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Research on Digital PCR Reference Measurement Method of PIK3CA Gene Mutation |
WANG Lei-lei1,2,WANG Xia2,XING De-chun1,DONG Lian-hua2,YANG Jing-ya1 |
1. College of Food Sciences & Technology, Shanghai Ocean University, Shanghai 201306, China
2. Frontier Metrology Science Center, National Institute of Metrology, Beijing 100029, China |
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Abstract PIK3CA mutation detection is an important predictor for realizing individualized treatment of tumors. Therefore, using PIK3CA as the target gene, three hotspot mutations, E542K, E545K, and H1047R, were selected to establish a droplet digital polymerase chain reaction (ddPCR) method for accurate and quantitative detection. The annealing temperature, primer probe concentration and other conditions were optimized, and the specificity, linear range and repeatability of the method were investigated. The results show that the established ddPCR detection method has good precision, the relative standard deviation value is between 0.392%~14.031% in the abundance range of 0.05%~82.75%, and the accuracy is high. The correlation coefficient with gravimetric method reach 99.91%, 99.98%, 99.94%, when the mutation abundance is above 0.2%, the repeatability of the method can reach within 5%, and when the mutation abundance is below 0.2%, the repeatability remains below 15%. The limit of blank of the three hot spot mutations in the 20μL reaction system are 0.03%, 0.04%, 0.04%, and the lower limit of detection and lower limit of quantification are both 0.05%. Therefore, the ddPCR reference measurement of PIK3CA gene E542K, E545K and H1047R mutations has high sensitivity and good repeatability, and has good applications in cancer diagnosis, individualized medication guidance and prognosis.
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Received: 02 February 2021
Published: 08 March 2023
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|
|
[9] |
Han G Y, Xu Z, Li Q S, et al. Detection of hereditary hearing loss gene by DNA microarray [J]. Eur Rev Med Pharmacol Sci, 2017, 21: 3538-3542.
|
[1] |
Myers M B, Banda M, Mckim K L, et al. Breast Cancer heterogeneity examined by high-sensitivity quantification of PIK3CA, KRAS, HRAS, and BRAF mutations in normal breast and ductal carcinomas [J]. Neoplasia, 2016, 18 (4): 253-263.
|
[2] |
Madsen R R, Vanhaesebroeck B, Semple R K. Cancer-associated PIK3CA mutations in overgrowth disorders [J]. Trends in molecular medicine, 2018, 24 (10): 856-870.
|
[6] |
Kodahl A R, Ehmsen S, Pallisgaard N, et al. Correlation between circulating cell-free PIK 3 CA tumor DNA levels and treatment response in patients with PIK 3 CA-mutated metastatic breast Cancer [J]. Molecular Oncology, 2018, 12 (6): 925-935.
|
[8] |
Kim T E, Jung E S, Jung C K, et al. DHPLC is a highly sensitive and rapid screening method to detect BRAFV600E mutation in papillary thyroid carcinoma [J]. Experimental and Molecular Pathology, 2013, 94 (1): 203-209.
|
[11] |
Han N, Cheng Q Y, Chen B, et al. PIK3CA mutations in resected small cell lung Cancer [J]. Advances in Clinical and Experimental Medicine, 2016, 25 (3): 397-402.
|
|
Dong L H, Wang J, Fu B Q, et al. Comparability study of digital PCR and next-generation sequencing methods used in KRAS gene mutation detection [J]. Acta Metrologica Sinica, 2018, 39 (3): 436-441.
|
[16] |
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.
|
[15] |
李慧调, 潘建章. 数字PCR技术的发展及应用 [J]. 化学进展, 2020, 32 (5): 581-593.
|
[36] |
李达, 王军, 杨忠, 等. 人类EGFR基因突变核酸标准物质的研制 [J]. 中国医学装备, 2020, 17 (2): 33-36.
