摘要：
目的 探讨利用肽核酸夹提高孕妇血浆胎儿父源性β珠蛋白突变基因的检出率,为地中海贫血(地贫)无创性产前诊断提供依据.方法 选择妊娠7 ～ 20周的孕妇共38例,丈夫基因型为41-42M/N,而孕妇基因型正常或为其他β地贫基因突变.以羊水、脐带血或出生后婴儿外周血标本的检测结果作为金标准对照.用QIAamp DNA Blood Mini试剂盒提取孕妇血浆总游离DNA,再将总游离DNA经过10 g/L琼脂糖凝胶电泳后回收长度100 ～ 300 bp的DNA,然后应用回收后的DNA并加肽核酸夹进行PCR扩增,最后采用反向斑点杂交确定基因型并与金标准检测结果进行比较.同时设置扩增方法不同的A、B对照组,对照组A应用总游离DNA并加肽核酸夹,对照组B应用回收后的DNA不加肽核酸夹进行验证.结果 共对38例孕妇外周血浆DNA标本的PCR产物进行杂交检测并和金标准结果进行比较.21例金标准基因型为41-42M/N的标本中,实验组、对照组A和B分别有19、8、12例检出41-42M,敏感度分别为90.5％、38.1％和57.1％;17例金标准基因型正常(N)的标本中,实验组、对照组A和B分别有1、1、2例出现假阳性,特异度分别为94.1％、94.1％和88.2％;准确度分别为92.1％、63.2％和71.1％.进行McNemar x2检验两两比较,实验组与对照组A和B的敏感度和准确度差异均有统计学意义(P＜0.05).结论 利用肽核酸夹对孕妇外周血浆中小片段游离胎儿DNA检测胎儿父源性β-珠蛋白基因突变的方法,不仅具有较高的敏感度、特异度和准确度,而且方法简便、实用.%Objective To study improvement of detection of paternally herited fetal mutant genes for β-globin in maternal plasma by PNA clamp to seek a noninvasive prenatal diagnostic method for β-thalassemia.Method A total of 38 maternal blood samples were collected at 7 to 20 weeks of gestation,samples in which the father carried CD41-42 mutation and mother carried normal gene or the other point mutation for β-thalassemia were examined.The results of fetal DNA in amniotic fluid,cord blood or peripheral blood of newboms were used as the gold standard for comparison.In the study group,the total cell-free DNA was extracted from maternal plasma using QIAamp DNA Blood Mini Kit.After extraction,the total cell-free DNA was separated by agarose gel (1％) electrophoresis,and the cell-free DNA with a size of 100-300 bp was retrieved from the gel slice.Then,the retrieved DNA-free cell underwent PCR amplified with a PNA clamp.The genotype was confirmed by the conventional method (reverse dot blot hybridization),and the results were compared to gold standard.Simultaneously,two control groups with different PCR procedures were set up.The PCR procedure of control group A was amplified with the extracted total cell-free DNA and PNA clamp,and the PCR procedure of control group B was amplified with the retrieved size-fractionated DNA-free cell without PNA clamp.Result Plasma samples from 38 pregnant women were detected using PCR products for hybridization,the results were compared with the gold standard.Regarding the 21 samples confirmed by gold standard with fetal genotype 41-42M/N,19,8,12 cases were detected as fetal genotype 41-42M in study group,control group A and control group B respectively,the sensitivity was 90.5％ (19/21),38.1％ (8/21) and 57.1％ (12/21)respectively;Concerning the 17 samples confirmed by gold standard with fetal normal genotype,the amount of false positive cases were 1,2 and 1 respectively.The respective specificity was 94.1％(16/17),94.1％ (16/17) and 88.2％ (15/17) respectively.The respective accuracies were 92.1％(35/38),63.2％ (24/38) and 71.1％ (27/38) respectively.The difference in sensitivity and specificity was pairwise compared by means of McNemar' s test.There was significant difference between new study group and control group A or control group B (all P ＜ 0.05).Conclusion The method of detection of paternally inherited fetal mutation genes for β-thalassemia using small size of fetal DNA-free cell in maternal plasma with PNA clamp had several advantages of reliable sensitivity,specificity and accuracy,indicating its potential of clinical practicality.
