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

The objective of this study was to establish and verify a digital PCR assay for the detection of 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 gene, and housekeeping gene 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 with 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 gene could be effectively detected at the concentration ratio of 50%, 10%, 5%, 1%, 0.5% and 0.1%, and the linear value was 0.9998. Finally, the preliminary results of digital PCR in clinical samples indicated that copy numbers were higher in advanced breast cancer patients than healthy controls. The above results demonstrated the advantages of our 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.