EMA/PMA-qPCR技术检测活肠炎沙门氏菌方法的对比研究

Comparison of propidium monoazide-qPCR with ethidium monoazide-qPCR for differentiation of live vs. dead Salmonella enteritidis

将DNA染料EMA(Edthidium Monoazide Bromide叠氮溴化乙啶)和PMA(Propidium Monoazide Bromide叠氮溴化丙啶)与实时荧光定量PCR技术相结合,通过对曝光时间、染料浓度进行优化,验证EMA/PMA-qPCR技术对于肠炎沙门氏菌活/死菌鉴别方法的可行性;对比2种试剂的作用效果,确定最为适宜的预处理方式。结果表明,使用浓度为10μg/mL的EMA,曝光时间为15 min,可以完全抑制浓度为5×107 cfu/mL的热致死肠炎沙门氏菌的DNA扩增。对于相同浓度的菌悬液,PMA则无法保证与热致死菌DNA分子的有效交联,对于死菌扩增的抑制效果不理想。降低菌液浓度为5×106 cfu/mL,提高PMA的浓度对于活菌的DNA模板有损耗,对热致死菌的抑制效果无改善。EMA-qPCR方法能克服普通PCR无法区别活死菌的缺点,更适宜作为沙门氏菌检测的一种初筛方式。

A quick and efficient method to detect viable Salmonella enteritis cells by using DNA dyes EMA（Edthidium Monoazide Bromide） and PMA （Propidium Monoazide Bromide） in combination with realtime quantitative PCR technology. By optimizing the exposure time, the concentration of EMA and PMA to verify the feasibility of the method on differentiation live vs dead salmonella enteritis. And compared the effects of two means to determine which is better to be used as a pretreatment. Results show that the concentration of EMA that can completely inhibit the DNA amplification derived from heat-killed Salmonella enteritis cells at a concentration of 5×10^7 cfu/mL was 10 μg/mL, while the optimal exposure time was 15 min. Under the same experiment conditions, PMA cannot make a perfect covalent bonding with the DNA of dead cells, which means PMA cannot manage to inhibit the amplification of DNA derived from heat-killed Salmonella enteritis cells. When reducing the concentration of suspension to 5×10^6 cfu/mL, increase the concentration of PMA would cause a loss of DNA templates derived from viable cells and show no improvement on inhibition of heat-killed cells. EMA-qPCR method can overcome the common shortcoming of traditional PCR technology and be used as a novel way to identify live and dead cells in further study. EMA-qPCR can be an effective indicator of cell viability.