中华血液学杂志
中華血液學雜誌
중화혈액학잡지
Chinese Journal of Hematology
2013年
3期
233-236
,共4页
胎儿DNA%β-地中海贫血%肽核酸夹%反向斑点杂交
胎兒DNA%β-地中海貧血%肽覈痠夾%反嚮斑點雜交
태인DNA%β-지중해빈혈%태핵산협%반향반점잡교
cffDNA%β-thalassemia%PNA clamps%Reverse dot blot
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.
目的 探討利用肽覈痠夾提高孕婦血漿胎兒父源性β珠蛋白突變基因的檢齣率,為地中海貧血(地貧)無創性產前診斷提供依據.方法 選擇妊娠7 ～ 20週的孕婦共38例,丈伕基因型為41-42M/N,而孕婦基因型正常或為其他β地貧基因突變.以羊水、臍帶血或齣生後嬰兒外週血標本的檢測結果作為金標準對照.用QIAamp DNA Blood Mini試劑盒提取孕婦血漿總遊離DNA,再將總遊離DNA經過10 g/L瓊脂糖凝膠電泳後迴收長度100 ～ 300 bp的DNA,然後應用迴收後的DNA併加肽覈痠夾進行PCR擴增,最後採用反嚮斑點雜交確定基因型併與金標準檢測結果進行比較.同時設置擴增方法不同的A、B對照組,對照組A應用總遊離DNA併加肽覈痠夾,對照組B應用迴收後的DNA不加肽覈痠夾進行驗證.結果 共對38例孕婦外週血漿DNA標本的PCR產物進行雜交檢測併和金標準結果進行比較.21例金標準基因型為41-42M/N的標本中,實驗組、對照組A和B分彆有19、8、12例檢齣41-42M,敏感度分彆為90.5％、38.1％和57.1％;17例金標準基因型正常(N)的標本中,實驗組、對照組A和B分彆有1、1、2例齣現假暘性,特異度分彆為94.1％、94.1％和88.2％;準確度分彆為92.1％、63.2％和71.1％.進行McNemar x2檢驗兩兩比較,實驗組與對照組A和B的敏感度和準確度差異均有統計學意義(P＜0.05).結論 利用肽覈痠夾對孕婦外週血漿中小片段遊離胎兒DNA檢測胎兒父源性β-珠蛋白基因突變的方法,不僅具有較高的敏感度、特異度和準確度,而且方法簡便、實用.
목적 탐토이용태핵산협제고잉부혈장태인부원성β주단백돌변기인적검출솔,위지중해빈혈(지빈)무창성산전진단제공의거.방법 선택임신7 ～ 20주적잉부공38례,장부기인형위41-42M/N,이잉부기인형정상혹위기타β지빈기인돌변.이양수、제대혈혹출생후영인외주혈표본적검측결과작위금표준대조.용QIAamp DNA Blood Mini시제합제취잉부혈장총유리DNA,재장총유리DNA경과10 g/L경지당응효전영후회수장도100 ～ 300 bp적DNA,연후응용회수후적DNA병가태핵산협진행PCR확증,최후채용반향반점잡교학정기인형병여금표준검측결과진행비교.동시설치확증방법불동적A、B대조조,대조조A응용총유리DNA병가태핵산협,대조조B응용회수후적DNA불가태핵산협진행험증.결과 공대38례잉부외주혈장DNA표본적PCR산물진행잡교검측병화금표준결과진행비교.21례금표준기인형위41-42M/N적표본중,실험조、대조조A화B분별유19、8、12례검출41-42M,민감도분별위90.5％、38.1％화57.1％;17례금표준기인형정상(N)적표본중,실험조、대조조A화B분별유1、1、2례출현가양성,특이도분별위94.1％、94.1％화88.2％;준학도분별위92.1％、63.2％화71.1％.진행McNemar x2검험량량비교,실험조여대조조A화B적민감도화준학도차이균유통계학의의(P＜0.05).결론 이용태핵산협대잉부외주혈장중소편단유리태인DNA검측태인부원성β-주단백기인돌변적방법,불부구유교고적민감도、특이도화준학도,이차방법간편、실용.
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.
