将一种DNA染料(ethidium bromide monoazide,EMA)与传统的PCR技术相结合,建立了一种能有效检测纯培养条件下副溶血弧菌死活菌细胞的新方法(EMA-PCR)。研究结果表明,当用1.4μg/mL或更高浓度的EMA渗透处理含有108 CFU/mL的副溶血弧菌死细胞菌悬液后再经20 min曝光处理,其PCR结果呈阴性,而不经EMA处理的对照组其PCR结果则呈阳性;当EMA的用量等于或者小于2μg/mL时,副溶血弧菌活细胞的PCR扩增不会受到抑制。经EMA处理,含有不同比例的副溶血弧菌死细胞和活细胞的混合液中活的副溶血弧菌能够通过PCR被选择性的定量,最小的检测水平为10 CFU/PCR。而且,经研究发现在(10～2×105)CFU/PCR范围内,DNA相对荧光强度与死活细胞混合液中活细胞的对数具有线性关系。

A new and efficient method for detection of the viable and dead cell of pure cultured of Vibrio parahaemolyticus was developed by using a DNA dye of ethidium bromide monoazide(EMA) in combination with the traditional polymerase chain reaction(EMA-PCR).The results showed that,under the light exposure for 20 minutes to photolyse the EMA in cell suspension of 108 CFU/mL Vibrio parahaemolyticus treated with EMA at the concentration of 1.4μg/mL,the PCR results were negative,but the control without treatment by using EMA,the PCR results were positive.The PCR for viable cell of Vibrio parahaemolyticus was not inhibited with the maximum concentration of EMA at 6μg/mL.After EMA treatment,the number of viable Vibrio parahaemolyticus cells in varying ratios of viable to dead cells could be selectively quantified by PCR.The minimum level of detection was 10 CFU/PCR reaction.A linear relationship was found between the relative fluorescent intensity of the DNA bands and the log of genomic targets derived from the viable cells in mixtures of viable and dead cells in the range of 10 to 2 × 105 CFU/PCR.