多重连接探针扩增技术及其应用

多重连接探针扩增是一种建立在PCR技术七的新型相对定量检测技术,该技术具有高效率、高分辨率、操作简便、设备要求低等诸多优势,目前已被广泛应用于基因片段缺失与重复、点突变及甲基化检测等相关疾病的分子生物学研究.其与基因芯片结合而建立的多重连接探针扩增微阵列芯片技术不但真正具备了高通量的检测能力,还使该技术的可靠性获得了很大的提高.本文就多重连接探针扩增的技术方法 、应用进展及目前还存在的局限性作一综述.     

应用多重连接依赖探针扩增技术快速检测胎儿染色体非整倍体异常

目的 探讨多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术在常见染色体非整倍体异常检测及其在产前诊断中的应用价值.方法 应用MLPA技术检测561份产前诊断样本和20例已知染色体异倍体标本,所有样本均进行常规染色体核型分析,比较MLPA结果和染色体核型分析结果,评价MLPA技术的临床符合率.结果 MLPA技术能够在48 h内出具检测结果,共检测出38例染色体异倍体,包括20例21-三体,10例18-三体,1例13-三体,4例Turner综合征,1例Klinefelter综合征,1例超雄综合征,1例48,XYY,+18双三体综合征.MLPA结果与染色体核型结果一致,检测结果100％准确.在561份产前诊断样本中,有550份样本的MLPA分析结果和细胞染色体核型的结果一致,临床符合率达到98.04％.结论 MLPA技术具有快速、简单、稳定等优点,可检测常见非整倍体异常,能作为产前诊断的可靠方法,具有临床实际应用价值.     

应用多重连接依赖式探针扩增技术快速高通量诊断胎儿染色体非整倍体异常

目的 评价多重连接依赖式探针扩增技术(multiplex ligation-dependent probe amplification,MLPA)在染色体非整倍体诊断中的应用价值,为我国羊水染色体诊断提供一种快速、特异、高通量的分子诊断手段.方法 应用MLPA技术检测了500份羊水标本,所有标本均进行荧光原位杂交(fluorescence in situ hybridization,FISH)技术检测和常规染色体核型分析,应用RH-MLPA-v511数据分析软件获得MLPA结果,比较MLPA技术与FISH和染色体核型分析结果的准确性,总结MLPA技术临床应用过程中的关键要点.结果 在500份羊水标本中,MLPA检测成功率97%.3个工作日完成结果的为92%,需重复检测的为5%,失败为3%.对染色体非整倍体异常检测敏感性和准确性100%.证实38例非整倍体病例探针信号比值＞正常二倍体4s,2例疑似三体结果＞2s.分析了21号染色体8条探针的杂交效率,21三体患者8条探针中平均4条探针比值＞1.3.结论 MLPA技术具有快速、特异、敏感、高通量、成本低等特点,可用于产前染色体非整倍体数目的快速检测,是传统染色体培养方法的补充,临床应用价值较高.

Abstract：

Objective To assess the diagnostic value of multiplex ligation-dependent probe amplification (MLPA) for detection of common chromosome aneuploidy in amniotic fluid (AF) cells in order to obtain an accurate, rapid, cost-effective and high-throughput method in routine prenatal clinical practice.Methods The MLPA test was performed on 500 AF samples by using kit P095 and the results were obtained by using analysis software RH-MLPA-v511. The results were compared with that from fluorescence in situ hybridization (FISH) and traditional karyotyping (TK). The technical critical issues were analyzed in routine diagnostic application. Results The absolute specificity and sensitivity of the MLPA test to detect the aneuploidy were 100%. For the 500 AF samples, the success rate of the MLPA tests was 97%. Among them 92% were finished within three working days and 5% required more days for repeating. The test failure rate was 3%. The results confirmed that for the 38 detectable aneuploid samples,the probe reliability weighted mean ratio values were more than 4SD compared to normal diploids and the 2 suspected trisomy samples were more than 2SD. In this study, authors analyzed hybridization efficiencies of 8 probes for chromosome 21, and the presence of a trisomy was considered if at least 4 of the 8 probes gave probe ratio of ＞1.3. Conclusion The data suggested that MLPA is a rapid, simple and reliable method for large scale testing for aneuploidy of chromosomes 13, 18, 21, X, or Y in AF. The MLPA technology is complementary to AF culture and valuable for prenatal diagnosis.     

