用不提取核酸PCR扩增孕妇血浆中胎儿游离DNA的SRY和FMR1基因

1001-764X201304-253-03··PCRDNASRYFMR1＊?陈斌a严伟a张琼a邹晓b周小棉a钟志敏a广州医学院附属广州市第一人民医院a．检验科b．妇产科广州510180建立一种不提取核酸的孕妇血浆中胎儿游离DNA的检测方法。构建不提取核酸PCR法用巢式PCR扩增Y染色体上的SRY基因序列和X染色体上的FMR1基因序列检测44例孕妇血浆中游离DNA。根据随访判断结果的准确性。44例孕妇经随访确认24例有男性胎儿20例有女性胎儿。24例孕男性胎儿的孕妇血浆标本中有21例扩增出SRY基因和FMR1基因敏感性为87．5%21/2420例孕女性胎儿的孕妇血浆标本中有17例扩增出FMR1基因敏感性为85．0%17/20。建立的不提取核酸PCR法具有快速、简便等优点可用于性连锁遗传病的产前诊断。胎儿游离DNASRY基因FMR1基因不提取核酸聚合酶链反应R714．55AExtraction-freePCRforamplificationofSRYandFMR1genefromfreefetalDNAinmaternalplasmaCHENBin1YANWei1ZHANGQiong1ZOUXiao2ZHOUXiao-mian1ZHONGZhi-min11．DepartmentofLaboratoryMedicine2．DepartmentofObstetricsandGynecologyGuangzhouFirstMunicipalPeople'sHospitalAffiliatedofGuangzhouMedicalCollegeGuangzhou510180ChinaAbstractObjectiveTodeveloparapidandsimplemethodforthedetectionoffreefetalDNAinmaternalplasma．MethodsADNAextraction-freePCRmethodwasdeveloped．Boththegene-specificsequencesofFMR1onXchromosomeandSRYonYchromo-somewereamplifiedsimultaneouslybymultiplexnestedPCR．ThefreefetalDNAinmaternalplasmafrom44pregnantwomenwasde-tected．Onthebasisoffollow-upresultstheaccuracyofthedevelopeddirectPCRmethodwasevaluated．ResultsBoththeFMR1andSRYgene-specificsequencesweredetectablein21of24maternalplasmasamplesobtainedfromthepregnantwomencarryingmalefe-tus．SingleFMR1gene-specificsequencewasdetectablein17of20plasmasamplesofpregnantwomencarryingfemalefetus．Theac-curacyrateofthemethodforgenderpredictionoffetuswere87．5%21/24formalefetusand85%17/20forfemalefetusrespec-tivelycomparedwiththegenderidentificationafterdelivery．ConclusionTheDNAextraction-freePCRdevelopedinthisstudywasrapidsimplefordetectionoffreeDNAinmaternalplasmathusitshouldbeusefulforprenataldiagnosisofsex-linkedgeneticdisease．KeywordsfreefetalDNASRYgeneFMR1geneDNAextraction-freePCR、、1。DNA2DNADNADNADNA、。PCRDNAYSRYXFMR1、。11．12010102011124420～35。8～16Hb＞90g/L。。55。。1．2PCR、MightyAmpDNA、DNAMarkerDL2000SYBGreenⅠMagaBioDNA·352·20134314ChinJClinLabSciApr．2013Vol．31No．4*81201163、812717302010B080702018201102A212028、20121A011035。1983。[e-mail]。/BioFluxMyCyclerPCRBio-RadUVIUltrospec2100proGE。1．34mLEDTA1500r/min10min－20℃。MagaBioDNA/55DNA。DNAA260/280nm1．7～1．9。1．4PCRXFMR1YSRY3-4GenBankNT_011681．16NT_011878．