Universal method for detecting copy number of target gene and titer of virus and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to a universal method for detecting the copy number of a target gene, a universal method for detecting the titer of alentivirus, application of the universal method, a PCBP2 gene detection kit and a universal kit for detecting the copy number of the target gene and the titer of the lentivirus. A PCBP2 gene is adopted as a calibration factor, and the nucleotide sequence of a PCBP2 gene template is shown as SEQ ID: NO 1 or SEQ ID: NO 2. The PCBP2 gene solves the problems that a traditional method can only detect ahuman sample and has universality; the traditional method for detecting the titer of a virus is poor in coincidence with a method for detecting the titer of the virus through a flow cytometry, and the universality of detecting the titer of the virus in practical clinical application is not high; and the PCBP2 gene is adopted as the calibration gene, the result stability is good, detection of thetiter of the lentivirus is more accurate, the detection result of the lentivirus is more coincident with the detection result of the flow cytometry, and clinical application is facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a general method for detecting the copy number of a target gene, a general method for detecting lentivirus titer and its application, a PCBP2 gene detection kit, and a general method for detecting the copy number of a target gene and virus titer. Reagent test kit. Background technique [0002] Lentiviral vectors have been widely used in the research of animal model gene therapy and the preparation of transgenic animals, and the accurate determination of the titer and infection efficiency of recombinant lentivirus is the key step, because the transduction efficiency of viral vectors mainly depends on the viral titer , which makes the detection of virus titer particularly important. [0003] Commonly used methods for virus titer or copy number detection are ELISA, Q-PCR, flow cytometry, RNA-Dot-blot, TCID50 assay and Real-time PCR (DNA quantitative detection method), wherei...

AI Technical Summary

Problems solved by technology

However, the workload of flow cytometry detection is large and will be affected by antibody affinity, batches, etc., resulting in poor repeatability and large error values ​​for detected virus titers. At the same time, for high-titer viruses, virus titers will be underestimated. Multiple conditions have been explored, and it is only applicable to the situation where the expressed gene can be detected by flow cytometry; while the titer detection is carried out by detecting the number of vector DNA copies after the infected cells are quantified by the DNA quantitative method based on RT-PCR, and the results are more stable and the most accurate. Close to the real infection titer, the current RT-PCR method based on gene therapy virus titer or copy number detection is more researched on the detection of human cells or human samples, but there is little research on non-human animal samples such as mouse samples. few

In the preclinical screening of gene therapy or modified cell therapy, animal experiments are required to evaluate the maintenance and residual status of viral vectors such as gene vectors, CAR expression vectors, or TCR-T expression vectors in vivo. It does not exist in non-human genomes such as mice, so it can only be detected by the method without internal reference, but the method without internal reference has poor stability and accuracy

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