The Establishment Of A Digital PCR Assay For Detection Of CK19 And Its Application In Quantitative Analysis Of CTC

Objective:We established and verified a digital PCR assay for the detection of CK19gene expression,aiming to use it in the quantitative analysis of circulating tumor cells(CTC)in cancer patients by the virtues of high sensitivity and absolute quantitationMethods:At first,the assay was preliminarily established.The primers and probes were designed according to the m RNA sequence of the CK19 gene,and the housekeeper gene ABL1 was used as the internal reference.The human breast cancer cell line MCF7 and the white blood cells in the peripheral blood of healthy people were used to screen the desired primers and probes.After the primers and probes were selected and their reaction conditions were confirmed,the performance test of the digital PCR reaction was carried out,including(1)Limit of blank(LOB)analysis using the m RNA of healthy people as template;(2)Limit of detection(LOD)analysis using the premixed templates of MCF7 cell lines and healthy human leukocytes at different concentrations.The initial validation with clinical samples then carried out.Secondly,the reaction conditions of digital PCR were further optimized according to the results of clinical samples.In addition to finish the performance tests of the above two aspects,the sensitivity test was performed using different concentrations of MCF7 cell m RNA as templates.Besides,different concentrations of healthy human m RNA were used as templates to test the specificity of the method.Meanwhile,the LOD analysis was carried out with different numbers of MCF7 cells that were pre-mixed with the background of 10~6healthy human leukocytes.Based on above results,the detection threshold was preliminarily established.Finally,the established digital PCR assay was used to analyze healthy people and cancer patients(mainly were breast cancer).The application prospects in early detection,prognosis assessment and efficacy monitoring of our assay was evaluated by comparing the result in differences patients with different stages.Results:The best primers and probe are screened out and the amplified products were confirmed correct by direct sequencing.After the reaction condition was optimized,the preliminary performance test demonstrated the LOB of CK19 was 9.24,8.93,3.12,3.17and 2.53 copies when the ABL1 gene copy number was 20,000,15,000,10,000,5000and 2500,respectively;in the LOD detection,the CK19 gene could be effectively detected at concentrations of 50%,10%,5%,1%,0.5%and 0.1%(linear R~2=0.9998).In the initial testing of clinical samples,the CK19 copy number in patients with advanced breast cancer was higher than in healthy people.In the performance test after further optimization,the CK19 gene can still be detected when the template input was0.00001ng,indicating that the method’s sensitivity is good.In the specificity test,a maximum of 20.2 CK19 copies were detected when the input amount of healthy human leukocytes template was increased to 100 ng.In contrast,107775.4 CK19 copies were detected when the input of MCF7 cell template was as low as 1ng,indicating that the specificity of this method was good.When reference genes copy numbers was 2000,5000,10000 and 18000,the LOB was 4.67,7.66,14.99 and 19.53,respectively.The minimum limit of LOD detection after template premixing of MCF7 with healthy human leukocyte can reach 0.1%(linear R~2=0.9995).In the test of cell premixing of MCF7 with healthy human leukocyte,one MCF7 cell could be detected,and the linearity is better when the mixed tumor cells are less than 100(linear R~2=0.9981).In the detection of clinical samples,the results of tumor patients showed a high degree of individualized differences compared with healthy people,and the CK19 copy number of typical cases was consistent with their clinical manifestations.In addition to blood samples,higher levels of CK19 copy number was also detected in cerebrospinal fluid samples from breast cancer patients and urine samples from bladder cancer patients,suggesting that these samples can also be used in patient’s dynamic monitoring.Conclusion:We have established a digital PCR assay for detection of CK19 gene,which demonstrated its advantages in sensitivity,specificity and accurate quantification.Besides,the preliminary verification in clinical samples shown good application prospect of our assay in quantitative analysis of CTC in cancer patient,especially breast cancer.