TP53-R273H/R273C基因突变数字PCR检测方法研究

doi:10.3969/j.issn.1000-1158.2024.08.22

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摘要 TP53基因是肿瘤的抑制基因,也是癌症中常用的靶向检测基因。R273H和R273C是TP53基因检测中常见的热点突变。为了建立这2个突变的微滴式数字聚合酶链式反应(ddPCR)检测方法,针对突变位点分别设计了2套ddPCR方法,验证了探针的特异性,并对退火温度和引物探针浓度进行了优化。结果显示,所建立的方法具有良好的特异性和重复性,ddPCR法与重量法之间的相关系数为0.9997和0.9996。R273H和R273C的检出限均为0.02%,定量限均为0.05%。这2套方法可用于准确检测TP53-R273H和TP53-R273C基因突变,为临床应用提供支持。

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	张洁洁
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关键词 ：生物计量, TP53;基因突变;R273H, R273C, 核酸检测;数字PCR

Abstract：TP53 gene is the tumor suppressor gene and commonly used as the targeted detection gene in cancer.R273H and R273C are common hot spot mutations in TP53 gene detection.In order to establish a droplet digital polymerase chain reaction (ddPCR) detection method for these two mutations, two sets of ddPCR methods were designed for the mutation sites, and the specificity of the probe was verified.The annealing temperature and primer probe concentration were optimized.The results showed that the established method had good specificity and repeatability, and the correlation coefficients between ddPCR and weight method were 0.9997 and 0.9996,.The limit of detection of R273H and R273C were both 0.02%, and the limit of quantitation were both 0.05%.The methods can be used to accurately detect TP53-R273H and TP53-R273C gene mutations, providing strong support for clinical application.

Key words： biometrology TP53;gene mutation R273H R273C nucleic acid testing;ddPCR

收稿日期: 2023-12-20 发布日期: 2024-09-05

PACS:

TB99

基金资助:中国计量科学研究院基本科研业务费重点领域专项(AKYZD202202)

通讯作者: 董莲华(1982-),河北涞水人,中国计量科学研究院研究员,博士,主要从事生物计量方面的研究。Email:lianhuadong@126.com 杨靖亚(1976-)陕西宝鸡人,上海海洋大学副教授,主要从事药理及分子生物学研究。Email:jyyang@shou.edu.cn E-mail: lianhuadong@126.com

作者简介: 张洁洁(1996- ),河南商丘人,上海海洋大学在读研究生,研究方向为核酸计量。Email:1748175004@qq.com

引用本文:

张洁洁,牛春艳,王霞,杨靖亚,董莲华. TP53-R273H/R273C基因突变数字PCR检测方法研究[J]. 计量学报, 2024, 45(8): 1242-1248.
ZHANG Jiejie,NIU Chunyan,WANG Xia,YANG Jingya,DONG Lianhua. Study on the Detection of TP53-R273H/R273C Gene Mutation by Digital PCR. Acta Metrologica Sinica, 2024, 45(8): 1242-1248.

