The application belongs to the technical field of molecular diagnosis, and particularly relates to a digital PCR kit and method for accurately detecting the copy number of a CAR
gene of a CAR-
T cell and a
lentivirus vector. The application first realizes the
absolute quantification of CAR, Rev and an internal
reference gene RNase P in a
single tube simultaneously, and has strong universality. Based on the principle of digital PCR, the absolute copy number is directly obtained without a
standard curve, and the result is accurate and highly repeatable. The detection
lower limit reaches 0.01%, and extremely low abundance CAR-T residues or microresidual diseases can be detected. Primers and probes are designed for the conservative regions of CD3zeta, Rev and
RNase P, and no cross reaction is verified, so that false positives are effectively avoided. The sample only needs gDNA, and there is no requirement for
cell activity, and the sample is compatible with freezing and transportation. The kit is premixed and optimized, the process is automated, and 4-5 hours are needed. The kit is suitable for the whole chain of CAR-T
drug research and development, production
process quality control, clinical patient monitoring and safety evaluation.