Establishment And Clinical Evaluation Of Melting Analysis Testing System For Deletional ?-thalassemia

Background and PurposeThalassemia(or TM)is one of the most common single-gene genetic diseases in the world which is mainly divided into ?-thalassemia and ?-thalassemia.There is no effective treatment for this disease.Through the application of appropriate analytical techniques on molecular screening and genetic diagnosis of the population,it can identify the high-risk couples that carry the disease-causing genes,and prenatally diagnose the fetus so as to avoid the birth of severe children,which is recognized as the preferred preventive measure at home and abroad.At present,the existing technical solutions have limitations for detecting a-detectional thalassemia.The high costs,cumbersome operation or pollution problems are the biggest limitation.The purpose of the study is to establish a new method which can detect a-globin deletion in rapid,accurate,simple and efficient,low-cost way,so as to solve the limitations of the current detection methods.Research MethodThe difference between different genotypes of deletional a-thalassemia lies in the size of the deletion fragment and the site of the cleavage,so the corresponding type of thalassemia can be accurate detection as long as the truncated sequence is detected.In this study,initially,we collected normal samples and common?-deletional thalassemia samples(--sEA/,--3.7/,--4.2/)and relatively rare a-deletional thalassemia samples(--Thailand/)from Chinese population as the research object.Then,we designed the primers and probes in appropriate position,according to the deletion fragment differences.Furthermore,we made a series of optimization and adjustment of the reaction conditions,such as primer concentration,probe concentration,PCR Buffer concentration,detection template quantity,DNA polymerase components and annealing temperature,number of cycles and so on.Finally,we determined a multiplex nested asymmetric fluorescent melting assay system for the detection of deletional a-thalassemia.In order to investigate the feasibility of its clinical application,the study also evaluated the sensitivity,specificity,accuracy and repeatability of the detection system.Research ResultsBased on the experimental purpose and experimental scheme,the feasibility of the detection of detectional ?-thalassemia fluorescent probe melting assay was verified and the detection system was established,this method allows for the accurate typing of deletional a-thalassemia samples of different genotypes at concentrations greater than or equal to 4 ng/ul(magnetic bead method,column method,phenol-chloroform extraction method)with an accuracy of 100%,compared with the traditional method(Gap-PCR,MLPA).The intra-batch repeatability of all four channels was less than 0.50%,and the inter-batch stability was less than 0.70%,meeting the in vitro diagnostic reagent quality requirements(coefficient of variation not exceeding 15%).It meets clinical application requirements.The evaluation of 2000 clinical samples showed that compared with the traditional method(Gap-PCR or MLPA),the detection method of detectional a-thalassemia fluorescent probe melting analysis method can achieve fast,efficient and accurate classification for different genotype detectional a-thalassemia samples.Research conclusionThrough the study,we established a new method for the diagnosis of deletional alpha-thalassemia using multiplex nested asymmetric fluorescent-melting analysis from the perspective of methodology,which effectively solved the problems of high cost and complicated operation of traditional methods and pollution caused during PCR operation.This method enables the detection of three types of common deletional a-thalassemia and a relatively rare deletional a-thalassemia in Chinese southern populations through a single-tube reaction,which greatly improves working efficiency and provides new technical options for the molecular diagnosis of deletional alpha-thalassemia and the prevention of the disease.Thalassemia,the most common monogenic disease,is the disease with the highest incidence in Guangxi Province.A patienfs results of a-thalassemia gene detection were found to be not consistent with that of blood test in daily screening.It was confirmed that there were multiple mutations at ?2-globin gene polyadenylation(polyA)signal site HBA2:c.* 64(T>C),68(A>C),71(G>A),74(C>A)582(G>A),92(A>G),98(T>C)combined with —SEA/?? by the sequencing of the proband and the core family members5 HBA: gene and HBA2 gene,which was a HBH disease caused by rare mutations.After that,we found two cases of unrelated patients with this type of mutation.Tt shows that mutation is not an accidental phenomenon,md has considerable incidence in the Guangxi,China.We analyzed the hematological manifestations of this type of thalassemia.It can cause moderate anemia.We should pay attention to it in the clinical diagnosis and genetic counseling.