9PrimerPremier5．01。FMR1．1、FMR1．2、SRY1SRY2PCRFMR1．1FMR1．2XFMR1ATL1STS301bpSRY1SRY2YSRY239bpFMR1．3、FMR1．2、SRY3SRY4FMR1．3FMR1．2XFMR1261bpSRY1SRY2YSRY198bp。。1SRYFMR15'→3'℃FMR1．1CCCTGATGAAGAACTTGTATCTC53FMR1．2GAAATTACACACATAGGTGGCACT54FMR1．3TCGCCTTTCTCAAATTCCAAG50SRY1CTAGACCGCAGAGGCGCCCAT60SRY2TAGTACCCACGCCTGCTCCGG60SRY3CATCCAGAGCGTCCCTGGCTT58SRY4CTTTCCACAGCCACATTTGTC52PCR25μL2μL1UMightyAmpDNA0．2μL0．2μmol/L2．5μL2×MightyPCRPoly-meraseMix12．5μLddH2O7．8μL。PCR25μLPCR2μL1UTaq0．2μL0．2μmol/L2．5μL250μmol/LdNTP2μL10×PCRbuffer2．5μLddH2O16．8μL。PCR95℃15min95℃10s55℃30s72℃30s4072℃2min。PCR20g/L。DNA。PCR1261bpXFMR1PCR2198bp261bpYSRYXFMR1。22．11、2、3、45μL1PCR20g/L。2、34μL2198bp261bp。2μL。2．2SRYFMR1711261bp2～72198bp261bp。1。MDNAmarkerDL20001～7112～728DNA9DNA。1PCR2．3442420。24212261bp198bp387．5%21/24。20171261bp32261bp198bp85．0%17/20。·452·20134314ChinJClinLabSciApr．2013Vol．31No．43DNA、7。DNA、5DNA、DNADNA。DNA。DNADNAQIAampDNA。DNA。DNAPCR。DNAPCR、6。PCR。YSRYXFMR1DNA。SRYFMR1。DNA2h。、。DNA。。41TaborAAlfirevicZ．Updateonprocedure-relatedrisksforprenataldiagnosistechniquesJ．FetalDiagnTher20102711-7．2SekizawaAPurwosunuYMatsuokaRetal．Recentadvancesinnon-invasiveprenatalDNAdiagnosisthroughanalysisofmaternalbloodJ．JObstetGynaecolRes2007336747-764．3TungwiwatWFucharoenGRatanasiriTetal．Non-invasivefetalsexdeterminationusingaconventionalnestedPCRanalysisoffetalDNAinmaternalplasmaJ．ClinChimActa20033341-2173-177．4MiuraKHigashijimaAShimadaTetal．Clinicalapplicationoffetalsexdeterminationusingcell-freefetalDNAinpregnantcarriersofX-linkedgeneticdisordersJ．JHumGenet2011564296-299．5．J．2007255395-396．6KermekchievMBKirilovaLIVailEEetal．MutantsofTaqDNApolymeraseresistanttoPCRinhibitorsallowDNAamplificationfromwholebloodandcrudesoilsamplesJ．NucleicAcidsRes20097540．2012-08-192013-01-24许晓蒙陈维忠····为维护本刊声誉和广大读者的利益对于一稿两投和一稿两用问题的处理声明如下。1．本声明中所说的“一稿两投”或“一稿两用”均指同一原始研究的报告“两投”或“两用”或虽然2篇文稿在文字的表达和讨论的叙述上有某些不同但其主要数据和图表是相同的。所指文稿不包括重要会议的纪要、疾病的诊断标准和防治指南、有关组织达成的共识性文件、新闻报道类文稿以及在一种刊物发表过摘要或初步报道而将全文投向另一种期刊的文稿即如果1篇文稿已以全文方式在某刊物发表除非文种不同否则不可再将该文投寄给他刊。2．读者举报文稿涉嫌一稿两投时编辑部将认真收集有关资料并仔细核实。确认属于一稿两投时将立即进行退稿处理。3．一稿两投或两用一经证实本刊将择期在杂志中发布撤销该论文的声明两年内拒绝刊登该文作者作为第一作者所撰写的一切文稿并向作者所在单位和该领域内的其他科技期刊进行通报。《》·552·20134314ChinJClinLabSciApr．2013Vol．31No．4