Full
Outline
To establish real-time PCR methods for the detection of residual SV40LTA&E1A and plasmids in the quality control of CAR-T cell therapy products, as well as the assay of reference plasmid calibration by microfluidic digital PCR technology.
SV40LTA&E1A were detected in one tube at the same time by multiplex TaqMan probe PCR. In view of the fact that it was hard to unify the standard of residue detection because multiple plasmid systems (four or five plasmids) existed in the current lentiviral packaging technologies, primers were designed from the significant sequence homology of replicon of those plasmids. Meanwhile, digital PCR method was validated.
Good specificity and quantitative limits of 2.80×10 copies·μL-1 (SV40LTA) and 2.98×10 copies·μL-1 (E1A) were achieved in the SV40LTA&E1A PCR method. The quantitative limit was 2.80×10 copies·μL-1 for plasmids detection method. The quantitative calculation results of plasmid copy number concentration of SV40LTA and E1A were 2.80×108 copies·μL-1 and 2.98×108 copies·μL-1, corresponding to 1.06×108 copies·μL-1 (SV40LTA) and 1.21×108 copies·μL-1 (E1A) by digital PCR method.
The SV40LTA&E1A and residual plasmid PCR detection methods have good specificity, sensitivity and accuracy. The calibration results of classical absorption method are about twice times larger than those by digital PCR method.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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