建立数字PCR检测非小细胞肺癌溶酶体相关的4次跨膜蛋白B基因拷贝数变异方法  

Establishment of Digital PCR Detection Method for Copy Number Variation of LAPTM4B Gene in Non-small Cell Lung Cancer

作　　者：王鲁[1] 徐国兵[1] 张青云[1] WANG Lu;XU Guo-Bin;ZHANG Qing-Yun(Department of Clinical Laboratory,Peking University Cancer Hospital,Beijing Institute for Cancer Research,Key Laboratory of the Ministry of Education for Malignant Tumor Pathogenesis and Translational Research,Beijing 100142,China)

机构地区：[1]北京大学肿瘤医院暨北京市肿瘤防治研究所检验科,恶性肿瘤发病机制及转化研究教育部重点实验室,北京100142

出　　处：《中国生物化学与分子生物学报》2023年第9期1356-1364,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：北京大学肿瘤医院科学研究基金(No.2020自主-19)资助。

摘　　要：建立芯片式数字PCR(dPCR)检测非小细胞肺癌(NSCLC)LAPTM4B基因拷贝数变异方法,并初步评估其基本性能和临床应用的可行性。设计LAPTM4B基因引物及特异性探针,建立dPCR反应体系,根据制备的不同浓度目的DNA样品验证该检测方法的最低检测限、精密度和线性范围。本研究首次建立并优化dPCR检测LAPTM4B基因拷贝数的反应体系,分析数据显示,该方法最低检测到12.5%的LAPTM4B基因拷贝数缺失,批间精密度变异系数CV<10%,且缺失比例在12.5%~100%范围内线性良好(R^(2)>0.99)。此外,收集2021年3月至7月于北京大学肿瘤医院就诊的6例NSCLC患者,采用自建方法检测其冰冻组织标本,探究其临床应用可行性。dPCR结果显示,其中5例患者的组织样本存在LAPTM4B基因拷贝数缺失,1例出现拷贝数增加,推测可能与其术前化疗有关。综上所述,本研究成功应用芯片式dPCR方法,建立非小细胞肺癌LAPTM4B基因拷贝数变异的检测体系,并在组织标本中初步证实其临床应用的价值。We aim to establish a chip-based digital PCR(dPCR)method for detecting copy number variation of the LAPTM4B gene in non-small cell lung cancer(NSCLC),and preliminarily evaluate its basic performance and clinical feasibility.The LAPTM4B gene primers and specific probes were designed to establish a dPCR reaction system.The detection limit,precision,and linearity of the method were verified according to the prepared target DNA samples of different concentrations.The reaction system of dPCR for LAPTM4B gene copy number detection was established and optimized for the first time.The results showed that 12.5%of LAPTM4B gene copy number deletion could be detected at the lowest level.The coefficient of variation of inter-batch precision was less than 10%,and the linearity of deletion ratio was good in the range of 12.5%-100%(R2>0.99).In addition,the copy numbers of LAPTM4B gene in frozen tissues from 6 NSCLC patients who were enrolled in Peking University Cancer Hospital from March to July 2021 were analyzed by the self-built dPCR method to explore the feasibility of this system.The dPCR results showed that there were deletions of LAPTM4B gene copy number in 5 cases and an increase in 1 case,which may be related to preoperative chemotherapy.In conclusion,this study successfully established a system to detect the copy number variation of LAPTM4B gene in non-small cell lung cancer by chip-based dPCR and preliminarily confirmed its clinical application value in tissue samples.

关 键 词：非小细胞肺癌 LAPTM4B 拷贝数变异 芯片式数字PCR 

分 类 号：R734.2[医药卫生—肿瘤]