低比例DNA污染对地中海贫血基因检测结果的影响  

Effect of low proportion of DNA contamination on the results of thalassemia gene testing

作　　者：许晨霞 王德刚[1] 张艳芳[1] 董兴盛[1] 唐海深[1] 梁睿[1] 彭建明[1] XU Chenxia;WANG Degang;ZHANG Yanfang;DONG Xingsheng;TANG Haishen;LIANG Rui;PENG Jianming(Prenatal Diagnosis Center,Bo'ai Hospital of Zhongshan,Zhongshan,Guangdong,China,528400)

机构地区：[1]中山市博爱医院产前诊断中心,广东中山528400

出　　处：《分子诊断与治疗杂志》2023年第3期409-412,421,共5页Journal of Molecular Diagnostics and Therapy

基　　金：中山市医学科研项目(2021A020096)。

摘　　要：目的 研究低比例DNA污染对地中海贫血基因检测结果判读的影响,以及荧光定量PCR(QF-PCR)技术的鉴定污染的能力。方法 2019年1月至2020年1月在中山市博爱医院进行地中海贫血基因检测的11 128名受检者中,选取其中7例确诊基因型分别为-a^(4.2)/aa、-a3.7/--^(SEA)、aWSa/--^(SEA)、a^(CS)α/aa、βCD41-42/βN、a^(CS)a/aa β^(IVS-Ⅱ-654)/βN、a^(QS)a/aa的患者为研究对象,提取外周血DNA,建立污染模型。用QF-PCR技术检测低比例污染样本。分别用PCR-流式荧光杂交方法和Gap-PCR+反向点杂交法检测污染模型。结果 当DNA污染比例小于10%(2 ng/μL)时QF-PCR技术易漏检,而PCR-流式荧光杂交法能提示3.7缺失型(Gap-PCR不提示);反向点杂交膜条法能提示WS、QS突变型α地贫和17种突变型β地贫(PCR-流式荧光杂交法不提示)。结论 两种方法相比较PCR-流式荧光杂交法对检测3.7缺失型更加灵敏,反向点杂交膜条检测法对α、β点突变的检测灵敏度更高。Objective To evaluate the effect of low proportion DNA contamination on the results of thalassemia gene testing and the ability of quantitative fluorescence PCR technique to identify contamination.Methods From January 2019 to January 2020,among the 11,128 subjects who underwent thalassemia gene testing at Zhongshan Bo'ai Hospital,7 of them were selected to be confirmed as⁃a^(4.2)/aa,⁃a3.7/⁃⁃Patients with^(SEA),awsa/⁃⁃^(SEA),a^(CS)a/aa,β^(CD41⁃42)/bN,a^(CS)a/aa,β^(IVS-Ⅱ-654)/βN,a^(QS)a/a were used as the research objects,and peripheral blood DNA was extracted to establish a contamination model.Low⁃proportion contamination samples were detected by QF⁃PCR technique.The contamination models were detected by PCR⁃flow fluorescence hybridization method and Gap⁃PCR+reverse dot hybridization method respectively.Results When the DNA contamination ratio is less than 10%(2 ng/μL),QF⁃PCR technology is easy to miss detection,while PCR⁃flow fluorescence hybridization method can prompt 3.7 deletion type(Gap⁃PCR does not prompt).Reverse dot blot strip method can reveal WS,QS mutantα⁃thalassemia and 17 kinds of mutantβ⁃thalassemia(PCR⁃flow cytometry fluorescence hybridization method does not prompt).Conclusion The PCR⁃flow fluorescent hybridization method is more sensitive to detect the 3.7 deletion type,and the reverse dot blot membrane strip detection method is more sensitive to the detection ofαandβpoint mutations.

关 键 词：地中海贫血 QF-PCR PCR-流式荧光杂交 Gap-PCR+反向点杂交 

分 类 号：R556.61[医药卫生—血液循环系统疾病] R440[医药卫生—内科学]