摘要
目的叶酸代谢的异常是导致反复流产、神经管畸形、先天性心脏病、高血压等多种疾病的高危因素,严重危害个体健康。个体携带MTHFR677、MTHFR1298或MTRR66致病变异时,会导致叶酸代谢通路受阻,是叶酸代谢异常的关键检测位点。本研究拟开发一种快速检测MTHFR677、MTHFR1298和MTRR66变异的实时荧光检测体系。方法采用位点特异性PCR(AS-PCR)结合Taqman探针法开发实时荧光检测体系,分两管完成检测。结果采用已知基因型样本和随机样本对构建的实时荧光分析系统进行验证。经采用商品化试剂盒或测序法进行验证,本研究组所开发的检测体系的检测结果与验证方法的检测结果一致。300例随机样本检测结果显示,在本区域随机人群中,各SNP对应的基因频率分别为MTHFR1298C 29.83%、MTHFR1298A 70.17%;MTHFR677T 19.00%、MTHFR677C 81.00%;MTRR66G27.00%、MTRR66A73.00%。结论本研究成功开发了一种快速检测MTHFR677、MTHFR1298和MTRR66基因多态性的检测方法。该方案简单易用,可以满足大部分已配备实时荧光检测系统的实验室的使用要求,检测速度快,自带内参基因、可以单样本检测,适用性较强。经验证,该体系检测结果与金标准(测序法)一致,是一种分析MTHFR677、MTHFR1298和MTRR66基因多态性的有效方法。
Objective Abnormal folic acid metabolism is a high-risk factor leading to recurrent miscarriage,neural tube malformation,congenital heart disease,hypertension and other diseases,which seriously endangers individual health.When individuals carry pathogenic variants of MTHFR677,MTHFR1298 or MTRR66,the folic acid metabolism pathway will be blocked,which is a key detection site for abnormal folate metabolism.This research intends to develop a real-time fluorescence detection system for rapid detection of MTHFR677,MTHFR1298 and MTRR66 mutations.Methods Allele-specific PCR(AS-PCR)combined with Taqman probe method was used to develop a real-time fluorescence detection system,and the detection was completed in two tubes.Results The constructed real-time fluorescence analysis system was verified with verified genotype samples and random samples.After verification using commercial kits or sequencing methods,the test results of the test system developed by this research group are consistent with those of the verification method.The test results of 300 random samples showed that in the random population of this region,the gene frequencies corresponding to each SNP were:MTHFR1298 C 29.83%,MTHFR1298 A 70.17%;MTHFR677 T 19.00%,MTHFR677 C 81.00%;MTRR66 G 27.00%,MTRR66 A 73.00%.Conclusion This study successfully developed a rapid detection method for MTHFR677,MTHFR1298 and MTRR66 gene mutations.This solution is simple and easy to use,and can meet the requirements of most laboratories equipped with real-time fluorescence detection systems.The detection speed is fast,with internal reference genes,single-sample detection,and strong applicability.It has been verified that the test results of this system are consistent with the gold standard(sequencing method),and it is an effective method for analyzing the polymorphisms of MTHFR677,MTHFR1298 and MTRR66 genes.
作者
龙驹
LONG Ju(Qinzhou Maternal and Child Health Care Hospital,Qinzhou,Guangxi 535099,China;Xi'an Jiaotong University,School of Basic Medical Sciences,Xi'an,Shaanxi 710061,China)
出处
《中国优生与遗传杂志》
2022年第1期133-137,共5页
Chinese Journal of Birth Health & Heredity
基金
钦州市科学研究与技术开发计划项目(20189914)
广西医学高层次骨干人才“139”计划培养人选(G201903017)。
