双重数字PCR法评价Luc2P系列报告基因细胞系的稳定性

Evaluation of stability of Luc2P reporter cell lines by duplex digital PCR

目的 建立双重数字PCR(digital PCR,dPCR)法评价Luc2P系列报告基因细胞系(CHO-K1、Hacat、HEK293和UT7)的萤光素酶(luciferase,Luc)基因稳定性。方法 提取Luc2P系列报告基因细胞系的基因组DNA,建立一种双重dPCR法,同时检测内参基因RPP30和目标基因Luc的拷贝数,以Luc相对拷贝数(copies Luc/copy RPP30)为指标评价Luc基因稳定性;参考《中国药典》三部(2020版)相关要求对建立的方法进行专属性、精密性、线性、准确性和耐用性验证,并分析方法的适用性。结果 未转染报告基因的原始细胞检测结果均为阴性,而4种报告基因细胞系中均可检测到阳性结果,且dPCR结果中阴性和阳性区可明显区分;以相对拷贝数为指标,采用芯片式dPCR法重复检测8次同一基因组DNA样品以及同一细胞6次独立提取的基因组DNA样品的相对标准偏差(RSD)均小于10%,Luc和RPP30的线性拟合R~2均大于0.99,建立的方法检测5组加标样本的加标回收率在50%~100%之间,且芯片式dPCR和液滴式dPCR检测结果一致。该方法可区分不同细胞克隆的Luc相对拷贝数,检测3个代次(P8、P12、P31)细胞Luc相对拷贝数的结果高度一致。结论 建立的双重dPCR法专属性、精密性、线性、准确性、耐用性和适用性良好,可用于Luc2P系列报告基因细胞系的Luc基因稳定性评价。

Objective To develop a duplex digital PCR(dPCR) for evaluation of the stability of luciferase(Luc) gene in Luc2P reporter cell lines(CHO-K1,Hacat,HEK293 and UT7).Methods Genomic DNAs of Luc2P reporter cell lines were extracted,a duplex dPCR was developed to determine the copies of reference gene RPP30 and the target gene Luc,and the relative copy number of Luc(copies Luc/copy RPP30) was employed as the indicator for the stability evaluation of Luc gene;The developed method was verified for the specificity,precision,linearity,accuracy and durability,and analyzed for the applicability,according to the related requirements in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition).Results All the original cells without reporter gene transfection were negative,while all the four reporter cell lines were positive,and the negative and positive regions in dPCR results were clearly distinguished;The relative standard deviation(RSD) of the eight repeated detections of the same genomic DNA sample and the six independent extractions of genomic DNA sample of the same cell were all less than 10%,and the linear fitting R~2 values were more than 0.99 for both Luc and RPP30.The spike recoveries of five groups of samples detected by the developed method were between 50% and 100%,and the results of chip-type dPCR and droplet-type dPCR were consistent.This method distinguished the relative copy number of Luc in different cell clones,and the results of detecting the relative copy number of Luc in three passages(P8,P12 and P31) were highly consistent.Conclusion The developed duplex dPCR method has good specificity,precision,linearity,accuracy,durability and applicability,and might be used to evaluate the stability of Luc gene in Luc2P reporter cell lines.