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飞行时间质谱与TaqMan探针技术在结核病易感性相关基因单核苷酸多态性筛选中的应用
引用本文:汪文斐,张国良,杨帆,杨辉,赵丽芳,徐发圣,张明霞,陈心春.飞行时间质谱与TaqMan探针技术在结核病易感性相关基因单核苷酸多态性筛选中的应用[J].中国防痨通讯,2014(6):434-439.
作者姓名:汪文斐  张国良  杨帆  杨辉  赵丽芳  徐发圣  张明霞  陈心春
作者单位:[1]深圳市第三人民医院广东省新发传染病诊治重点实验室,518112 [2]安徽省蚌埠医学院生命科学学院,518112
基金项目:国家自然科学基金(81341128);广东省自然科学基金(S2012040007213);深圳市科技计划项目(201202189)
摘    要:目的 对比分析飞行时间质谱技术(MALDI-TOF)和TaqMan探针筛选与结核病易感性相关单核苷酸多态性(SNP)位点的结果,以及联合应用的方法学和效果评价.方法 选取2010年10月至2011年4月在深圳市第三人民医院收治并确诊的结核病患者400例为结核病组,对照组为同时期收集的健康体检者300名,利用MALDI-TOF对选取的7个SNP位点(rs2227476、rs1800795、rs56077270、rs1800797、rs2227484、rs2227472和rs2227473)同时进行基因分型,初步筛选易感SNP位点;与结核病易感相关的单个SNP位点,采用基于TaqMan探针技术的实时荧光定量PCR对同样的标本再进行基因分型,比较两种方法的准确性与敏感度;以rs2227473位点为实例对分型结果的基因频率进行分析,确定肺结核的易感SNP.结果 MALDI-TOF分型成功率为99.7%(698/700),TaqMan探针技术为98.4%(689/700);在基因分型过程中,MALDI-TOF与TaqMan探针方法对1例标本的分型结果不一致,经过对此分型结果进行了测序验证,MALDI-TOF的分型结果正确,MALDI-TOF在准确性和敏感度比TaqMan法稍高.位点rs2227473基因频率分析中,结核病组G等位基因频率(90.3%,722/800)明显高于对照组(82.5%,495/600)(x2=6.911,P=0.009).结论 上述肺结核易感基因的筛选方法是可行的;实例分析中,将两种方法联合应用,发现了IL-22基因rs2227473位点等位基因G可能与肺结核发病相关,两位点中等位基因A可能为保护性基因.

关 键 词:结核    多态性  单核苷酸  白细胞介素类  光谱法  质量  基质辅助激光解吸电离  DNA探针  疾病遗传易感性

Methodological comparison of MALDI-TOF and TaqMan probe technology in SNP genotyping and their application in screening SNP with susceptibility to tuberculosis
Institution:WANG Wen-fei,ZHANG Guo-liang,YANG Fan,YANG Hui,ZHAO Li-fang XU Fa-sheng ZHANG Ming-xia( 1.Third People's Hospital of Shenzhen, Guangdong Key Laboratory for Emerging infectious Disease, Shenzhen 518112, China;)
Abstract:Objective To develop a new method based on MALDI-TOF and TaqMan probe for screening the single nucleotide polymorphisms (SNP) with susceptibility to tuberculosis and evaluate its clinical application value.Methods At first,total 7 sites we targeted were detected by MALDI-TOF simultaneously.The single SNP site was detected by using TaqMan probe technology.The two methods were compared by accuracy and sensitivity.We determined a susceptibility gene site by comparing genotype and gene frequency between 400 TB patients and 300 healthy controls by using this method.Results The success rate of genotyping by MALDI-TOF was 99.7% (698/700),that of TaqMan probe technology was 98.4% (689/700).The frequency of G allele at rs2227473 in patient groups (90.3%,722/800) was significantly higher than that in control groups (82.5%,495/600) (x2=6.911,P =0.009).The other 6 SNP sites had no significant difference between two groups.Conclusion We successfully established a new method for screening the SNPs with susceptibility to tuberculosis.The polymorphism of IL22 rs2227473 allele G may be associated with the susceptibility to pulmonary tuberculosis,allele A may be the protective gene.
Keywords:Tuberculosis  pulmonary  Polymorphism  single nucleotide  Interleukins  Spectrometry  mass  matrix-assisted laser desorption-ionization  DNA probes  Genetic predisposition to disease
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