Research And Application Of Highly Sensitive Techniques In Allogeneic DNA Detection Based On Digital PCR

The development of PCR has experienced three generations of technological innovations.As the third generation of PCR technology,digital PCR is based on the statistical principle of Poisson distribution and limited dilution of the reaction system to achieve absolute quantitative analysis of target nucleic acid molecules.With the popularization and progress of digital PCR technology and its advantages of high sensitivity,high precision,tolerance to inhibitors,etc.,digital PCR widely used in liquid biopsy of tumors,noninvasive prenatal testing,pathogenic microorganism detection,genetically modified organism detection,gene expression,next generation sequencing library preparation,genome editing and cell therapy,etc.The thesis focuses on the research and application of highly sensitive technique in allogeneic DNA detection based on digital PCR.Importantly,we developed the clinically appropriate workflow for chromosomal aneuploidies detection,which is applied to disease screening of amniotic fluid samples,chorionic villus sampling and non-invasive prenatal diagnosis samples.At the same time,we developed the rapid genotyping and quantitative detection assay for DNA genetic markers,which is suitable for quantitative analysis and dynamic monitoring of allogeneic DNA in clinical.Chapter 1 described the development process of digital PCR technology and introduced the principles and characteristics of various technical platforms,as well as the application of digital PCR technology in the fields of liquid biopsy,molecular diagnosis and food safety.Chapter 2 focused on the detection of chromosomal aneuploidy abnormalities.Segmental duplication as the high-fidelity biomarker,we developed a computational program,ChAPDes,which integrates segmental duplication searching,refinement,and design of specific PCR primer/probe sets in a pipeline.The generated primer/probe sets were first tested in a multiplex multicolor melting curve analysis for the detection of five common aneuploidies.The clinical cohort study of 463 prenatal diagnosis samples verified the specificity and sensitivity were 99.64%and 100%.Then,the primer/probe sets were then tested in a digital PCR assay for the detection of trisomy 21.Finally,a digital PCR protocol was established to quantify maternal plasma DNA sequences for the noninvasive prenatal detection of fetal trisomy 21.The specificity and sensitivity were 100%.Chapter 3 focused on genotyping and quantification of DNA genetic markers,we used multiple asymmetric PCR system and multiplex multicolor melting curve analysis to detect 48 InDel and SNP polymorphic loci.It could accurately identify the genotype of each locus at the same time.Based on digital PCR,we established the InDel/SNP quantification assay for determination different allele percentage.The detection range was 5 to 50,000 copies per reaction,and the limit of detection was 0.01%.In additional,an optional preamplification enrichment assay was proposed for low-concentration or fragmented samples.A total of 1～10 ng DNA was added to a single reaction to complete the preamplification of target nucleic acid molecules by about 100-fold within 1 hour.The limit of blank of the preamplification enrichment assay was 0.1675%,and the accuracy and stability of preamplification assay for allogeneic DNA detection were significantly better than that of direct detection.Chapter 4 focused on application of allogeneic DNA determination by DNA genetic marker genotyping and quantitative detection assay,such as the maternal cell contamination assessment by identifying low-content maternal DNA from high-content fetal DNA.We collected three types of 67 prenatal diagnosis samples for maternal cell contamination assessment.The results showed the chorionic villus sampling were more likely to be contaminated by maternal cells.The application has excellent clinical applicability and prospects in prenatal diagnosis field.Also,DNA genetic marker genotyping and quantitative detection assay applied in determination of cell free fetal DNA fraction.We collected 52 fetal free DNA samples from maternal serum.The results showed that the fetal fraction in the first trimester ranged from 3.5%to 17.6%,with a median of 8.06%,and the second trimester ranges from 2.6%to 16.7%,with a median of 8.62%,and the third trimester was 24.3%.The application can improve the accuracy of noninvasive prenatal testing in the first trimester.Furthermore,DNA genetic marker genotyping and quantitative detection assay also applied in allograft allogeneic DNA detection.We collected and monitored 2 cases hematopoietic stem cell transplantation,17 cases kidney transplantation and 8 cases liver transplantation patients.With dynamic monitoring of the donor chimerism percentage and dd-cfDNA percentage,the application provides a noninvasive monitoring workflow for transplant injury and rejection evaluation after organ transplantation.This rapid,noninvasive and effective workflow does not require any invasive pathological sampling,and not rely on quantitative analysis of donor DNA.It has low cost,accurate and directly digital quantitative results.