应用多重连接依赖式探针扩增技术快速高通量诊断胎儿染色体非整倍体异常

Objective To assess the diagnostic value of multiplex ligation-dependent probe amplification (MLPA) for detection of common chromosome aneuploidy in amniotic fluid (AF) cells in order to obtain an accurate, rapid, cost-effective and high-throughput method in routine prenatal clinical practice.Methods The MLPA test was performed on 500 AF samples by using kit P095 and the results were obtained by using analysis software RH-MLPA-v511. The results were compared with that from fluorescence in situ hybridization (FISH) and traditional karyotyping (TK). The technical critical issues were analyzed in routine diagnostic application. Results The absolute specificity and sensitivity of the MLPA test to detect the aneuploidy were 100%. For the 500 AF samples, the success rate of the MLPA tests was 97%. Among them 92% were finished within three working days and 5% required more days for repeating. The test failure rate was 3%. The results confirmed that for the 38 detectable aneuploid samples,the probe reliability weighted mean ratio values were more than 4SD compared to normal diploids and the 2 suspected trisomy samples were more than 2SD. In this study, authors analyzed hybridization efficiencies of 8 probes for chromosome 21, and the presence of a trisomy was considered if at least 4 of the 8 probes gave probe ratio of ＞1.3. Conclusion The data suggested that MLPA is a rapid, simple and reliable method for large scale testing for aneuploidy of chromosomes 13, 18, 21, X, or Y in AF. The MLPA technology is complementary to AF culture and valuable for prenatal diagnosis.     

应用多重连接依赖式探针扩增技术快速高通量诊断胎儿染色体非整倍体异常

Objective To assess the diagnostic value of multiplex ligation-dependent probe amplification (MLPA) for detection of common chromosome aneuploidy in amniotic fluid (AF) cells in order to obtain an accurate, rapid, cost-effective and high-throughput method in routine prenatal clinical practice.Methods The MLPA test was performed on 500 AF samples by using kit P095 and the results were obtained by using analysis software RH-MLPA-v511. The results were compared with that from fluorescence in situ hybridization (FISH) and traditional karyotyping (TK). The technical critical issues were analyzed in routine diagnostic application. Results The absolute specificity and sensitivity of the MLPA test to detect the aneuploidy were 100%. For the 500 AF samples, the success rate of the MLPA tests was 97%. Among them 92% were finished within three working days and 5% required more days for repeating. The test failure rate was 3%. The results confirmed that for the 38 detectable aneuploid samples,the probe reliability weighted mean ratio values were more than 4SD compared to normal diploids and the 2 suspected trisomy samples were more than 2SD. In this study, authors analyzed hybridization efficiencies of 8 probes for chromosome 21, and the presence of a trisomy was considered if at least 4 of the 8 probes gave probe ratio of ＞1.3. Conclusion The data suggested that MLPA is a rapid, simple and reliable method for large scale testing for aneuploidy of chromosomes 13, 18, 21, X, or Y in AF. The MLPA technology is complementary to AF culture and valuable for prenatal diagnosis.     

多重链接探针扩增技术在染色体非整倍体畸变诊断中的应用

目的 探讨多重连接探针扩增(multiplexligation-dependent probe amplification,MLPA)技术在13、18、21、X、Y染色体非整倍体畸变诊断中的应用价值.方法 收集本院经染色体核型分析确诊包括有上述5种染色体数目异常及正常的样本共44份,其中外周血30份、胎儿脐带血10份、羊水4份,提取标本DNA,采用MLPA技术对样本染色体数目进行分析,并与染色体核型分析结果进行比对.结果 42例样本检测结果与染色体核型分析结果一致,1例染色体核型分析未能作出判断的标记染色体片段被识别为Y染色体片段,1例21-三体嵌合体未能做出明确判断,临床检出率97.7%(43/44).结论 MLPA技术通过单管反应同时检测40多个不同靶基因序列的拷贝数,具有高通量、特异、便捷及成本较低等特点,可应用于常见染色体非整倍体畸变的临床诊断和产前诊断.

