摘要
目的 构建子宫内膜癌PTEN基因968delA突变检测技术平台,为临床分析PTEN基因968delA突变与子宫内膜癌的相关性提供技术支持。方法 设计PTEN基因968delA位点的特异性硫化修饰引物,以本实验室前期构建的包含PTEN基因968delA位点的突变型质粒和野生型质粒为模板,进行高保真聚合酶介导的双向引物延伸反应,采用正交设计进行PCR条件优化,再将其优化后采用突变敏感性分子开关技术检测从南华大学附属第一医院收集的62例临床患者子宫内膜组织样本PTEN基因968delA位点突变。结果 突变敏感性分子开关技术检测PTEN基因968delA位点最佳PCR反应条件为:30个循环、模板DNA(10 ng/μL)0.3μL、引物(10μmol/L)0.5μL、退火温度61℃,灵敏度达10^(-3)ng/μL。62例子宫内膜癌组织与21例正常子宫内膜组织标本均未发现PTEN基因968delA突变。结论 成功构建了子宫内膜癌PTEN基因968delA突变检测技术平台,为该技术在PTEN基因968delA的突变筛查中的应用提供了依据。
Objective To construct a technical platform for detecting the 968delA mutation of PTEN gene in endometrial cancer,and provide the technical support for clinically analyzing the correlation of PTEN gene 968delA mutation with endometrial cancer.Methods The specific 3′-phosphorothioate-modified primers for the 968delA site of PTEN gene were designed.Using wild-type plasmids and the mutant-type plasmids containing the 968delA site of PTEN gene previously constructed in our laboratory as templates,the high-fidelity polymerase mediated bidirectional primer extension reaction was performed.Then,the conditions of PCR were optimized by the orthogonal design.The optimized mutation-sensitive molecular switch technology was used to detect the 968delA mutation of PTEN gene in 62 clinical endometrial tissue samples collected from the First Affiliated Hospital of Nanhua University.Results The optimal reaction conditions of PCR for detecting the 968delA site of PTEN gene by the mutation-sensitive molecular switch technology were as follows:30 cycles,0.3μL of DNA template(10 ng/μL),0.5μL of primers(10μmol/L)and annealing temperature of 61℃,and its sensitivity was up to 10^(-3) ng/μL.The 968delA mutation of PTEN gene was not found in 62 clinical endometrial cancer tissue and 21 normal endometrial tissue samples.Conclusion The technical platform for detecting the 968delA mutation of PTEN gene in endometrial cancer is successfully constructed,which provides a basis for the application of this technology in the 968delA mutation screening of PTEN gene.
作者
李赛
龚咏晴
向花花
董巍檑
马艳
郭紫芬
LI Sai;GONG Yongqing;XIANG Huahua;DONG Weilei;MA Yan;GUO Zifen(Department of Obstetrics and Gynecology,the First Affiliated Hospital of University of South China,School of Pharmaceutical Science,Hengyang Medical College,University of South China,Hengyang 421001,Hunan,China;Institute of Pharmacy and Pharmacology,School of Pharmaceutical Science,Hengyang Medical College,University of South China,Hengyang 421001,Hunan,China)
出处
《临床检验杂志》
CAS
2023年第3期171-175,共5页
Chinese Journal of Clinical Laboratory Science
基金
湖南省教育厅重点项目(19A419)。
