叠氮溴乙锭-环介导等温扩增法快速检测空肠弯曲菌活菌

Rapid detection of viable Campylobacter jejuni by ethidium monoazide bromide loop-mediated isothermal amplification

中文关键词:  空肠弯曲菌  环介导等温扩增  叠氮溴乙锭  食源性致病菌  活菌检测

英文关键词:Campylobacter jejuni  loop-mediated isothermal amplification  ethidium bromide monoazide  food borne pathogens  viability detection

中文摘要:

      目的 建立叠氮溴乙锭(ethidium monoazide bromide, EMA)-环介导等温扩增(loop-mediated isothermal amplification, LAMP)快速检测空肠弯曲菌活菌的方法。方法 以空肠弯曲菌mapA基因为检测靶基因, 纯培养物提取模板进行LAMP反应, 检测空肠弯曲菌LAMP灵敏度、EMA曝光照射时间试验和使用浓度试验。结果 空肠弯曲菌LAMP检测灵敏度为800 CFU/mL; 曝光照射时间为5 min; EMA终浓度50 μg/mL以下时不会抑制空肠弯曲菌活菌的DNA扩增, 终浓度为1 μg/mL EMA能有效抑制4×104 CFU/mL空肠弯曲菌死菌扩增; 1%活菌混合体系中EMA-LAMP检测结果为阳性。结论 EMA-LAMP方法能有效快速检测空肠弯曲菌活菌。

英文摘要:

      Objective To establish a rapid method for detecting Campylobacter jejuni viable bacteria by ethidium monoazide bromide-loop-mediated isothermal amplification (EMA-LAMP). Methods The mapA gene was used as the target gene for LAMP assay of Campylobacter jejuni, and LAMP assay was carried out by utilizing its isolation culture as the template. The sensitivity test for LAMP, the light exposure time and EMA concentration were optimized. Results LAMP detection sensitivity of Campylobacter jejuni was 800 CFU/mL. The exposure time was 5 min. When the final concentration of EMA was below 50 μg/mL, the DNA amplification of viable Campylobacter jejuni was not inhibited. When the final concentration of EMA was 1 μg/mL, and the amplification of dead Campylobacter jejuni was effectively inhibited at 4×104 CFU/mL. The results of EMA-LAMP in 1% of the live bacteria mixture were positive. Conclusion The method of EMA-LAMP can detect Campylobacter jejuni efficiently and rapidly.