摘要
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-dependent isothermal 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0.075μmol/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。
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.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2015年第10期206-210,共5页
Food Science
基金
苏州科技局科技支撑计划项目(SS201338)
关键词
单核细胞增生李斯菌
hly基因
赖解旋酶恒温扩增
实时荧光HDA
快速检测
Listeria monocytogenes
hemolysin gene
helicase-dependent isothermal amplification
real-time fluorescence HDA
rapid detection
