, 11 wells and 12 wells of each dilution are negative controls. Cell survival was monitored by adding 100 μ l DMEM 2% FBS to negative control wells. The sample was loaded starting from the highest dilution.
V: virus diluent D: negative control: DMEM 2% FBS medium
（4）37℃，5%CO2Cell culture box culture and observation:
1) MOI: after 24 hours incubation, microscopic observation, wells with all cells floating, and minimal viral fluid loading volume was recorded.
2) GTU: culturing for 24-48 hours, and observing and counting the number of the fluorescent cells under a fluorescent microscope.
3) TCID 50: on the 10 th day of culture, observation was performed under an inverted microscope, and the number of wells in each row in which CPE (cytopathic effect) appeared was counted. If positive, a small spot or some cells showing CPE, if undetermined, can be compared to the negative control. The number of positive wells in each row was counted. The test is valid if the negative control has no CPE and the cells grow well, the lowest dilution is 100% positive and the highest dilution is 100% negative.
And (IV) uniformly converting PFU:
MOI result conversion: the calculation is calculated with reference to MOI =10, i.e. 1 cell is infected with 10 viruses, and the value of the viral titer PFU/ml is scaled, if 5 μ Ι wells start drifting all over, then the viral titer is: 10X 5X 105/5μl =1×106 PFU/μl。
GTU result conversion: virus titration (PFU/ml) = fluorescent cell number × 109. Mean values were obtained from replicate infection samples.
TCID50 result calculation and conversion: as in the following examples, the appearance of a positive well is designated "+" and the appearance of a negative well is designated "-".
1) The lowest dilution at which 100% positivity occurs was -710The highest dilution at which 100% negativity occurred was -910。
2)100ul of viral stock titer T1: accurately calculating by adopting a KARBER statistical method: for 100ul of virus stock, 10-fold gradient dilutions were made with titers of: t =101+d ( s-0.5 )Wherein d = log10[ degree of dilution]=1 (for 10-fold dilution, d = log)1010= 1; d = log3[ dilution degree ] if 3-fold gradient dilution is adopted]) S = sum of positive ratios (10 dilution 10 from 1 st fold)-1Start) =1+1+1+1+1+1+1+0.4=7.4. Although some low dilutions are omitted from the assay (e.g., 10)-1And 10-2) But should still be calculated with a positive ratio10:10= 1.
3) Therefore, the titer T in 100ul of virus stock solution1= 1+1×(7.4-0.5)10= 1×107.9Diluting 100ul of virus stock solution to -7.910After the gradient (i.e. dilution) 7.910After doubling) the amount of virus contained in 100ul was taken as 1TCID 50.
4) Titer T in 1ml of viral stock2= T1×10=1×108.9TCID50/ml。
5) Conversion of TCID50/ml to PFU/ml:
the formula I is as follows: 1X 10N TCID50 = 1×10( N - 0.7) PFU/ml, (N is an index of the value of TCID 50)
1X 10 in the above example8.9TCID50/ml=1×10( 8.9 - 0.7) PFU /ml=1×108.2PFU /ml
The formula II is as follows: TCID50 × 0.7= PFU/ml
1X 10 in the above example8.9TCID50/ml=0.7×108.9 PFU/ml=7×107.9 PFU/ml
Formula one from AdEasyTMThe manual of operations "titer d = log10 values according to TCID50 were 0.7 higher than the standard plaque method. Equation two is from the ATCC interpretation "multiplex the TCID50 titer (per ml) by 0.7 to predict the mean number of PFU/ml"
6) The titer values obtained from two repeated experiments should differ by less than or equal to 0.7 log10 titer values (10)0.7）。
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (4)
1. A method for comprehensively evaluating titer of adenovirus pAd-M3C by combining TCID50, MOI, GTU and PFU, which comprises the following steps:
firstly, amplifying the adenovirus pAd-M3C by using a tool cell 293A;
(II) purifying the adenovirus harvested after amplification by using a concentration kit;
and (III) performing titer detection on the purified virus solution: diluting the purified adenovirus, infecting cells in vitro by using adenovirus solutions with different dilutions, culturing the cells, and calculating MOI, GTU and TCID50 values;
and (IV) uniformly converting the PFU.
