ABSTRACT
<p><b>OBJECTIVE</b>To establish a rapid method for detection of alpha-globin gene αααbased on droplet digital PCR (ddPCR) technique.</p><p><b>METHODS</b>The differential sequence between the X1 and Y1 box of α1 gene was selected as the amplicon of the target gene with β-actin as the reference gene. The specific primers and TaqMan probes were designed, and then a quantitative method for detecting the copy number was established based on ddPCR technique. The sensitivity and accuracy of the method were evaluated by detecting 28 samples of known genotypes and 60 clinical samples.</p><p><b>RESULTS</b>The ddPCR-based method accurately identified the genotypes of all the 28 samples with known genotypes and detected 5 cases of αα/αααfrom the 60 clinical samples, and the results were verified by MLPA. The sensitivity and accuracy of this method were both 100% for detecting alpha-globin gene ααα.</p><p><b>CONCLUSION</b>This ddPCR-based method for detecting αααtriplet can be applied for population screening and in routine clinical molecular diagnosis with simple operation, rapid analysis and accurate results.</p>
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the multiplex ligation-dependent probe amplification (MLPA) technology in the detection of gene deletion and prenatal diagnosis of α-thalassaemia.</p><p><b>METHODS</b>Phenotypes were analyzed by whole blood cell counting and hemoglobin component detection of peripheral blood samples from the subjects. The gene deletions and point mutations of α- thalassaemia were detected with regular gap-PCR and reverse dot blot (RDB) method. At last, the MLPA method was applied for detection of α-globin gene deletion. All the prenatal diagnosis samples were detected with both gap-PCR and MLPA method.</p><p><b>RESULTS</b>α-thalassaemia phenotype was found in 75 samples from 1256 (628 couples) peripheral blood samples for pre-pregnancy or prenatal thalassemia gene screening. Among them, 71 samples carrying α-gene mutations and consistent with phenotypes were detected by routine methods. In the other 3 samples with no α-gene mutations detected and 1 sample with HbH phenotype but genotype of ﹣α(4.2)/αα were analyzed by MLPA and found each one samples of whole α-globin gene cluster deletion, respectively. Seventeen high risk couples were screened. Among the 17 prenatal diagnosis samples, 2 villus samples contaminated by exogenous DNA were confirmed by MLPA method.</p><p><b>CONCLUSION</b>MLPA is an effective complement for α-thalassaemia gene deletion detection. The molecular diagnosis strategy and process of gap-PCR combined with MLPA for α- thalassaemia gene deletion detection can prevent the missing of gene deletion, and false-positive or false-negative misdiagnosis of α-thalassaemia in prenatal diagnosis.</p>