干血斑标本珠蛋白生成障碍性贫血基因检测方法的建立∗

摘要

目的：建立并优化干血斑标本基因组DNA提取方法和流程,以适用于临床进行珠蛋白生成障碍性贫血(又称地中海贫血,以下简称“地贫”)基因诊断,并对干血斑打孔标本间可能的交叉污染和保存的稳定性进行分析。方法收集150份血液标本制备干血斑后,采用打孔仪打孔,用洗脱裂解液对血斑进行洗脱,并对洗脱方法进行优化,采用磁珠法提取血斑 DNA,再进行地贫基因检测,判断干血斑和全血的地贫基因检测结果是否相符。干血斑采用2种地贫基因检测方法进行检测,以验证其是否适用于多种方法。地贫阳性标本间打空白孔,对空白孔进行地贫基因检测确定打孔是否存在交叉污染。将干血斑常温干燥保存6、9个月后进行地贫基因检测,以判断其稳定性。结果采用5个3 mm 直径的干血斑在55℃振荡洗脱1 h,可以获得 DNA 浓度10~20 ng/μL(50μL DNA 溶解液),DNA 质量好。干血斑和全血的地贫基因检测结果完全一致,干血斑的2种地贫基因检测方法的结果也完全一致。打孔仪连续对干血斑打孔,地贫基因未检测出交叉污染。干血斑标本存放6、9个月后依然能够稳定地进行地贫基因检测。结论干血斑标本可以准确、方便、稳定地进行地贫基因检测,是地贫基因检测标本转诊的理想方式。%Objective To devolope a method for extracting DNA from dried blood spots (DBS)and optimizing the operating procedure,which could be applied to clinical gene diagnosis of thalassemia.And the cross contamination of DBS punching and the storage stability of DBS were studied.Methods A total of 1 50 blood specimens were collected,and DBS were prepared.Circles (3 mm in diameter)were punched in the DBS,and eluted with lysis buffer.The eluting method and operating procedure were opti-mized.Genomic DNA extracted from the elution solution by magnetic beads,and were performed thalassemia gene test.Finally jud-ging whether the results of DBS and whole blood were consistent.Two methods of thalassemia gene test were used in DBS and the compatibility of DBS processing method was verified.Judging whether there was cross contamination of DBS punching by the thalassemia gene test results of blank hole which were punched in the blank filter paper between thalassemia positive DBS.The DBS storage stability in thalassemia gene test was verified by detecting the DBS which were dry stored at room temperature for 6 and 9 months.Results 5 circles (3 mm in diameter)DBS were vibrating eluted at 55 ℃ for 1 hour,the DNA concentration extracted from the elution solution was 10-20 ng/μL,which was dissolved in 50 μL solution,and the DNA quality was good.The thalassemia gene test results of DBS and whole blood were the same,and the DBS results of two thalassemia gene test methods were the same too. The cross contamination of DBS punching was not detected in thalassemia gene test.The DBS which were dry stored at room tem-perature for 6 and 9 months could be stably performed thalassemia gene test.Conclusion DBS could be used to perform thalassemia gene test,which is accurate,convenient and stable.It is an ideal way for specimen referral of thalassemia gene test.