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Method for comprehensively evaluating adenovirus pAd-M3C titer by combining TCID50, MOI, GTU and PFU

A comprehensive evaluation and virus titer technology, applied in the field of molecular biology and cellular immunity, can solve the problems of no clear standards for mutual conversion, achieve the effect of reducing experimental costs, improving reliability and stability, and reducing repeated experiments

Active Publication Date: 2022-03-22
FUJIAN ACAD OF MEDICAL SCI
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Problems solved by technology

However, in scientific research exchange, literature reference, and project research, these methods often need to be compared and referenced, and there is no clear standard for the conversion between several main methods. If various evaluation methods can be used comprehensively to form a Standard experimental operation, fast and concise conversion, improve the effectiveness of titer evaluation, and lay the foundation for the quantification and standardization of subsequent experiments

Method used

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  • Method for comprehensively evaluating adenovirus pAd-M3C titer by combining TCID50, MOI, GTU and PFU
  • Method for comprehensively evaluating adenovirus pAd-M3C titer by combining TCID50, MOI, GTU and PFU
  • Method for comprehensively evaluating adenovirus pAd-M3C titer by combining TCID50, MOI, GTU and PFU

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Embodiment 1

[0050] Example 1 TCID50, MOI, GTU combined with PFU method for comprehensive evaluation of adenovirus pAd-M3C titer

[0051] (1) The adenovirus pAd-M3C (purchased from Addgene) was massively amplified using the tool cell 293A.

[0052] (2) The adenovirus harvested after amplification was purified using a concentration kit (Vivapure®AdenoPACK™ SARTORIUS (Sartorius, Cat. No. VS-AVPQ 022)).

[0053] (3) Carry out titer detection to the purified virus solution:

[0054] (1) 293A cells were seeded in 6-, 12-, and 96-well plates according to the following counting requirements, at 37°C, 5% CO 2 The cells were cultured overnight in an incubator.

[0055] 1) Use DMEM 10wt% FBS medium to prepare at least 3×10 7 The cells were prepared with DMEM 2wt% FBS medium for 10 5 cells / ml at least 20 ml.

[0056] 2) Adding samples: Add 293A cells to each well of 6, 12, and 96-well plates according to the following requirements, and repeat for 1 plate:

[0057] 6-well plate: 1×10 6 cells / 3 ...

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Abstract

The invention provides a method for comprehensively evaluating the titer of adenovirus pAd-M3C by combining TCID50, MOI, GTU and PFU. Three detection methods of TCID50, MOI and GTU are integrated, after experimental operation steps are improved and refined, the adenovirus pAd-M3C titer is detected at the same time, conversion and comparison are carried out through PFU, and the TMGP method is called for short, so that an original single method can be optimized and improved in repeatability, stability, sensitivity, usability and time consumption, the effectiveness of adenovirus titer detection is improved, and the method is suitable for popularization and application. And a foundation is laid for subsequent experimental standard operation. The TMGP method can be used for estimating the MOI value of the virus titer within 24-48 hours, verifying GTU data within 36-48 hours and comparing the accurate titer of TCID50 on the most critical tenth day, so that repeated experiments are reduced, the experiment cost is reduced, and the effectiveness is improved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology cell immunity. It specifically relates to a method for comprehensively evaluating the titer of adenovirus pAd-M3C by combining TCID50, MOI, and GTU with PFU. Background technique [0002] Currently, virus titer determination methods mainly include two categories: physical methods (VP) and biological methods (GTU, PFU, TCID50 and MOI). [0003] The method of measuring VP ((Virus Particles) or OPU (Optical Particle Unit, Optical ParticleUnit)) is to measure the absorbance of virus particles at 260nm (the total absorbance of viral DNA and protein is mainly DNA), 1 OD value Equivalent to 1.1×10 12 virus particles. Assays performed by this method are relatively stable across laboratories, but it does not distinguish between infectious and defective virus particles. Therefore, this method can only provide the amount of virus, as for the quality, such as whether it contains defective partic...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/06G01N15/10C12R1/93
CPCC12Q1/06G01N15/10G01N2333/075G01N2015/1006G01N2015/1022G01N2015/1024
Inventor 王坤韩俊永陈金烟金静君薛士杰
Owner FUJIAN ACAD OF MEDICAL SCI
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