Abstract：

Objective To investigate the application value of the multiplex ligation-dependent probe amplification (MLPA) technique in diagnosis and prenatal diagnosis of chromosomes 13, 18, 21, X and Yaneuploidy. Methods Forty-four cases including 30 peripheral blood samples, 10 fetal cord blood samples,and 4 amniotic fluid samples were collected in this study. DNA was isolated from the samples and detected by MLPA, followed by analyzing in ABI310 Genetic Analyzer. Analysis of copy number changes for chromosomes 13, 18, 21, X and Y was carried out with RH-MLPA-analysis software. The routine karyotype analyses were also done for all the samples. Results Of 44 samples, the results of 42 by MLPA method was consistent with that by chromosome karyotyping. Only one case with trisomy 21 chimerism was failed to reach conclusion. In addition, one case of mark chromosome segment was identified as Ychromosome segment by MLPA, while karyotyping failed to make judgment. The accurate rate of MLPA was 97. 7% (43/44). Conclusion The MLPA technique can simultaneously detect dozens of different target sequences and their copy number changes in a single reaction. It showed high specificity, good reproducibility, was fast and high-throughput. The MLPA technique can be applied to diagnosis and prenatal diagnosis of the common chromosomal aneuploidy.     

多重链接探针扩增技术在染色体非整倍体畸变诊断中的应用

目的 探讨多重连接探针扩增(multiplexligation-dependent probe amplification,MLPA)技术在13、18、21、X、Y染色体非整倍体畸变诊断中的应用价值.方法 收集本院经染色体核型分析确诊包括有上述5种染色体数目异常及正常的样本共44份,其中外周血30份、胎儿脐带血10份、羊水4份,提取标本DNA,采用MLPA技术对样本染色体数目进行分析,并与染色体核型分析结果进行比对.结果 42例样本检测结果与染色体核型分析结果一致,1例染色体核型分析未能作出判断的标记染色体片段被识别为Y染色体片段,1例21-三体嵌合体未能做出明确判断,临床检出率97.7%(43/44).结论 MLPA技术通过单管反应同时检测40多个不同靶基因序列的拷贝数,具有高通量、特异、便捷及成本较低等特点,可应用于常见染色体非整倍体畸变的临床诊断和产前诊断.     

多重链接探针扩增技术在染色体非整倍体畸变诊断中的应用

目的 探讨多重连接探针扩增(multiplexligation-dependent probe amplification,MLPA)技术在13、18、21、X、Y染色体非整倍体畸变诊断中的应用价值.方法 收集本院经染色体核型分析确诊包括有上述5种染色体数目异常及正常的样本共44份,其中外周血30份、胎儿脐带血10份、羊水4份,提取标本DNA,采用MLPA技术对样本染色体数目进行分析,并与染色体核型分析结果进行比对.结果 42例样本检测结果与染色体核型分析结果一致,1例染色体核型分析未能作出判断的标记染色体片段被识别为Y染色体片段,1例21-三体嵌合体未能做出明确判断,临床检出率97.7%(43/44).结论 MLPA技术通过单管反应同时检测40多个不同靶基因序列的拷贝数,具有高通量、特异、便捷及成本较低等特点,可应用于常见染色体非整倍体畸变的临床诊断和产前诊断.     

多重连接探针扩增检测Williams综合征微缺失

MLPA是一种简便快速检测缺失及重复突变的方法,该技术弥补了荧光原位杂交技术的不足,可用于实验室对Williams综合征的快速检测,具有一定临床诊断价值.     

采用多重连接探针扩增技术检测智力低下儿童的基因缺陷

目的 探讨原因不明智力低下儿童的发病与染色体亚端粒基因重组间的关系.方法 采用多重连接探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测30名原因不明的综合征性智力低下患儿的染色体亚端粒区域.结果 检测到5例患儿存在染色体亚端粒的基因缺失或重复突变,分别为4p缺失,21q重复,10p重复、4p缺失,15p重复,3p重复、9p缺失.结论 不明原因智力低下儿童的发病与染色体亚端粒基因重组密切相关.MLPA技术可以作为一种高效、特异的方法对智能障碍儿童进行基因缺陷筛查.     

