摘要
目的 构建可同时检测多个辐射相关基因单核苷酸多态性(single nucleotide polymorphism, SNP)的高通量、高分辨率、快速的方法。方法 选取2020年4~6月火箭军特色医学中心75例年满18周岁的健康男性为研究对象,采集外周血并提取DNA。文献筛选辐射相关17种基因28个SNP,设计多重PCR引物和单碱基延伸引物,通过PCR扩增和延伸,应用DP-TOF型飞行时间质谱检测系统对SNP位点进行基因分型,并对其中7份样本用Sanger测序法进行验证。结果 构建的28个SNP均分型成功。75份DNA样本28个SNP的基质辅助激光解吸电离飞行时间质谱(martix-assisted laser desorption/ionization time of flight mass spectrometry, MALDI-TOF-MS)检测结果显示基因分型检出率为100.0%。7份DNA样本28个SNP的基因型与Sanger测序结果完全符合,其准确度为100.0%。结论 成功构建可同时检测17种基因28个辐射相关SNP的核酸质谱检测技术平台,该方法具有高通量、快速准确的优点。
Objective To establish a high-throughput,high-resolution and fast method that can simultaneously detect multiple single nucleotide polymorphisms(SNPs)of genes associated with radiation.Methods 75 healthy males over the age of 18 at the PLA Rocket Force Characteristic Medical Center from April 2020 to June 2020 were selected as the research subjects to collect peripheral blood and extract DNA.17 genes with 28 SNPs were screened from literature.Multiple PCR primers and single base extension primers were designed.Genotypes of SNPs were tested with the DP-TOF time-of-flight mass spectrometry detection system after PCR amplification and extension,and 7 of them were verified by Sanger sequencing method.Results The 28 SNPs constructed were genotyped successfully.28 SNPs in 75 DNA samples detected by(martix-assisted laser desorption/ionization time of flight mass spectrometrym,MALDI-TOF-MS)showed a genotyping detection rate of 100.0%.The genotyped of 28 SNPs from 7 samples were completely consistent with Sanger’s sequencing results,with an accuracy of 100.0%.Conclusion The nucleic acid mass-spectrometric detection technology platform which could simultaneously detect 28 SNPs of 17 genes associated with radiation was successfully constructed.It has the advantages of high-throughput,fast and accuracy.
作者
郝文霞
张阳东
宋丽杰
高峥
牛冬梅
楚瑞雪
HAO Wenxia;ZHANG Yangdong;SONG Lijie;GAO Zheng;NIU Dongmei;CHU Ruixue(the PLA Rocket Force Characteristic Medical Center,Beijing 100088,China;Rocket Army 96606 Military Hospital,Henan Luoyang 471003,China;Lishilu Clinic,Central Medical Branch of PLA General Hospital,Beijing 100820,China)
出处
《现代检验医学杂志》
CAS
2023年第5期5-11,共7页
Journal of Modern Laboratory Medicine
基金
军队后勤面上项目(CEP21J009)
中心创新课题(ZXKC19019)。
关键词
电离辐射
单核苷酸多态性
核酸质谱技术
ionizing radiation
single nucleotide polymorphism
nucleic acid mass spectrometry technology
