本研究旨在针对水产品中活的副溶血性弧菌建立一种快速定量的PCR检测方法。基于叠氮溴化丙锭（PMA）在一定光照条件下能抑制死亡菌DNA扩增，以及微滴式数字PCR技术能将检测精度扩展至单分子目标基因，并实现绝对定量的特点，以副溶血性弧菌tlh基因为目的片段设计及筛选适合的特异性引物与探针，优化反应体系，通过对PMA浓度及曝光条件等优化，建立了一种联用PMA-ddPCR技术快速检测水产品中活的副溶血性弧菌的定量检测方法。研究结果显示：选择16 μg/mL作为PMA工作浓度，曝光时间为8 min，此条件下能够完全抑制副溶血性弧菌死菌的DNA扩增并对活菌扩增无影响。通过对比PMA-ddPCR和PMA-qPCR的检测低限分别为2×101 cfu/mL、2×102 cfu/mL，PMA-ddPCR法的灵敏度比PMA-qPCR法的高。应用PMA-ddPCR定量方法检测人工污染的基围虾和小帆立贝这两种海产品，在基围虾中最低可检出1.9×101 cfu/g的副溶血性弧菌、在小帆立贝中最低可检出8.9 cfu/g的副溶血性弧菌。该研究为将PCR技术实际应用于水产品中低量污染、活的副溶血性弧菌的定量检测奠定了基础。

A rapid quantitative PCR detection method for live Vibrio parahaemolyticus in aquatic products was studied. As propidium monoazide (PMA) can inhibit the death of bacteria DNA amplification under at certain lighting condition, and the droplet digital PCR technology can extend detection accuracy to single molecule target genes and achieve absolute quantification, a new method called PMA-ddPCR (combined PMA and ddPCR ) was applied to detect live Vibrio parahaemolyticus. Specific primers and probes targeting the tlh gene of Vibrio parahaemolyticus were designed, and the PCR reaction system was optimized. The PMA concentration and exposure conditions of PMA-ddPCR for target gene were also tested. The results showed that the working concentration of PMA was 16 μg/mL and the exposure time was 8 min. The expansion of dead bacteria could be completely inhibited and the activity of live bacteria was not affected under this condition. The sensitivity of PMA-ddPCR method was higher than that of PMA-qPCR method by comparing the detection limits of PMA-ddPCR and PMA-qPCR (2×101 cfu/mL and 2×102 cfu/mL, respectively). The live Vibrio parahaemolyticus in artificially contaminated marine products (sea shrimp and small scallop) were detected by PMA-ddPCR quantitative method, and the lowest detectable Vibrio parahaemolyticus in sea shrimps was 1.9×101 cfu/g, and the lowest detectable of Vibrio parahaemolyticus in small sail was 0.89×101 cfu/g. This study provided the foundation for the application of PCR technology to the quantitative detection of low-level pollution of live Vibrio parahaemolyticus in aquatic products.

广东省科技计划项目（2016A040403093）；广州市科技计划项目（201804010500）