ABSTRACT
Objective To investigate the clinical diagnostic value of circulating tumor cells (CTCs)and circulating cell-free DNA (cfDNA) in peripheral blood samples in breast cancer. Methods From July 2017 to April 2018, 47 patients with BMC (7 in stage Ⅱ, 19 in stage Ⅲ and 21 in stage Ⅳ), 24 patients with benign breast diseases and 28 healthy people were selected. After collecting peripheral blood samples, serum and blood cells were separated. The size-based high-throughput microfluidic chip was used to capture CTCs. The real-time fluorescent quantitative PCR based on Alu sequence was used to detect the length of cfDNA(247 bp, 115 bp)in the serum, and the ratio of amplified products of long and short fragments was used as the index of DNA integrity. The Mann-Whitney U test or Kruskal-Wallis H test was used to compare the differences between the groups and analyze the relationship between CTCs and cfDNA and clinical parameters of breast cancer. The ROC curve was drawn and the area under the curve (AUC) was used to evaluate the feasibility of blood cell CTCs and plasma cfDNA detection as diagnostic criteria. Results The CTCs and cfDNA of 47 BMC patients were analyzed. The CTCs and cfDNA integrity index (Alu 247/115) of BMC patients were significantly higher than those of physical examination patients[(13.98± 12.36)cells / ml vs (1.14 ± 1.35) cells / ml; 0.7687 ± 0.3868 vs 0.5094 ± 0.2456], and the difference was statistically significant(the U value was 126.5,359.0;P<0.001), the area under ROC curve of CTCs was 0.885 (95％CI: 0.805-0.965), cut-off value was 7.68/ml, sensitivity was 80.4％, specificity was 96.4％. The area under ROC curve of Alu 247/115 was 0.727(95％CI: 0.608-0.847), cut-off value was 0.431, sensitivity was 71.7％, specificity was 71.4％. The AUC of CTCs and Alu 247/115 was 0.919 (95％CI 0.854-0.984), which was higher than the single test of each indicator. Conclusions CTCs and cfDNA may be the potential biological indicators for breast cancer diagnosis. The combined detection of CTCs and cfDNA maybe improve the diagnosis rate of breast cancer patients.
ABSTRACT
We report here a novel membrane transfer-based DNA detection method, in which alkaline phosphatase labeled gold nanoparticle (AuNP) probes were used as a means to amplify the detection signal. In this method, the capture probe P1, complimentary to the 3' end of target DNA, was immobilized on the chip. The multi-component AuNP probes were prepared by co-coating AuNPs with the detecting probe P2, complimentary to the 5' end of target DNA, and two biotin-labeled signal probes (T10 and T40) with different lengths. In the presence of target DNA, DNA hybridization led to the attachment of AuNPs on the chip surface where specific DNA sequences were located in a "sandwich" format. Alkaline phosphatase was then introduced to the surface via biotine-streptavidin interaction. By using BCIP/NBT alkaline phosphatase color development kit, a colorimetric DNA detection was achieved through membrane transfer. The signal on the membrane was then detected by the naked eye or an ordinary optical scanner. The method provided a detection of limit of 1 pmol/L for synthesized target DNA and 0.23 pmol/L for PCR products of Mycobacterium tuberculosis 16S rDNA when the ratio of probes used was 9:1:1 (T10:T40:P2). The method described here has many desirable advantages including high sensitivity, simple operation, and no need of sophisticated equipment. The method can be potentially used for reliable biosensings.