Yili Xu*, Zhen Sun, Xin Li, Xiaodong Yin, Guowei Zhang
Hangzhou Qingxin Gene Co., Ltd., Hangzhou 310000, Zhejiang, China.
*Corresponding author: Yili Xu
Abstract
Cell therapy has emerged as a novel therapeutic approach, demonstrating significant advancements in the treatment of various diseases, particularly those that are refractory to conventional therapies. This field has rapidly gained prominence in global research. However, the genetic modification of clinical-grade cells presents challenges, particularly the need to avoid screening tags such as resistance and fluorescent markers, which complicate the monoclonal screening process and increase the associated workload. In this study, we introduce an improved quantitative polymerase chain reaction (qPCR) screening method designed to expedite the identification of positive monoclonal clones that have undergone gene editing. By incorporating multiplex fluorescence coding alongside optimized primer design, this approach enables the simultaneous detection of edited loci and endogenous controls within a single reaction. The use of programmable cycle threshold (Ct) delays and low-dimer primer panels guarantees 100% specificity and sensitivity, while effectively minimizing the incidence of false positives resulting from primer-dimer artifacts. This workflow is compatible with standard qPCR instruments, providing a cost-effective alternative to next-generation sequencing (NGS) or digital PCR for the large-scale validation of clones.
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How to cite this paper
An Improved qPCR Strategy for Fast Screening of Positive Monoclonal Genes for Gene Editing
How to cite this paper: Yili Xu, Zhen Sun, Xin Li, Xiaodong Yin, Guowei Zhang. (2025) An Improved qPCR Strategy for Fast Screening of Positive Monoclonal Genes for Gene Editing. International Journal of Clinical and Experimental Medicine Research, 9(3), 353-357.
DOI: http://dx.doi.org/10.26855/ijcemr.2025.05.019