CAJ | 학술논문

目的建立应用高分辨率熔解曲线(high resolution melting curve method,HRM)和焦磷酸测序技术快速筛查亚甲基四氢叶酸还原酶基因677C＞T位点多态性的方法.方法收集唐氏综合征患者样本155例、健康儿童样本182名,应用3种HRM技术(常规法、内标法和异源双链法)进行677C＞T位点多态性检测,并比较3种方法的优劣.采用焦磷酸测序技术快速测定样本中677C＞T位点等位基因频率,并与HRM检测的结果进行比较.结果常规的HRM可区分纯合子与杂合子,但分辨基因型CC和TT的效果不佳.内标法和异源双链法能够有效分辨3种基因型.对焦磷酸测序结果的分析提示,其检测混合样本中等位基因频率的准确度随样本数增加而提高.当样本数超过30时,结果与实际值的差异小于4％.统计结果还提示,唐氏综合征患者组基因型TT的频率明显高于对照组(25.2％ vs.14.3％,P＜0.05),而等位基因T的频率仅略高于对照(44.9％ vs.40.1％,P＞0.05).结论内标法HRM分析可作为检测等位基因型的首选方法,而焦磷酸测序则可快速测定群体中的等位基因频率.
목적 건립응용고분변솔용해곡선(high resolution melting curve method,HRM)화초린산측서기술쾌속사사아갑기사경협산환원매기인677C＞T위점다태성적방법.방법 수집당씨종합정환자양본155례、건강인동양본182명,응용3충HRM기술(상규법、내표법화이원쌍련법)진행677C＞T위점다태성검측,병비교3충방법적우렬.채용초린산측서기술쾌속측정양본중677C＞T위점등위기인빈솔,병여HRM검측적결과진행비교.결과 상규적HRM가구분순합자여잡합자,단분변기인형CC화TT적효과불가.내표법화이원쌍련법능구유효분변3충기인형.대초린산측서결과적분석제시,기검측혼합양본중등위기인빈솔적준학도수양본수증가이제고.당양본수초과30시,결과여실제치적차이소우4％.통계결과환제시,당씨종합정환자조기인형TT적빈솔명현고우대조조(25.2％ vs.14.3％,P＜0.05),이등위기인T적빈솔부략고우대조(44.9％ vs.40.1％,P＞0.05).결론 내표법HRM분석가작위검측등위기인형적수선방법,이초린산측서칙가쾌속측정군체중적등위기인빈솔.
Objective To establish a rapid method for detecting MTHFR gene 677C ＞ T polymorphisms with high resolution melting curve method (HRM) and pyrosequencing.Methods Peripheral blood samples were collected from 155 Down syndrome patients and 182 normal controls from Children's Hospital of Shanghai.The accuracy of three methods including regular HRM,internal-control HRM and artificial heterozygosity HRM was compared.Meanwhile,allele frequencies in 10,30 and 50 mixed samples were measured with pyrosequencing,and the results were compared with that of HRM.Results Heterozygosity of 677C ＞ T polymorphism could be distinguished by various HRM methods.However,homozygotes CC and TT were only identifiable by internal-control HRM and artificial heterozygosity HRM.The accuracy of pyrosequencing for allele frequency has improved with increased sample number.When the number of mixed samples exceeded 30,the difference between pyrosequencing results and actual values became less than 4 ％.TT genotype was more frequent in Down syndrome patients than controls (25.2％ vs.14.3％).No significant difference was found in T allele frequency between the two groups (44.9％ vs.40.1％).Conclusion Respectively,internal-control HRM and pyrosequencing may be ideal methods for determination of genotype and allelic frequencies.