LDR—ULP结合荧光定量PCR快速检测β-地中海贫血的应用研究  

Rapid detection of beta-thalassemia by LDR-ULP combined with real-time PCR

作　　者：徐欢[1] 杨成[1] 李发科[1] 罗杰[1] 蒋文斌[1] 张峰领[1] 汪超[1] 颜保松[1] 唱凯[1] 陈鸣[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所检验科,重庆400042

出　　处：《中华检验医学杂志》2016年第10期766-770,共5页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金重点项目（81430053）

摘　　要：目的 以6种临床常见突变型为研究对象，建立一种快速检测β－地中海贫血的新方法。方法 收集重庆市大坪医院2015年1至12月正常野生型标本50份和6种临床常见突变型标本42份，PCR扩增外周血β－珠蛋白基因，扩增产物与独立设计的连接检测反应－制式连接探针（LDR－ULP）体系杂交后，荧光定量PCR检测突变类型，琼脂糖电泳验证检测结果，标本梯度稀释法分析LDR－ULP结合荧光定量PCR方法的灵敏度，Kappa检验分析与反向膜杂交法的检测一致性。结果 LDR－ULP杂交反应与多重连接探针扩增技术（MLPA）相比可提高杂交效率2.53倍，经荧光定量PCR扩增后，6种临床常见突变型均出现了明显的扩增曲线，而正常野生型则在40个循环内均无明显扩增曲线。本方法在DNA标本大于5 pg范围内均能得到明显的扩增曲线，对92份临床外周血标本DNA进行检测，本研究方法与反向膜杂交方法结果一致率为100%。结论 本研究通过LDR－ULP技术的特异性，结合荧光定量PCR方法的灵敏性和实时快速的特点，建立了一种操作简便、价格低廉、检测快速的β－地中海贫血的检测新方法。Objective To establish a new method for rapid detection of β-thalassemia by investigating six clinical common mutation types. Methods Fifty cases of clinical wild-type samples and 42 cases of β-thalassemia samples were collected, and β-globin gene was amplified by PCR. Uniform ligation probe （ULP）specific probes were designed for hybridization reaction to increase the reaction specificity and real-time PCR was performed to increase the sensitivity. After that, PCR products were verified by agarose electrophoresis. After examining the specificity and sensitivity, Kappa test between LDR-ULP method and reverse dot blot（RDB） method was conducted. Results Hybridization efficiency was improved 2.53 times by LDR-ULP hybridization. Each mutant type showed a significant amplification curve, whereas the wild- type had no significant curve within 40 cycles. The limit of determination of this method was 5 pg. The results of 92 eases of peripheral blood samples detected by the method of LDR-ULP and RDB were completely consistent. Conclusion In this study, a simple, inexpensive, rapid new method to detect β-thalassemia were established.

关 键 词：Β地中海贫血 实时聚合酶链反应 连接酶检测反应 制式连接探针 

分 类 号：R440[医药卫生—诊断学] R556.61[医药卫生—临床医学]