MBL基因突变检测新方法及RRTI患者的突变研究

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:MBL 54G>A突变与RRTI发病风险相关,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。

Development of a fluorescence polarization method for rapid detecting the MBL mutants and the detection in recurrent respiratory tract infections children

Objective： To develop a rapid and sensitive method for detecting MBL genetic mutants and evaluate the association with recurrent respiratory tract infections（RRTI）in children.Methods： The MBL exon-1 region was amplified by PCR,the non-incorporated primers and excesses dNTPs were removed by enzymatic digestion.The dye-terminator（R110-acyC or TAMRA-acyT） was incorporated on the 3′-end of the primer via template directed dye-terminator incorporation reaction（TDI） and the mutations were determined by fluorescence polarization（FP） assay.The MBL mutants were analyzed among 50 healthy and 46 RRTI children using this new method.Results： The accuracy and sensitivity were evaluated and verified by standard sequencing method.The 54 GA mutant was found among both healthy and RRTI children.The genotypes among healthy group were： 39 homozygote wild-type G/G（78.00%）,8 heterozygote G/A（16.00%） and 3 homozygote mutant A/A（6.00%）.The genotypes among the RRTI children were： 18 homozygote wild-type G/G（39.13%）,22 heterozygote G/A（47.83%） and 6 homozygote mutant T/T（13.14%）.The frequency of MBL mutation was significant higher in RRTI children than in healthy controls.The 52CT and 57GA mutants were not found in both groups.Conclusions： The MBL mutation associates with the risk of RRTI among children.This new MBL genotype can be used to help the diagnosis and screening the high risk children for RRTI.