应用多重连接依赖性探针扩增技术进行脊髓性肌萎缩症的基因诊断及产前诊断

目的 探讨多重连接依赖性探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术在脊髓性肌萎缩症(spinal muscular atrophy,SMA)基因诊断及产前诊断中的应用.方法 选择来自8个SMA家系的患者4例,父母16例,胎儿4例,应用MLPA技术进行分析,对患者同时应用聚合酶链反应-限制性片段长度多态性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)方法进行分析.结果 对患者的检测,MLPA分析结果与PCR-RFLP检测结果相符,4例患者的运动神经元存活基因(survival motor neuron gene,SMN)1的第7和第8外显子均为纯合缺失.除家系1、4母亲的SMN1基因MLPA检测结果与其他家系不同外,其余各家系14名父母均明确诊断为SMN1基因杂合缺失突变携带者.结论 MLPA技术是一种准确可靠的基因定量分析方法 ,适合于SMA患者、携带者的基因诊断及产前诊断.     

多重连接探针扩增技术的原理及其在儿科学中的应用

多重连接探针扩增技术（mu ltip lex ligation-dependentprobe amp lification,MLPA）于2002年由Schouten等首先报道,是近几年发展起来的一种针对待检DNA序列进行定性和半定量分析的新技术。该技术高效、特异,在一次反应中可以检测45个核苷酸序列拷贝数的改变,目前已经应用于多个领域、多种疾病的研究。     

应用多重连接探针扩增技术行脊髓性肌萎缩症产前诊断的结果分析与遗传咨询指导

目的:对脊髓性肌萎缩症(spinal muscular atrophy, SMA)家系进行产前诊断,为SMA产前分子诊断提供遗传咨询指导意见。方法:纳入2016年至2019年在本院产前诊断中心就诊的21个家系开展研究,应用多重连接探针扩增(multiplex ligation-dependent probe ampli...     

应用甲基化特异性多重连接依赖性探针扩增和甲基化特异性PCR检测Prader-Willi综合征

目的比较甲基化特异性多重连接依赖性探针扩增和甲基化特异性PCR两种方法检测Prader–Willi综合征。方法应用细胞遗传学、甲基化特异性多重连接依赖性探针扩增和甲基化特异性PCR方法检测1个Prader–Willi综合征家系。结果甲基化特异性多重连接依赖性探针扩增和甲基化特异性PCR方法均能对患者进行检测,为缺失型致病,而其父母未见异常。结论甲基化特异性多重连接依赖性探针扩增方法检测Prader–Willi综合征比甲基化特异性PCR方法提供更多的致病信息。     

胎儿心脏结构异常与羊水染色体非整倍体及拷贝数变异的相关性

目的:探讨胎儿心脏结构异常与羊水染色体非整倍体及拷贝数变异(copy number variation,CNV)的相关性。方法:对328例孕妇进行胎儿超声检查及染色体微阵列分析(chromosomal microarray analysis,CMA),根据胎儿的心脏结构将其分为正常组( n＝273)与异常组...     

MLPA技术在检测胎儿非整倍体异常中的应用

目的探讨多重探针连接依赖式扩增（multiplex ligation—dependent probe amplification,MLPA）在快速检测胎儿染色体非整倍体异常中的应用价值。方法344例产前诊断标本同时进行MLPA检测及核型分析,所有样本进行MLPA获得的扩增产物信息经Coffalyserv9．4软件（Holland—MRC公司）进行定量分析,观察样本DNA拷贝数的变化,并将所得结果与染色体核型分析结果进行比较,计算其检测的敏感度、特异度及阳性预测值。结果MLPA分析在标本接收后24h内即可得出结果,共检出染色体倍体异常产前诊断标本5例,包括唐氏综合征（Downsyndrome,47,＋21）6例、爱德华氏综合征（Edwardssyndrome,47,＋18）l例。MLPA检测24h报告结果临床符合率为97．7％。结论MLPA检测21、18、13、X、Y等染色体非整倍体疾病时,与核型分析相比较,MLPA是一种快速、高效的分析非整倍体的产前诊断的方法,具有临床应用价值。     

