中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2012年
5期
407-412
,共6页
严提珍%罗世强%唐宁%钟青燕%聂昌君%李伍高%王秋华%蔡稔
嚴提珍%囉世彊%唐寧%鐘青燕%聶昌君%李伍高%王鞦華%蔡稔
엄제진%라세강%당저%종청연%섭창군%리오고%왕추화%채임
β地中海贫血%聚合酶链反应%熔解曲线分析%基因诊断%产前诊断
β地中海貧血%聚閤酶鏈反應%鎔解麯線分析%基因診斷%產前診斷
β지중해빈혈%취합매련반응%용해곡선분석%기인진단%산전진단
beta-Thalassemia%Polymerase chain reaction%Melting curve analysis%Genetic diagnosis%Prenatal diagnosis
目的 对荧光PCR熔解曲线法用于β-地中海贫血(β-地贫)基因诊断和产前诊断进行临床评价.方法 收集2011年1至8月柳州市妇幼保健院外周血标本451份,其中经血液学表型分析为β-地贫阳性表型标本372份,β-地贫阴性表型标本79份.同时收集2011年6至9月夫妇双方经PCR-RDB探针法确诊为β-地贫基因携带者,在我院行β-地贫产前诊断的胎儿绒毛、羊水及脐带血标本共84份,其中孕10～ 13周胎儿绒毛标本16份、孕16 ～24周羊水标本64份、孕17周以上胎儿脐血标本4份.按双盲对照试验,分别采用荧光PCR熔解曲线法(可检测24种β珠蛋白基因突变)、PCR-反向点杂交(RDB)探针法(可检测17种β珠蛋白基因突变)和DNA测序法同时对451份外周血标本和84份胎儿绒毛、羊水、脐带血产前诊断标本进行检测.计算荧光PCR熔解曲线法和PCR-RDB探针法、DNA基因测序法检测结果的野生型符合率、突变型符合率和总符合率.结果 利用荧光熔解曲线法在451份外周血标本中共检出13种突变类型、19种基因型,其中有447份标本与PCR-RDB探针法的基因型检测结果相符,符合率为99.1％ (447/451),902个等位基因位点检出符合率为99.6％(898/902),有4份标本与PCR-RDB探针法的基因型检测结果不相符,经DNA测序法验证,有3份标本与荧光熔解曲线法的检测结果完全一致,1份标本的β珠蛋白基因突变不在荧光熔解曲线法检测范围而未被检出.450份外周血标本的荧光熔解曲线法检测结果与DNA测序法相符,符合率为99.8％ (450/451).利用荧光熔解曲线法在84份产前诊断胎儿标本中共检出8种突变类型、18种基因型,168个等位基因位点的检测结果与PCR-RDB探针法和DNA基因测序法检测结果符合率为100％.结论 荧光PCR熔解曲线法可同时检测多份待测标本,并可准确检出多种基因突变类型,可用于β-地贫的基因诊断和产前诊断.
目的 對熒光PCR鎔解麯線法用于β-地中海貧血(β-地貧)基因診斷和產前診斷進行臨床評價.方法 收集2011年1至8月柳州市婦幼保健院外週血標本451份,其中經血液學錶型分析為β-地貧暘性錶型標本372份,β-地貧陰性錶型標本79份.同時收集2011年6至9月伕婦雙方經PCR-RDB探針法確診為β-地貧基因攜帶者,在我院行β-地貧產前診斷的胎兒絨毛、羊水及臍帶血標本共84份,其中孕10～ 13週胎兒絨毛標本16份、孕16 ～24週羊水標本64份、孕17週以上胎兒臍血標本4份.按雙盲對照試驗,分彆採用熒光PCR鎔解麯線法(可檢測24種β珠蛋白基因突變)、PCR-反嚮點雜交(RDB)探針法(可檢測17種β珠蛋白基因突變)和DNA測序法同時對451份外週血標本和84份胎兒絨毛、羊水、臍帶血產前診斷標本進行檢測.計算熒光PCR鎔解麯線法和PCR-RDB探針法、DNA基因測序法檢測結果的野生型符閤率、突變型符閤率和總符閤率.結果 利用熒光鎔解麯線法在451份外週血標本中共檢齣13種突變類型、19種基因型,其中有447份標本與PCR-RDB探針法的基因型檢測結果相符,符閤率為99.1％ (447/451),902箇等位基因位點檢齣符閤率為99.6％(898/902),有4份標本與PCR-RDB探針法的基因型檢測結果不相符,經DNA測序法驗證,有3份標本與熒光鎔解麯線法的檢測結果完全一緻,1份標本的β珠蛋白基因突變不在熒光鎔解麯線法檢測範圍而未被檢齣.450份外週血標本的熒光鎔解麯線法檢測結果與DNA測序法相符,符閤率為99.8％ (450/451).利用熒光鎔解麯線法在84份產前診斷胎兒標本中共檢齣8種突變類型、18種基因型,168箇等位基因位點的檢測結果與PCR-RDB探針法和DNA基因測序法檢測結果符閤率為100％.結論 熒光PCR鎔解麯線法可同時檢測多份待測標本,併可準確檢齣多種基因突變類型,可用于β-地貧的基因診斷和產前診斷.
