Application Of CND Detection Based On MeltArray Technology In Chimerism

Chimerism detection has a variety of clinical applications that can provide important diagnostic information in different clinical contexts,such as fetal microchimerism identification in autoimmune diseases,transplant damage and transplant rejection testing,and vertical monitoring,etc.The early detection method of chimera is mainly to distinguish donor and recipient cells based on phenotypic characteristics or genetic differences.At present,the more commonly applicable method is to use inherent genetic variations on autosomes,such as STR,SNP,and DIP.However,these methods have their inherent shortcomings and are not suitable for widespread clinical application.Therefore,it is necessary to explore a simple,fast,and more suitable detection scheme for clinical promotion and application in order to achieve early real-time monitoring of the patient's condition.In first chapter,we elaborated the basic concept of chimeras,analyzed the clinical application significance of chimera detection,outlined the current common chimera detection methods,and summarized the selection of polymorphic genetic markers suitable for chimera detection.Finally,the research content of this thesis is proposed.The second chapter,we summarized the guidelines for selection of CND genetic markers suitable for chimera detection,and screened 40 CND loci suitable for chimera detection in Chinese population through literature search and SYTO 9 dye method.Using 40 CND sites as the detection object,a relatively simple and fast method to find the difference between donor and pre-transplant recipients was established using fusion array technology,which greatly simplified the search for effective markers before quantitative detection of chimeras.The system is two tubes of four colors and forty weights,and the whole closed-tube detection can distinguish the genotype of CND locus by simple melting curve analysis,and the sensitivity can reach 150 copies/reaction.The system was used to detect 650 unrelated individual specimens,and the homozygous deletion frequency of each point was counted,which was similar to the homozygous deletion frequency calculated by the SYTO 9 dye method.Finally,the amount of information in this system is analyzed.The probability of finding at least one valid site in a donor-acceptor pair consisting of any two unrelated individuals can reach 99.99%;in 870 donor-acceptor groups consisting of 30 unrelated individuals of the donor pairs,100%of the donor-acceptor pairs have at least one effective site;among the 342 donor-acceptor pairs composed of first-degree relatives,98.83%of the donoracceptor pairs have at least one site.This indicates that the 40 CND loci of this system have close to 100%information potential and can effectively distinguish donor and recipient cells.In third chapter,based on CND loci,we use a combination of vector probe technology and qPCR to establish a single-quantitative detection system for 40 CND loci and the reference gene GAPDH.The closed-loop detection is performed throughout the process,using only the reference gene standard the content of the effective sites in the unknown template can be obtained from the curve and the Cq value corresponding to each effective site,and the chimeric rate is calculated by the formula.The CND quantitative detection system has a sensitivity of 300 copies/reaction.Except that there are four sites with relatively low amplification efficiency,the amplification efficiency of the other sites is basically the same,all fluctuating between 85.40%and 98.05%,and the linear relationship is good(R2>0.995),which has high stability and repeatability.The chimera rate measured by the simulated chimera sample experiment has good agreement with the theoretical value(R2>0.99),the repeatable sensitivity of the quantitative test can reach 1%,and most of it can reach 0.1%.In the quantitative monitoring of donor chimerism rate in a patient after hematopoietic stem cell transplantation,the patient's postoperative chimeric state gradually changed from a mixed chimeric state(MC)to a fully chimeric state(CC).The actual clinical results are consistent.Compared with the gold standard STR-PCR analysis technology for chimera detection,the quantitative detection system is simpler and faster in the quantitative detection of chimera,the experimental cost is low,the workload is less,no special instrument is needed,and the degree of automation is high,which is more suitable for promote and apply clinically.