CAJ | 학술논문

目的建立一种TaqMan real-time PCR探针杂交法,用于快速检测人rs12979860位点多态性.方法从人各不同组织器官中提取基因组DNA,利用小沟结合蛋白(MGB)探针的高度特异性,设计相应的引物探针,根据实时荧光定量PCR过程中产生的信号在2个检测通道的强弱变化来鉴别等位基因差异.最后所测样本经DNA测序验证结果的准确性.采用x2检验进行统计分析.结果利用TaqMan MGB蛋白作为探针,能够区分只有1个碱基差异的目标基因组片段.该方法最低检测浓度为1.5 ng/μl,扩增有效率达97.6％.不同组织器官(肾、心脏、子宫、血浆)及分泌物(鼻咽部)的DNA均可以用于检测.在随后对40例高加索人和50例黄种人的检测中亦证实,rs12979860位点的基因型及等位基因频率存在差异,高加索人以CT基因型为主(26/40),而黄种人以CC基因型为主(40/50),差异有统计学意义(x2=18.75,P＜0.05).在两种族人群中,抗病毒治疗血清学转换与患者基因型之间差异无统计学意义(P均＞0.05).结论 TaqMan MGB法能快速并准确地区分rs12979860位点多态性,具有高度特异性和灵敏度,适用于多种样本的临床检测,是对丙肝患者抗病毒个体化治疗进行遗传分析的理想工具.
목적 건립일충TaqMan real-time PCR탐침잡교법,용우쾌속검측인rs12979860위점다태성.방법 종인각불동조직기관중제취기인조DNA,이용소구결합단백(MGB)탐침적고도특이성,설계상응적인물탐침,근거실시형광정량PCR과정중산생적신호재2개검측통도적강약변화래감별등위기인차이.최후소측양본경DNA측서험증결과적준학성.채용x2검험진행통계분석.결과 이용TaqMan MGB단백작위탐침,능구구분지유1개감기차이적목표기인조편단.해방법최저검측농도위1.5 ng/μl,확증유효솔체97.6％.불동조직기관(신、심장、자궁、혈장)급분비물(비인부)적DNA균가이용우검측.재수후대40례고가색인화50례황충인적검측중역증실,rs12979860위점적기인형급등위기인빈솔존재차이,고가색인이CT기인형위주(26/40),이황충인이CC기인형위주(40/50),차이유통계학의의(x2=18.75,P＜0.05).재량충족인군중,항병독치료혈청학전환여환자기인형지간차이무통계학의의(P균＞0.05).결론 TaqMan MGB법능쾌속병준학지구분rs12979860위점다태성,구유고도특이성화령민도,괄용우다충양본적림상검측,시대병간환자항병독개체화치료진행유전분석적이상공구.
Objective Establishment and development of a novel Single-Nucleotide-Polymorphism TaqMan Real-Time PCR assay for rapid detection of rs12979860 that predicts HCV therapy response.Methods Human genomic DNA were extracted from solid tissues,secretion and plasma before allelic discrimination.With the property of minor groove binding protein (MGB) binding to minor groove of DNA with strong specificity and affinity,primers and MGB probes were particularly designed for differentiation of human genomic frequencies.MGB probe-based real-time PCR was established to increase allelic discrimination using two probes that only differ in one nucleotide of IL28B rs12979860.The specificity was evaluated by fluorescence signal emissions which were selected from two signal channels.And DNA sequencing was used to confirm the genomic polymorphisms.Results TaqMan probe-based SNP real-time PCR increased allelic discrimination using two probes that only differ by one nucleotide of amplicon,which indicated this assay was easily performed regardless of genomic DNA concentration and quality,minimizes sources of error.The sensitivity was as low as 1.5 ng/μl,the amplification efficacy was 97.6％.The genotype frequencies of CC,CT were remarkably different between Caucasian and Mongolian.The dominated genotype of Caucasian was CT,while most Mongolian was carried CC (26/40 vs.40/50,x-2 =18.75,P value ＜ 0.05).However,the genotype between two population showed no relationship with their virological clearance clinically (P value ＞ 0.05).Conclusions This TaqMan MGB assay shows highly specificity,which has potential as a routine diagnostic test for the detection of rs12979860 from various types of samples.This robust assay would be widely used clinically to predict patients' response before anti-HCV treatment in personalized medicine.