목적 대형광PCR용해곡선법용우β-지중해빈혈(β-지빈)기인진단화산전진단진행림상평개.방법 수집2011년1지8월류주시부유보건원외주혈표본451빈,기중경혈액학표형분석위β-지빈양성표형표본372빈,β-지빈음성표형표본79빈.동시수집2011년6지9월부부쌍방경PCR-RDB탐침법학진위β-지빈기인휴대자,재아원행β-지빈산전진단적태인융모、양수급제대혈표본공84빈,기중잉10～ 13주태인융모표본16빈、잉16 ～24주양수표본64빈、잉17주이상태인제혈표본4빈.안쌍맹대조시험,분별채용형광PCR용해곡선법(가검측24충β주단백기인돌변)、PCR-반향점잡교(RDB)탐침법(가검측17충β주단백기인돌변)화DNA측서법동시대451빈외주혈표본화84빈태인융모、양수、제대혈산전진단표본진행검측.계산형광PCR용해곡선법화PCR-RDB탐침법、DNA기인측서법검측결과적야생형부합솔、돌변형부합솔화총부합솔.결과 이용형광용해곡선법재451빈외주혈표본중공검출13충돌변류형、19충기인형,기중유447빈표본여PCR-RDB탐침법적기인형검측결과상부,부합솔위99.1％ (447/451),902개등위기인위점검출부합솔위99.6％(898/902),유4빈표본여PCR-RDB탐침법적기인형검측결과불상부,경DNA측서법험증,유3빈표본여형광용해곡선법적검측결과완전일치,1빈표본적β주단백기인돌변불재형광용해곡선법검측범위이미피검출.450빈외주혈표본적형광용해곡선법검측결과여DNA측서법상부,부합솔위99.8％ (450/451).이용형광용해곡선법재84빈산전진단태인표본중공검출8충돌변류형、18충기인형,168개등위기인위점적검측결과여PCR-RDB탐침법화DNA기인측서법검측결과부합솔위100％.결론 형광PCR용해곡선법가동시검측다빈대측표본,병가준학검출다충기인돌변류형,가용우β-지빈적기인진단화산전진단.
Objectives To investigate the clinical value of the melting curve analysis-based PCR assay for the clinical genetic diagnosis and prenatal diagnosis of β-thalassemia.Methods A total of 451 peripheral blood samples,including 372 cases with β-thalassemia phenotypes and 79 cases without β-thalassemia phenotypes,were collected by Liuzhou Municipal Maternity and Child Healthcare Hospital between January 2011 and August 2011.Moreover,another 84 cases,including 16 fetal villi samples (10 - 13 weeks),64 amniotic fluid samples (16 -24 weeks ) and 4 umbilical cord blood samples (above 17 weeks),whose parents were β-thalassemia carriers,were also collected for this assay between June 2011 and September 2011.A double-blind test was done to compare the detection reliability of the melting curve analysis-based assay (24 β-thalassemia mutations can be detected) with PCR-RDB probe assay (17 β-thalassemia mutations can be detected ) and DNA sequencing using these samples.The wildtype,mutant and total concordance rates of the genotyping results were calculated separately among the melting curve analysis based assay,PCR-RDB probe assay and DNA sequencing.Results Among the 451 peripheral blood samples,thirteen mutations and nineteen genotypes were obtained by using melting curve analysis-based assay.447 samples had the same detection results and 4 samples had different detection results by comparing melting curve analysis-based assay with PCR-RDB probe assay,thus,the concordance rate of the sample detection result was 99.1％ (447/451),and the concordance rate of the allele detection result was 99.6％ (898/902).DNA sequencing results of the 4 samples showed that 3 samples had the same genotyping result with melting curve analysis-based assay,and 1 sample had the same genotyping result with PCR-RDB probe assay.A rare β-globin mutation which was not included by melting curve analysis-based assay was not detected.Thus,the genotypes of 450 samples were detected accurately by melting curve analysis-based assay,and the concordance rate of the sample detection between the melting curve assay and DNA sequencing assay was 99.8％ (450/451).Among 84 fetal villi,amniotic fluid,and umbilical cord blood samples,8 mutation types and 18 genotypes were obtained by using melting curve analysis-based assay.All the samples have the same detection results by comparing melting curve analysis-based assay with PCR-RDB probe assay and DNA sequencing,so the concordance rate of the genotyping results was 100％ among the melting curve analysis-based assay,PCR-RDB probe assay and DNA sequencing.Conclusions The melting curve analysis-based PCR assay can detect multiple unknown samples simultaneously,and detect multiple mutations accurately.It is very useful for the genetic diagnosis and prenatal diagnosis of β-thalassemia.