|
[14] |
马丹, 魏咏新, 李丹, 等. 水产品中创伤弧菌ddPCR定量方法的建立 [J]. 食品科学, 2020, 41 (12): 305-311.
|
|
Li H D, Pan J Z. The development and application of digital PCR technology [J]. Progress in Chemistry, 2020, 32 (5): 581-593.
|
[22] |
刘晓, 朱鹏宇, 王垚, 等. 数字PCR在功能核酸精准检测中的研究进展 [J]. 生物技术通报, 2018, 34 (9): 149-162.
|
[25] |
EP17-A Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline [S].
|
[3] |
Chen L, Yang L, Yao L, et al. Characterization of PIK3CA and PIK3R1 somatic mutations in Chinese breast cancer patients [J]. Nature communications, 2018, 9 (1): 1-17.
|
[4] |
Riccardi A, Lemos C, Ramos R, et al. PIK3CA mutational analysis of parathyroid adenomas [J]. JBMR Plus, 2020, 4 (6): e10360.
|
[7] |
Zheng H L, Wang Y, Tang C N, et al. TP53, PIK3CA, FBXW7 and KRAS mutations in esophageal Cancer identified by targeted sequencing [J]. Cancer Genomics-Proteomics, 2016, 13 (3): 231-238.
|
[12] |
Alowiri N H, Hanafy S M, Haleem R A, et al. PIK3CA and PTEN genes expressions in breast Cancer [J]. Asian Pacific Journal of Cancer Prevention: Apjcp, 2019, 20 (9): 2841.
|
|
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.
|
[17] |
王迪,王志栋,吴枭, 等. SARS-CoV-2基因组RNA标准物质的研制[J]. 计量学报, 2021, 42(2): 259-264.
|
[19] |
张雨. 非小细胞肺癌外周血游离DNA及肿瘤细胞EGFR基因突变检测方法的研究进展 [J]. 中国肺癌杂志, 2016, 19 (11): 766-772.
|
|
Zhang Y. Research progress in detection methods of non-small cell lung cancer peripheral blood free DNA and tumor cell EGFR gene mutations [J]. Chinese Journal of Lung Cancer, 2016, 19 (11): 766-772.
|
[21] |
Vogelstein B, Kinzler K W. Digital PCR [J]. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96 (16): 9236-9241.
|
[23] |
Luks V L, Kamitaki N, Vivero M P, et al. Lymphatic and other vascular malformative/overgrowth disorders are caused by somatic mutations in PIK3CA [J]. The Journal of pediatrics, 2015, 166 (4): 1048-1054. e5.
|
[24] |
刘二龙, 卢丽, 凌莉, 等. 红薯源性成分微滴式数字PCR的检测与定量分析 [J]. 现代食品科技, 2019, 35 (7): 273-277, 28.
|
[26] |
刘铭, 苗娜, 李俊芝, 等. PIK3CA及PDK1在乳腺浸润性导管癌中的表达及临床意义 [J]. 诊断病理学杂志, 2020, 27 (8): 573-577.
|
|
Liu M, Miao N, Li J Z, et al. The expression and clinical significance of PIK3CA and PDK1 in invasive ductal carcinoma of the breast [J]. Journal of Diagnostic Pathology, 2020, 27 (8): 573-577.
|
|
Hao L X, Pan C, Cai J C. PIK3CA gene mutation frequency and hotspot mutation pattern in gastric cancer tissues [J]. Basic Medicine and Clinical Medicine, 2014, 34 (12): 1671-1675.
|
[30] |
黄志雄. 卵巢透明细胞癌PIK3CA基因突变与扩增研究 [D]. 武汉: 武汉科技大学, 2015.
|
[33] |
Nyaruaba R, Mwaliko C, Kering K K, et al. Droplet digital PCR applications in the tuberculosis world[J]. Tuberculosis, 2019, 117: 85-92.
|
[5] |
Alvarez-Garcia V, Bartos C, Keraite I, et al. A simple and robust real-time qPCR method for the detection of PIK3CA mutations [J]. Scientific Reports, 2018, 8 (1): 1-10.
