The Establishment And Application Of High Throughput Blood Genotyping Technology

Background and PurposeABO blood group system is one of the most important clinical type,ABO blood group identification of clinical blood transfusion safety and security organ transplantation have important significance,the domestic and foreign clinical ABO blood group identification mainly serological method to the traditional methods,the identification of ABO phenotype,the method detection speed,easy operation,low price,but detect vulnerable to red blood cells surface antigen activity and secretion of individual status and the effects of microbial contamination,etc.Since the blood group gene was cloned in 1990,the ABO blood group genotyping technique based on DNA detection has greatly compensated for the deficiency of serological methods.However,the current methods of genotyping of blood groups are not high enough,flexible enough,slow speed,high cost and unsuitable for Chinese population.Therefore,this study establishes a set of genome-wide automatic nucleic acids extraction system,at the same time the asymmetry PCR-SSP technology and molecular beacon(molecular beacons,MB)detection technology,to create a new kind of ABO blood group genotyping technique.Currently,the blood group system has two blood group systems with 36 routine tests,and the detection rate is 5.6%.Abnormal blood infusion other test type system,the risk of patients with autoimmune antibodies,especially need a long-term transfusion patients,such as: sickle anemia,abnormal blood related antibodies,for a long time,resulting in no blood to lose.Female patients,such as inputting foreign blood,can produce the corresponding antibody,resulting in neonatal hemolysis or infertility.Therefore,it is urgent to develop high-flux identification method.A high-fluxes genotyping method of ABO,RH,KEL,FY,JK and MNS was established in the early stage of the expansion of blood group genotyping.MethodAn automated whole-genome nucleic acid extraction system would be established by improving the existing Hamiltonian and Thermo's nucleic acid extractor in the department and optimized the system.The comparison of extraction effect of three different samples of total blood,white film layer and red blood cells would be verified and found the best model.ABO blood group system via the NCBI SNP loci of different blood groups,to find the site design asymmetry PCR-SSP primer,and then to asymmetric PCR-SSP amplification,the amplification products sequencing,in view of the molecular beacon amplification product design,then using molecular beacon for rapid detection of amplification products.ResultsBy adding the sample equipment the reasonable layout of mesa,the optimization of the extraction reagent and sample amount,according to different fluid properties by use of different sample model,effectively avoid the pollution between samples,to avoid the liquid caused by improper model instrument hanging bead,and sample weight,through the improvement in oscillation frequency and oscillation time,cracking,washing time,washing and elution time method,the whole genome DNA extracting high purity and concentration.Optimized asymmetric PCR-SSP reaction conditions and molecular beacon detection conditions.96 tests can be completed within 300 min,and the detection of 96 blood samples shows that the detection sensitivity is lower than the PM and the specificity is 100%.ConclusionIn this study,a set of ABO blood group genotyping technology with high automation and high fluxes in China is established,which can detect the A1 of ABO blood group system.A2.O1.O2.B alleles,can detect 21 kinds of blood type genotype ABO blood group system,the automatic full genome DNA extraction apparatus,is to improve on existing instruments,this method is suitable for most of the blood system,saving the cost of the instrument,with the advantages of quick,easy,in order to adapt to the characteristics of molecular beacon detection single,this research adopts the asymmetry PCR-SSP extend,can not only detect the SNP but also meet the requirements of the late detection of target molecules.Molecular beacon detection target molecule has the advantages of fast,specific and sensitive.This study first to molecular beacon technology used for ABO blood group genotyping,convenient and flexible for molecular beacon detection,its application to Kell,Kidd,Diego,Duffy system has excellent effect on the detection of blood type.