磷酸甘露糖异构酶基因快速初筛方法的建立

Development of Rapid Screen Method of Phosphomannose Isomerase

建立磷酸甘露糖异构酶(PMI)基因环介导等温扩增快速检测方法。设计了两对内引物和两对外引物,优化后的反应条件为:FIP,BIP浓度为0.8μmol/L,F3,B3浓度为0.2μmol/L,镁离子浓度为2.0 mmol/L,甜菜碱浓度为1.6 mmol/L,反应时间为40 min,反应温度为62.0℃。SYBR Green法检测需2 h,电泳法检测共需2.5 h。与实时荧光和普通PCR比较特异性良好,转基因成分检测限达到0.01%。结果表明该检测方法简单且快速。

A loop mediated isothermal amplification（LAMP） assay was developed for the rapid detection of gene PMI.A set of sepcific primers,two inner primers and outer primers were designed.The concentrations： FIP and BIP,both 0.8 μmol/L,F3 and B3,both 0.2 μmol/L,Mg2＋,2 mmol/L,Betaine,1.6 mmol/L.The reaction time and temperatures were optimized for 40 min at 62.0 ℃.SYBR Green I detection method can be finished in 2 h.Electrophoresis detection method can be finished in 2.5 h.The LAMP assay has a higher specificity compared with Real Time PCR and conventional PCR.The detection limit of GM elements reaches 0.01 %.The results indicated the suitability and simplicity of the test as a rapid method for PMI specific detection.