|
[13] |
董莲华, 王晶, 傅博强, 等. 数字PCR和下一代测序方法用于KRAS基因突变检测中的可比性研究 [J]. 计量学报, 2018, 39 (3): 436-441.
|
|
Wang D,Wang Z D,Wu X, et al. Development of Reference Material of SARS-CoV-2 Genomic RNA[J]. Acta Metrologica Sinica, 2021, 42(2): 259-264.
|
|
Liu E L, Lu L, Ling L, et al. Detection and quantitative analysis of sweet potato-derived components by microdrop digital PCR [J]. Modern Food Science and Technology, 2019, 35 (7): 273-277, 28.
|
[31] |
Hui Y, Wu Z, Qin Z, et al. Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus [J]. Virologica Sinica, 2018, 33 (3): 270-277.
|
[32] |
任怡菲, 高琴, 邓婷婷, 等. 基于数字PCR的转基因水稻LL62品系精准定量检测方法建立 [J]. 生物技术通报, 2016, 32 (8): 69-76.
|
[34] |
He L, Simpson D, Gnzle M G. Detection of enterohaemorrhagic Escherichia coli in food by droplet digital PCR to detect simultaneous virulence factors in a single genome [J]. Food Microbiology, 2020, 90: 103466.
|
[10] |
Franken A, Rivandi M, Yang L, et al. A multiplex PCR-Based next Generation Sequencing-Panel to identify mutations for targeted therapy in breast Cancer circulating tumor cells [J]. Applied Sciences, 2020, 10: 3364.
|
|
Ma D, Wei Y X, Li D, et al.Establishment of ddPCR quantitative method for Vibrio vulnificus in aquatic products [J]. Food Science, 2020, 41 (12): 305-311.
|
[20] |
Pinheiro L B, Coleman V A, Hindson C M, et al. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification [J]. Analytical Chemistry, 2012, 84 (2): 1003-1011.
|
[27] |
郝兰香, 潘超, 蔡建春. 胃癌组织中PIK3CA基因突变频率及热点突变模式 [J]. 基础医学与临床, 2014, 34 (12): 1671-1675.
|
[28] |
Milbury C A, Li J, Liu P, et al. COLD-PCR: improving the sensitivity of molecular diagnostics assays [J]. Expert Review of Molecular Diagnostics, 2011, 11 (2): 159-169.
|
[35] |
Michaelidou K, Koutoulaki C, Mavridis K, et al. Detection of KRAS G12/G13 mutations in cell Free-DNA by droplet digital pcr, offers prognostic information for patients with advanced Non-Small cell lung Cancer [J]. Cells, 2020, 9 (11): 2514.
|
|
Li D, Wang J, Yang Z, et al. Development of human EGFR gene mutation nucleic acid reference materials [J]. Chinese Medical Equipment, 2020, 17 (2): 33-36.
|
[18] |
Matsuda K. PCR-based detection methods for single-nucleotide polymorphism or mutation: real-time PCR and its substantial contribution toward technological refinement [J].Advances in clinical chemistry,2017,80: 45-72.
|
|
Liu X, Zhu P Y, Wang Y, et al. Research progress of digital PCR in the precise detection of functional nucleic acids [J]. Biotechnology Bulletin, 2018, 34 (9): 149-162.
|
[29] |
Xiang Z, Wan R, Zou B, et al. Highly sensitive and specific real-time PCR by employing serial invasive reaction as a sequence identifier for quantifying EGFR mutation abundance in cfDNA [J]. Analytical and Bioanalytical Chemistry, 2018, 410 (26): 6751-6759.
|
|
Ren Y F, Gao Q, Deng T T, et al. Establishment of accurate quantitative detection method for genetically modified rice LL62 line based on digital PCR [J]. Biotechnology Bulletin, 2016, 32 (8): 69-76.
|
|
|
|