单核细胞增生李斯特氏菌依赖解旋酶DNA恒温扩增检测方法的建立

摘要

为建立单核细胞增生李斯特氏菌(Lm)的依赖解旋酶DNA恒温扩增(HDA)检测方法,本研究以Lm的iap基因为目的片段设计特异性引物,建立了可在65℃恒温扩增快速(90 min)检测Lm的HDA检测方法,分别对反应体系中的MgAc2、Bst聚合酶、UvrD解旋酶、dNTPs以及引物等的浓度进行优化,进行了特异性和灵敏度试验,并与普通PCR方法进行了比较.结果表明,所建立起的检测法最低检测限为7.8 × 103cfu/mL,灵敏度与普通PCR方法相当.Lm的HDA检测方法具有普通PCR的特异、灵敏等特点,并且对仪器要求更低,具有良好的应用前景.%In this paper, a new method for detection of Listeria monocytogenes was established based on helicase-dependent isothermal DNA amplification (HDA). A pair of primers to target the iap gene (AF532297) of L. monocytogenes was synthesized and the amplification was conducted under 65℃ for 90 minutes after initial denature and annealing to amplify a 105 bp product from L. monocytogenes, but not from other bacteria. The results showed that the sensitivity of HDA was 7.8 × 103 cfu/mL which was similar to the result of PCR method. Therefore, the rapid and easy-to-use method for detecting L. monocytogenes has a potential application.