A Screening Method For Non-common Deletional Alpha-globin Gene Mutation

Objective:Thalassemia is also called mediterranean anemia,is a serious health hazard,it is one of the main causes of birth defects.In China,Guangxi and Guangdong are the regions where the incidence rate of thalassemia rate are relatively high.Although the"Guangxi model"for the prevention and control of thalassemia has been highly recognized by the leaders including Chen Zhu,chairman of the Central Committee of the agricultural and labor party,the gene carrying rate of thalassemia is still high.It shows that the current screening methods for thalassemia are insufficient,incapable of finding out static alpha-thalassemia and the non-common gene type of alpha-thalassemia mutation.The purpose of this study is toestablish deletional alpha-thalassemia detection system,this is a method that can be used to explore the deletional alpha thalassemia gene,high throughput and high sensitivity on a large scale,to provide scientific basis for the country or region to formulate the prevention and control policy ofthalassemia,to reduce the birth rate of children with thalassemia major,finally,to reduce the pressure and burden of family and society.Methods:To establish a triple Tap Man real-time fluorescence quantitative PCR detection system,combined with the principle of relative quantitative analysis of 2^-??ct,and to quickly diagnose the number of copies of alpha-thalassemia gene,according to the difference of the proportion of alpha-globin gene and the number of copies of the donor gene.Through a series of optimization and adjustment of the reaction conditions,such as primer concentration,probe concentration,PCR reaction solution concentration,detection template quantity,DNA polymerase and other components and circulation number,the detection system of the triple Taq Man real-time fluorescence PCR of deletional alpha-thalassemia was finally determined.In order to explore the feasibility of its clinical application,accuracywere evaluated,the methods established in this study were detected by alpha-thalassemiageno types,which were verified by Gap-PCR,which is considered as the gold standard.Results:?1?This study confirmed and established the triple fluorescence quantitative PCR detection method of deletion type?–thalassemia.The method to determine the copy number of HBA as follows:the judgment value of 2^-??ct=1 indicates that the number of copies of sample HBA are normal,2^-??ct?0.75 indicates that the number of copies of HBA are lower than the normal value,that is,the positive value is 0-0.89,and the negative value is0.9-1.1.The judgment values of HBZ are as follows:2^-??ct=1 indicates that the copy numbers of HBZ in the sample are normal,2^-??ct?0.5,indicats that the copy numbers are lower than normal.?2?The genotypes of alpha-thalassemia in 500 clinical samples were detected by the methods of this experiments,there were 422 cases of four copies with HBA,33 cases of three,43 cases of two,and 2 cases of one.Among them,we have explored the non-common deletional alpha-globin gene mutationthat can not be found in clinical routine test:thalassemia gene type of Thailand deletion type.?3?the results were verified by Gap-PCR,the accuracy of this method is 99.60%,and thesensitivity is 100%.The accuracy of clinical routine detection technology is 98.60%,thesensitivity is 93.42%.The detection rate of the method established in this study is higher than that of the clinical routine detection method,and the difference between the test method of this study and the clinical routine test method is statistically significant,the detection rate and the sensitivityof the method established in this study is significantly higher than that of the routine clinical detection method.Conclusion:In this study,we established a method-based method for the detection of alpha-thalassemia deficiency based on the relative quantitative technique of globin gene copy numbers,it can find out static,light and non-common alpha-thalassemia.This method has high accuracy and good sensitivity.It can detect the common alpha-thalassemia genotypes and non-common alpha-thalassemia genotypes which are not detected by clinical routine test,especially in those laboratories in which testing is restricted to a panel of common deletions,reduce or avoid birth defects.