The Establishment And Application Of Real-time Fluorescence PCR For Fast Detection Of Mutation In β-globin Gene

This research is to set up a real-time fluorescence PCR technique on the fast detection of the six most common gene mutants ofβ- globin gene in Chinese population and to apply it in the gene diagnosis and prenatal diagnosis ofβ-thalassemia.First of all, a real-time fluorescence PCR technique is set up for the detection of mutation site of the six most commonβ- globin gene mutants [eg.CD41-42(-CTTT) deletion mutation, TATA box nt-28(A→G)mutation, IVSII654(C→T) mutation, CD71-72(+A) code shifting mutation, CD17(A→T) nonsense mutation, and CD26(G→A) mutation] in Chinese population based on allele-specific PCR by using SYBR green I fluorescent dye. Positive samples of the six most commonβ- globin gene mutants mentioned above have been detected by using the real-time fluorescence PCR technique. The consequence of genotype is totally in accordance with DNA sequencing, the specificity and accuracy of which is 100%.By using the real-time fluorescence PCR technique, I have succeeded in diagnosing 68 blood samples of 20 lineages which had been suffering severeβ-thalassemia. 64 out of 66β-thalassemia carrier are found to carry one or two of the sixβ- globin gene mutants mentioned above. Rate of detection is 97%. 13 kinds ofβ- globin gene mutants are detected. Among which, 25 cases are CD41-42/N, counting for 37.88%; 8 cases are homozygotes of CD41-42, counting for 12.12%; 7 cases are nt-28/N and another 7 cases are IVSII654/N, counting for 10.61% respectively; 5 cases are dual heterozygotes of CD41-42/ IVSII654, counting for 7.58%. 3 cases are dual heterozygotes of CD41-42/nt-28, counting for 4.55%; 2 cases are CD71-72/N and 2 cases are dual heterozygotes of CD41-42/ CD71-72, counting for 3.03% respectively; sample with CD17/N,CD26/N and dual heterozygotes of CD41-42/CD17,nt-28/ IVSII654,or CD71-72/CD26 is respectively 1 case and each counting for 1.52%. 2 cases are unknown mutation, counting for 3.03%. The results of this research show that the method set up for detecting six most commonβ- globin gene mutants based on real-time fluorescence PCR technique has the properties of high specificity, low cost, and rapidness. Thus it is fitted for the screening ofβ-thalassemia gene mutants in large population. This method provides a platform for a highly efficient, rapid, and accurate gene diagnosis before and after delivery as well.By using the real-time fluorescence PCR technique, we analyze the distribution characteristics of genotype and gene frequency of the six most commonβ- globin gene mutants in1872 cases of Hainan Han population and 2248 cases of Hainan Li population. The findings of this research show thatβ-thalassemia is in high incidence in Han and Li populations in Hainan. The rate ofβ-thalassemia carrier in Han population is 2.08%. The frequency of allele is 0.0104. CD41-42 (-CTTT) deletion mutation is the most common mutant genotype and TATA box nt-28(A→G) mutation, IVSII654(C→T)mutation and CD71-72(+A)code shifting mutation follows after. The mutant genotypes are complicated and heterogeneity is very high. The rate ofβ-thalassemia carrier in Hainan Li population is 8.10%. The frequency of allele is 0.0405. The mutant genotype is quite simple, with almost all of which is CD41-42(-CTTT) deletion mutation. None of the other five types of mutation was detected. This research provides a primary data concerning the distribution characteristics of genotype and gene frequency ofβ-thalassemia in Hainan Han and Li populations. The result of the research can be a scientific evidence for the prevention and cure ofβ-thalassemia. In addition, it provide a molecular genetics evidence for the research of population genetics, anthropology and headstream of ethnic group.