用于循环肿瘤细胞定量分析的CK19数字PCR检测方法的建立及性能验证

doi:10.16288/j.yczz.22-358

遗传 ›› 2023, Vol. 45 ›› Issue (3): 250-260.doi: 10.16288/j.yczz.22-358

用于循环肿瘤细胞定量分析的CK19数字PCR检测方法的建立及性能验证

马春辉^1,²(), 胡海旭², 张丽娟², 刘毅^1,²(), 刘天懿²

1.安徽医科大学基础医学院，合肥 230032
2.中国人民解放军总医院第五医学中心，北京 100071

收稿日期:2022-11-15 修回日期:2023-01-03 出版日期:2023-03-20 发布日期:2023-01-10
通讯作者: 刘毅 E-mail:mch964409611@163.com;liuyi790114@163.com
作者简介:马春辉，在读硕士研究生(联合培养)，专业方向：病理生理学。E-mail: mch964409611@163.com
基金资助:
国家自然科学基金项目(81672278);国家自然科学基金项目(82241230)

Establishment and verification of a digital PCR assay for the detection of CK19 expression in quantitative analysis of circulating tumor cell

Chunhui Ma^1,²(), Haixu Hu², Lijuan Zhang², Yi Liu^1,²(), Tianyi Liu²

1. School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China
2. The Fifth Medical Center of People′s Liberation Army General Hospital, Beijing 100071, China

Received:2022-11-15 Revised:2023-01-03 Online:2023-03-20 Published:2023-01-10
Contact: Liu Yi E-mail:mch964409611@163.com;liuyi790114@163.com
Supported by:
the National Natural Science Foundation of China(81672278);the National Natural Science Foundation of China(82241230)

摘要/Abstract

摘要：

本研究拟建立检测CK19基因表达的数字PCR(digital PCR，dPCR)方法并测试其性能，以期利用该方法的超高敏感性和绝对定量优势进行循环肿瘤细胞(circulating tumor cells, CTC)的定量分析。首先，根据CK19基因的mRNA序列进行引物探针设计，以看家基因ABL1作为内参，采用人乳腺癌MCF7细胞和健康人白细胞从13套引物探针之中筛选出了最佳的引物探针，测序结果证实扩增序列无误。其次，针对选定的引物探针进行反应条件的优化，以不同浓度的健康人白细胞cDNA为模板进行空白检测限(limit of blank，LOB)的分析，证实当ABL1基因拷贝数为20,000、15,000、10,000、5000、2500时，CK19的LOB分别为9.24、8.93、3.12、3.17和2.53拷贝。再次，采用MCF7细胞和健康人白细胞的cDNA模板进行不同浓度预混的检测限(limit of detection，LOD)分析，证实50%、10%、5%、1%、0.5%和0.1%的浓度配比下均可以有效检出CK19基因，线性R²值为0.9998。最终，临床样本的数字PCR检测结果发现晚期乳腺癌患者的CK19拷贝数高于健康人对照。上述结果说明本研究所建立的检测CK19基因的数字PCR方法具有敏感、特异和定量准确等优势，未来经过进一步验证之后有望用于乳腺癌的CTC定量分析，具有良好的应用前景。

关键词: 循环肿瘤细胞, 细胞角蛋白19, 数字PCR, 乳腺癌, 定量分析

Abstract:

The objective of this study was to establish and verify a digital PCR assay for the detection of CK19 gene expression, and to use it to detect circulating tumor cells (CTC) by taking advantages of its ultra-high sensitivity and absolute quantitation. Firstly, the primers and probes were designed according to the mRNA sequence of CK19 gene, and housekeeping gene ABL1 was used as the internal control. The best candidate was screened by human breast cancer MCF7 cells and healthy human leukocytes from 13 sets of primer and probes and verified by direct sequencing. Secondly, after the reaction conditions of the selected primers and probes were optimized, limit of blank (LOB) analysis were performed with different concentrations of cDNAs as templates from healthy human leukocytes. The results revealed the LOB of CK19 with ABL1 copy numbers of 20,000, 15,000, 10,000, 5000 and 2500 were 9.24, 8.93, 3.12, 3.17 and 2.53 copies, respectively. Thirdly, the different concentrations of cDNAs from MCF7 cells and healthy human leukocytes were premixed and used in the limit of detection (LOD) analysis, which showed that the CK19 gene could be effectively detected at the concentration ratio of 50%, 10%, 5%, 1%, 0.5% and 0.1%, and the linear R² value was 0.9998. Finally, the preliminary results of digital PCR in clinical samples indicated that CK19 copy numbers were higher in advanced breast cancer patients than healthy controls. The above results demonstrated the advantages of our CK19 digital PCR assay in sensitivity, specificity, and accurate quantification. If verified further, the assay is expected to play significant roles in the quantitative analysis of CTC in breast cancer with a good application prospect.

Key words: circulating tumor cells, cytokeratin 19, digital PCR, breast cancer, quantitative analysis

马春辉, 胡海旭, 张丽娟, 刘毅, 刘天懿. 用于循环肿瘤细胞定量分析的CK19数字PCR检测方法的建立及性能验证[J]. 遗传, 2023, 45(3): 250-260.

Chunhui Ma, Haixu Hu, Lijuan Zhang, Yi Liu, Tianyi Liu. Establishment and verification of a digital PCR assay for the detection of CK19 expression in quantitative analysis of circulating tumor cell[J]. Hereditas(Beijing), 2023, 45(3): 250-260.

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编号	扩增长度	序列(5′→3′)	碱基数	起点	终点
CK19-1	136 bp	F-CAGCTGAGCATGAAAGCTGC	20	998	1017
		R-GCTCACTATCAGCTCGCACA	20	1133	1114
		P-CGCGCTTTGGAGCCCAGCTGGCGC	24	1047	1070
CK19-2	128 bp	F-AACCATGAGGAGGAAATCAG	20	710	729
		R-ATGACCTCATATTGGCTTCG	20	837	818
		P-AAGTGGGAGGCCAGGTCAGTGTGGA	25	744	768
CK19-3	91 bp	F-AGCCACTACTACACGACCAT	20	443	462
		R-CATTGTCGATCTGCAGGACA	20	533	514
		P-GCGGGACAAGATTCTTGGTGCCACCA	26	472	497

用于循环肿瘤细胞定量分析的CK19数字PCR检测方法的建立及性能验证

Establishment and verification of a digital PCR assay for the detection of CK19 expression in quantitative analysis of circulating tumor cell

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指标	不同ABL1拷贝数梯度
指标	20,000	15,000	10,000	5000	2500
∧FP	4.83	4.6	0.83	0.85	0.53
LOB	9.24	8.93	3.12	3.17	2.53

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