mbl基因突变检测新方法及rrti患者的突变研究

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1、MBL基因突变检测新方法及RRTI患者的突变研究作者：薛丽,赵新,杨芳,赵锦荣,张文红,白玉杰【摘要】目的：建立一种新的MBL基因突变检测方法，并分析反复呼吸道感染儿童中突变频率及发病相关性。方法：PCR扩增MBL基因第一外显子区段，核酸外切酶和碱性磷酸酶降解,清除PCR产物中残余引物和dNTPs，经引物延伸反应荧光素(R110或TAMRA)标记终止碱基特异性掺入引物的3′末端，测定荧光偏振值判断掺入碱基及突变类型。用所建立方法检测46例反复呼吸道感染(RRTI)患儿和50例健康对照的MBL突变。结果：所

2、建立方法检测MBL突变经测序验证完全正确。在临床RRTI患儿及对照组中发现54位密码子G>A突变，健康儿童中野生纯合型(G/G)39例(78.00%)、杂合型(G/A)8例(16.00%)、纯合突变(A/A)3例(6.00%);RRTI患儿中野生纯合型(G/G)18例(39.13%)、杂合型(G/A)22例(47.83%)、纯合突变(A/A)6例(13.04%);RRTI患儿组A等位基因频率显著高于对照组。患儿和对照组均未检测到52和57位密码子突变。结论：MBL54G>A突变与RRTI发病风险

3、相关，所建立的MBL基因多态性快速检测方法可用于小儿反复呼吸道感染辅助诊断和高风险人群的筛查。【关键词】甘露聚糖结合凝集素;基因突变;反复呼吸道感染;荧光偏振15　　[ABSTRACT]Objective:TodeveloparapidandsensitivemethodfordetectingMBLgeneticmutantsandevaluatetheassociationwithrecurrentrespiratorytractinfections(RRTI)inchildren.Methods:Th

4、eMBLexon1regionwasamplifiedbyPCR,thenonincorporatedprimersandexcessesdNTPswereremovedbyenzymaticdigestion.Thedyeterminator(R110acyCorTAMRAacyT)wasincorporatedonthe3′endoftheprimerviatemplatedirecteddyeterminatorincorporationreaction(TDI)andthemutati

5、onsweredeterminedbyfluorescencepolarization(FP)assay.TheMBLmutantswereanalyzedamong50healthyand46RRTIchildrenusingthisnewmethod.Results:Theaccuracyandsensitivitywereevaluatedandverifiedbystandardsequencingmethod.The54G>Amutantwasfoundamongbothhealthyand

6、RRTIchildren.Thegenotypesamonghealthygroupwere:39homozygotewildtypeG/G(78.00%),8heterozygoteG/A(16.00%)and3homozygotemutantA/A(6.00%).ThegenotypesamongtheRRTIchildrenwere:18homozygotewildtypeG/G(39.13%),22heterozygoteG/A(47.83%)and6homozygotemutantT/T(13

7、.14%).ThefrequencyofMBLmutationwassignificanthigherinRRTIchildrenthaninhealthycontrols.The52C>Tand57G>Amutantswerenotfoundinbothgroups.Conclusions:15TheMBLmutationassociateswiththeriskofRRTIamongchildren.ThisnewMBLgenotypecanbeusedtohelpthediagnosisa

8、ndscreeningthehighriskchildrenforRRTI.　　[KEYWORDS]Mannanbindinglectin;Geneticmutant;Recurrentrespiratorytractinfections;Fluorescencepolarization　　甘露聚糖结合凝集素(mannanbindinglectin,MBL)是血浆中的胶原凝集素，其多糖识别结构域与细菌、真菌及