一种DNA染料结合聚合酶链反应检测鉴别植物病原细菌死活细胞

Differentiation of Live and Dead Cell of Bacterial Plant Pathogen in Polymerase Chain Reaction Assays Using a DNA Binding Dye

建立了一种将DNA染料(EMA)结合PCR的新分析方法,用于有效检测区分植物病原细菌死活细胞.结果表明,当用2.0 mg/L或更高浓度的EMA渗透处理含有106cfu/mL的死细胞菌悬液后再曝光处理10 min,其PCR结果呈阴性,而未经过EMA渗透处理的对应样品PCR结果呈阳性;EMA渗透处理含有适当数量病菌活细胞的种子浸泡液后,更有助于特异性检测混合体系中靶标菌活细胞.分析认为,该方法避免了传统PCR无法区分细菌死活细胞的弊端,是一种快速、灵敏且能有效鉴别病原细菌死活细胞的新方法.

Polymerase chain reaction（PCR） assays are quicker and often just as sensitive as agar plating assays for detecting plant pathogenic bacteria. However, PCR can be used to detects both live and dead cells. Since dead cells do not cause disease, their detection can be a problem in seed-health testing. Ethidium monoazide（EMA） , a DNA binding dye, can prevent the amplification of DNA from dead cells of food-borne bacteria. To test the use of EMA for detecting live cells of Acidovorax avenae subsp, citrulli（Aac）, seed extracts, spiked with Aac containing 3 × 10^6, 3 × 10^7, and 3 × 10^8 cfu/mL, were treated with EMA at 0, 1, 2, 3, and 4 mg/L and incubated at room temperature and at 75 ℃ for 3 min to kill Aac. After 10 min in the dark, the mixtures were put on ice under light for 5, 10, and 15 min to allow EMA to be effective, then assayed by classical PCR and agar plating. The results show that heat-treated suspensions containing dead cells of Aac at a level of 3 × 10^6 cfu/mL were PCR negative when pretreated with 2 mg/L or greater EMA and 10 min or more exposures. However, all treatments without EMA containing Aac at 3 × 10^6 cfu/mL or greater were ineffective as PCR results were positive. These preliminary results show that the addition of EMA to seed extracts containing moderate numbers of Aac has a potential for the specific detection of live cells.