产前染色体微阵列分析结果为新发临床意义不明变异胎儿的随访结果

目的探讨产前染色体微阵列分析(CMA)结果为新发临床意义不明变异(VOUS)胎儿的临床预后。方法以2017年7月至2021年12月在南京大学医学院附属鼓楼医院产前诊断中心接受检测的6 826例胎儿为研究对象。回顾性分析其CMA检测的结果, 对判定为新发VOUS的胎儿进行随访。结果在6 826例胎儿中, 506例为VOUS, 其中237例进行了溯源检测, 24例为新发变异。有效随访20例, 随访时间范围为出生后4 ～ 26个月, 其中4例引产, 4例出生后出现临床表型, 12例未见异常。结论建议对VOUS进行溯源检测, 并对携带新发VOUS的胎儿进行持续随访, 以明确VOUS的临床意义。     

NIPT-plus在胎儿染色体拷贝数变异筛查中的应用

目的 分析新型无创产前基因检测(noninvasive prenatal testing-plus,NIPT-plus)在胎儿染色体拷贝数变异筛查中的应用价值.方法 回顾性分析104例实施胎儿染色体拷贝数变异筛查的孕妇资料,均分别采用NIPT-plus和无创产前基因检测(noninvasive prenatal tes...     

早孕期超声异常胎儿的染色体核型分析北大核心CSCD

目的探讨早孕期（孕11～14周）不同的超声异常指标对于筛查胎儿染色体异常的价值。方法对159例孕11～14周超声发现异常的胎儿进行绒毛或羊水核型分析,比较不同的超声异常指标所对应的胎儿染色体异常的检出率。结果最常见的早孕期超声异常指标依次为颈部透明层（nuchal translucency,NT）增厚、全身皮肤水肿、颈部淋巴水囊瘤、鼻骨缺失或显示不清、心脏畸形和静脉导管a波倒置。NT增厚的染色体异常检出率为34．5％（39／113）,主要为21三体、18三体和45,x。全身皮肤水肿的染色体异常检出率为76．7％（23／30）,以45,X、18三体和21三体为主。颈部淋巴水囊瘤的染色体异常检出率为62．5％（10／16）,以45,x和21三体为主。鼻骨缺失或显示不清的染色体异常检出率为71．4％（10／14）,以21三体和18三体为主。心脏畸形的染色体异常检出率为72．7％（8／11）,以21三体和18三体为主。静脉导管a波倒置的染色体异常检出率为50．0％（4／8）,其中21三体2例、18三体和45,X各1例。结论NT增厚、全身皮肤水肿、颈部淋巴水囊瘤、鼻骨缺失或显示不清、心脏畸形、静脉导管a波倒置是最常见的与染色体异常相关的早孕期超声异常指征。全身皮肤水肿、心脏畸形、鼻骨缺失或显示不清、颈部淋巴水囊瘤是染色体异常检出率最高的4种指征。这些指征同时出现得越多,染色体异常检出率越高。     

一例心脏缺陷胎儿染色体1p36．3微缺失的产前诊断北大核心CSCD

目的对1例心脏缺陷胎儿进行细胞遗传学诊断,明确其病因,为再生育复发风险评估及产前诊断提供依据。方法应用单核苷酸多态微阵列芯片（single nucleotide polymorphism—based arrays,SNP—array）对胎儿及其父母行全基因组DNA扫描分析,结合G显带核型分析及荧光原位杂交（fluorescence in situ hybridization,FIsH）验证胎儿染色体1p36．3微缺失的来源。结果高密度SNP—array芯片检测显示胎儿在1p36．33p36．23存在6．9Mb的微缺失,G显带核型分析和FISH检测提示其父亲核型为46,XY,t（1;14）（p36．3;p12）,胎儿携带一条由父亲1P末端和14p末端平衡易位形成的1号衍生染色体,核型为46,XY,der（1）t（1;14）（p36．3;p12）pat。结论SNP—array结合G显带和FISH技术有助于发现染色体末端隐匿性易位、微缺失或微重复,提高诊断准确率,对复发风险的评估有重要价值。