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［3］	LEVAV-COHEN Y, GOLDBERG Z, TAN K H, et al. The p53-Mdm2 loop: a critical juncture of stress response ［J］. Sub-cellular biochemistry, 2014, 85: 161-186.
［19］	王蕾蕾, 王霞, 邢德纯, 等. PIK3CA基因突变数字PCR参考测量方法研究［J］. 计量学报, 2023, 44(3): 368-376.
［2］	RELLI V, TREROTOLA M, GUERRA E, et al. Abandoning the Notion of Non-Small Cell Lung Cancer ［J］. Trends in molecular medicine, 2019, 25(7): 585-594.
［4］	NISHIKAWA S, IWAKUMA T. Drugs Targeting p53 Mutations with FDA Approval and in Clinical Trials ［J］. Cancers, 2023, 15(2): 429.
［1］	SARODE P, MANSOURI S, KARGER A, et al. Epithelial cell plasticity defines heterogeneity in lung cancer ［J］. Cellular signalling, 2020, 65: 109463.
［6］	SAINI H, CHOUDHARY M, SHARMA H, et al. Chloroquine induces transitory attenuation of proliferation of human lung cancer cells through regulation of mutant P53 and YAP ［J］. Molecular biology reports, 2023, 50(2): 1045-1058.
［8］	XIAO G, LUNDINE D, ANNOR G K, et al. Gain-of-Function Mutant p53 R273H Interacts with Replicating DNA and PARP1 in Breast Cancer ［J］. Cancer research, 2020, 80(3): 394-405.
［10］	ZHOU J, LUO J, LI P, et al. Triptolide promotes degradation of the unfolded gain-of-function Tp53(R175H/Y220C) mutant protein by initiating heat shock protein 70 transcription in non-small cell lung cancer ［J］. Translational lung cancer research, 2022, 11(5): 802-816.
［11］	FU Y, WANG A, ZHOU J, et al. Corrigendum: Advanced NSCLC patients with EGFR T790M harbouring TP53 R273C or KRAS G12V cannot benefit from osimertinib based on a clinical multicentre study by tissue and liquid biopsy ［J］. Front Oncol, 2023, 13: 1236311.
［12］	GARG A, HAZRA J P, SANNIGRAHI M K, et al. Variable Mutations at the p53-R273 Oncogenic Hotspot Position Leads to Altered Properties ［J］. Biophys J, 2020, 118(3): 720-728.
［13］	ZHU G, CAI J, ZHONG H. TP53 signal pathway confers potential therapy target in acute myeloid leukemia ［J］. Eur J Haematol, 2023, 110(5): 480-489.
［9］	LI L, LI X, TANG Y, et al. Common cancer mutations R175H and R273H drive the p53 DNA-binding domain towards aggregation-prone conformations ［J］. Phys Chem Chem Phys, 2020, 22(17): 9225-9232.
［14］	KOJABAD A A, FARZANEHPOUR M, GALEH H E G, et al. Droplet digital PCR of viral DNA/RNA, current progress, challenges, and future perspectives ［J］. J Med Virol, 2021, 93(7): 4182-4197.
［16］	CHEN B, JIANG Y, CAO X, et al. Droplet digital PCR as an emerging tool in detecting pathogens nucleic acids in infectious diseases ［J］. Clinica chimica acta; international journal of clinical chemistry, 2021, 517: 156-161.
	NIU C Y, ZHANG Y Z, YANG J Y, et al. Resrach on the Collaborative Value Assignment of Plasmid Nucleic Acid Refernce Materials Based on Digital PCR ［J］. Acta Metrologica Sinica, 2021, 42(11): 1522-1527.
［18］	SUO T, LIU X, FENG J, et al. ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens ［J］. Emerg Microbes Infect, 2020, 9(1): 1259-1268.
	WANG L L, WANG X, XING D C, et al. Research on Digital PCR Reference Measurement Method of PIK3CA Gene Mutation ［J］. Acta Metrologica Sinica, 2023, 44(3): 368-376.
［20］	IFCC. Protocols for Determination of Limits of Detection and LImits of Quantitation;Approved Guideline. NCCLS document EP17-A : ISBN 1-56238-551-8 ［S］. USA : NCCLS, 2004.
［22］	VADDAVALLIP L, SCHUMACHER B. The p53 network: cellular and systemic DNA damage responses in cancer and aging ［J］. Trends in genetics : TIG, 2022, 38(6): 598-612.
［7］	LV T, LV H, FEI J, et al. p53-R273H promotes cancer cell migration via upregulation of neuraminidase-1 ［J］. J Cancer, 2020, 11(23): 6874-6882.
［17］	牛春艳, 张永卓, 杨佳怡, 等. 基于数字PCR的质粒核酸标准物质合作定值研究［J］. 计量学报, 2021, 42(11): 1522-1527.
［5］	SALEH M M, SCHEFFLER M, MERKELBACH-BRUSE S, et al. Comprehensive Analysis of TP53 and KEAP1 Mutations and Their Impact on Survival in Localized- and Advanced-Stage NSCLC ［J］. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer, 2022, 17(1): 76-88.
［15］	OLMEDILLAS-LOPEZ S, OLIVERA-SALAZAR R, GARCIA-ARRANZ M, et al. Current and Emerging Applications of Droplet Digital PCR in Oncology: An Updated Review ［J］. Mol Diagn Ther, 2022, 26(1): 61-87.
［23］	PARK B, IM J, HAN K. Comparative Analysis of Gene Correlation Networks of Breast Cancer Patients Based on Mutations in TP53 ［J］. Biomolecules, 2022, 12(7): 979.
［21］	BARBOSA K, LI S, ADAMS P D, et al. The role of TP53 in acute myeloid leukemia: Challenges and opportunities ［J］. Genes Chromosomes Cancer, 2019, 58(12): 875-888.