CAJ | 학술논문

建立应用替氮溴化乙锭(ethidium monoazide,EMA)与环介导等温扩增(loop-mediated isothermal amplification,LAMP)相结合检测沙门氏死/活菌的方法.该方法具有快速、方便和灵敏度高等优点,以SYBR Green Ⅰ为显色剂、恒温65℃、反应1h便可得出检测结果.该检测系统中,得到241bp的目标片段和类似于梯子状的系列条带.该方法的检测限为10~(-10)g DNA/管,是EMA-PCR方法的1%.本方法实用性强,具有普遍的应用意义.
건립응용체담추화을정(ethidium monoazide,EMA)여배개도등온확증(loop-mediated isothermal amplification,LAMP)상결합검측사문씨사/활균적방법.해방법구유쾌속、방편화령민도고등우점,이SYBR Green Ⅰ위현색제、항온65℃、반응1h편가득출검측결과.해검측계통중,득도241bp적목표편단화유사우제자상적계렬조대.해방법적검측한위10~(-10)g DNA/관,시EMA-PCR방법적1%.본방법실용성강,구유보편적응용의의.
A combinatorial method, designated as ethidium monoazide (EMA)-loop-mediated isothermal amplification (LAMP) method, was established to detect five/dead Salmonella due m the selective penetration of EMA into dead cells in the presence of live cells. DNA covalently bound to EMA can not be amplified by LAMP. In this detect system, a series of primers were specially designed m recognize six distinct sequences on target invA gene in Salmonella. Totally 241 bp target DNA was amplified and visualized as ladder-like bands on agarose gel within 60 min under isothermal condition of 65 ℃. This detection method was rapid, convenient and highly sensitive. The detection limit of this LAMP assay was 10~(-10)g DNA/tube, which exhibited 100-fold higher sensitivity than EMA-PCR method. This novel EMA-LAMP method for detecting live/dead bacteria will have great potential.