2. The method of claim 1, wherein: performing titer detection on the purified virus solution in the step (three), wherein the method comprises the following steps:
(1) 293A cells were plated in 6, 12, 96-well plates at 37 ℃ with 5% CO according to the following counting requirements2Culturing the cells in a cell culture box overnight;
preparation of at least 3X 10 with DMEM 10wt% FBS Medium7The cells of (1), prepared with DMEM 2wt% FBS medium 105At least 20 ml of cells per ml;
sample adding: 6. 12, 96-well plates 293A cells were added per well as per the following requirements and 1 plate was repeated:
6, a pore plate: 1X 106Cells/3 ml medium MEM10% FBS;
12 orifice plate: 5X 105Cells/1.5 ml medium MEM10% FBS;
96-well plate: 1X 10 additions per well4Cells/100 μ l;
(2) diluting the purified adenovirus solution:
DMEM 2% FBS medium was added: adding 0.09ml into a tube, 0.9ml into a tube, 0.36ml into tubes from the third to the fifth, and 2.7ml into the rest tubes;
adding a purified adenovirus solution: adding 0.01ml of virus solution into the tube, sucking up and down for 5 times, and mixing uniformly;
dilution by multiple times: changing a new tip, sucking 0.1ml from the first tube, adding into the second tube, sucking up and down, beating for 5 times, and mixing uniformly; changing a new tip, sucking 0.04ml from a second tube, adding into a third tube, sucking and beating for 5 times up and down for uniformly mixing, and diluting the tubes for the fourth to fifth times; changing a new tip, sucking 0.3ml from a fifth tube, adding into a sixth tube, sucking and beating for 5 times, uniformly mixing, diluting the seventh to the ninth tubes for the same time, and repeatedly diluting to the highest dilution degree; the second 0- ⑬ tubes correspond to 10 tubes respectively-1—10-13Diluting;
(3) in vitro infection was performed by adding the diluted virus solution of table 1 to 293A cells:
second tube 10 was taken from the 6-well plate in which 293A cells were cultured-2Adding 0, 2, 5, 10, 25 and 50 mu l of diluted virus solution into 6 holes respectively;
adding the second tube 10 to the 12-well plate cultured with 293A cells-2Dilution and third tube 10-3Diluting virus solution, and adding 0, 2, 5, 10, 25 and 50 mu l into 6 wells of each dilution;
adding the last 8 dilutions into a 96-well plate cultured with 293A cells, and placing the tube 10 to the tube ⑬-6—10-13Adding the diluted virus solution into a 96-well plate according to 100 mu l of each well, wherein 10, 11 and 12 wells of each dilution are negative controls; cell survival was monitored by adding 100 μ l DMEM 2% FBS to negative control wells;
（4）37℃，5%CO2cell culture box culture and observation:
MOI: culturing for 24 hours, observing under a microscope, and recording the minimum virus solution sample adding volume in the hole with all the cells floating;
GTU: culturing for 24-48 hours, and observing and counting the number of fluorescent cells under a fluorescent microscope;
TCID 50: culturing for 10 days, observing under an inverted microscope, and calculating the number of holes with CPE in each row; the number of positive wells in each row was counted.
3. The method of claim 1, wherein: and (IV) uniformly converting the PFU, wherein the conversion method is as follows:
(1) MOI result conversion: the calculation is calculated with reference to MOI =10, i.e. 1 cell is infected with 10 viruses, and the value of the viral titer PFU/ml is scaled, if 5 μ Ι wells start drifting all over, then the viral titer is: 10X 5X 105/5μl =1×106 PFU/μl；
(2) And (3) converting a GTU result: viral titration = number of fluorescent cells × 109PFU/ml, mean values obtained from replicate infection samples;
(3) TCID50 results calculation and conversion:
1) TCID50 results calculation: TCID50 titer was T =10 for 100ul virus dilution1+d(s-0.5)D = log10 dilution, s = sum of positive ratios, positive ratio being the ratio CPE positive experimental well/10 experimental wells;
2) conversion of TCID50/ml to PFU/ml:
the formula I is as follows: 1X 10N TCID50 = 1×10( N - 0.7) PFU/ml, where N is an index of the TCID50 value T;
or the formula two: TCID50 × 0.7= PFU/ml.
4. Use of the method of claim 1 for adenovirus titer detection.
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