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。

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.