环介导等温扩增法快速检测白血病PML/RARα的方法学建立与初步应用  

Methodological Establishment and Preliminary Application of Loop-mediated Isothermal Amplification in the Rapid Detection of Leukemia PML/RARα

作　　者：仇立芳 施引 孙琳琳[1] 董琳[2] 姜国胜[1] QIU Li-fang;SHI Yin;SUN Lin-Lin(School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Shandong Jinan 25006)

机构地区：[1]济南大学,山东省医学科学院医学与生命科学学院,山东济南250062 [2]山东省千佛山医院血液科,山东济南250014

出　　处：《医学检验与临床》2018年第4期35-40,共6页Medical Laboratory Science and Clinics

摘　　要：目的：建立环介导等温扩增LAMP检测PML-RARα融合基因方法,进行APL患者微小残留病的快速检测.方法：登录NCBI在线软件设计多组LAMP引物,并设计验证阶段所需的PCR引物.根据反应时间及特异性对不同LAMP引物组的反应进程和结果进行检测、 筛选.根据同一核酸序列设计PCR引物,用于检测基因表达情况,根据反应液的颜色变化裸眼观察,判断扩增结果.同时,将外引物F3、B3作为PCR引物,用于LAMP方法和PCR方法的比较,分析LAMP方法的特异性和敏感性.结果：LAMP法检测PML-RARα融合基因不需要改变温度,包括变性步骤的全过程都能在等温条件下短时间内完成,可以检测扩增产物或者判定扩增反应发生与否.LAMP法具有不需要特殊试剂,扩增不需要改变反应温度,快速判断结果的特点.结论：LAMP法检测PML-RARα融合基因,具有较高的特异性、 敏感性,是传统RT-PCR和荧光定量PCR的替代方法.Objective： To establish a loop-mediated isothermal amplification of LAMP to detect the PML-RARα fusion gene for monitoring minimal residual disease （MRD） . Methods： Log NCBI access to the conserved gene sequence of the PML-RARα fusion gene, sequence alignment by software, design multiple sets of LAMP primers online and design PCR primers required for the validation phase. A total of 4 LAMP primers, including a pair ofirmer primers and a pair of external primers, and according to the reaction time and specificity of different LAMP primer sets of the reaction process and results were detected and screened. PCR primers were designed based on the same nucleic acid sequence to detect gene expression. At the same time, the outer primers F3 and B3 were used as PCR primers for the comparison of the LAMP method and the PCR method. The expression of PML-RARα gene was detected by LAMP assay, the specificity and sensitivity of the method were analyzed. The amplification result was observed with the naked eye according to the color change of the reaction solution. Results： The detection of PML-RARα fusion gene by LAMP method did not need to change the temperature. The whole process including the denaturation step can be completed in a short time under isothermal condition, and the amplification product can be detected or the amplification reaction can be judged. The LAMP method does not require special reagents, amplification does not need to change the reaction temperature, the characteristics of the results can be detemfined quickly. Conclusion： The detection of PML-RARα fusion gene by LAMP method has high specificity and sensitivity. It is an alternative method to traditional RT-PCR and fluorescence quantitative PCR.

关 键 词：白血病 微小残留病 环介导等温扩增方法 融合基因 

分 类 号：R51[医药卫生—内科学]