实时荧光HDA法快速检测单核细胞增生李斯特菌

目的：建立一种用于单核细胞增生李斯特菌的实时荧光赖解旋酶恒温核酸扩增（helicase-dependentisothermal DNA amplification，HDA）快速检测方法。方法：针对单核细胞增生李斯特菌hly基因序列设计引物对，基于荧光定量聚合酶链式反应（polymerase chain reaction，PCR）仪的平台，提取单核细胞增生李斯特菌基因组DNA，以此作为模板，优化反应温度、反应时间及引物浓度。利用单核细胞增生李斯特菌及10 株对照菌株，并与实时荧光PCR对比，来验证实时荧光HDA方法的特异性和灵敏度，并初步用于样品检测。结果：实时荧光HDA体系的最适引物浓度为0.075 μmol/L，反应温度及反应时间为65 ℃、80 min（40 个循环），具有良好的特异性和灵敏度。结论：建立了一种特异性强、灵敏度高的实时荧光HDA检测单核细胞增生李斯特菌的方法。

Real-Time Fluorescence Helicase-Dependent Isothermal DNA Amplification Method for Rapid Detection of Listeria monocytogenes in Foods

The purpose of this study was to develop a real-time helicase-dependent isothermal DNA amplification (HDA)
method for the rapid detection of Listeria monocytogenes. Based on the platform of real-time PCR, pairs of primers targeting
the hemolysin gene (hly) of Listeria monocytogenes were designed, and genomic DNA was extracted from a standard strain
of L. monocytogenes for use as the template. The reaction temperature, primers and template DNA concentration were
optimized. Compared with real-time PCR method, the specificity and sensitivity of the real-time HDA method were
evaluated with L. monocytogenes and 10 bacteria control strains, and then this developed method was used to detect
L. monocytogenes in real samples. The results showed that the optimal primer concentration, reaction temperature and
time for real-time HDA system were 0.075 mol/L, 65 ℃ and 80 min (40 cycles), respectively. This system showed a
high specificity and sensitivity. Thus a real-time HDA method for rapid and specific detection of L. monocytogenes has
been successfully established.