Abstract Vibrio parahaemolyticus, a worldwide pathogen, has been proved to be capable of entering into viable but nonculturable (VBNC) state and survive under stressful conditions. In this study, a method of real-time quantitative PCR coupled with propidium monoazide (PMA-qPCR) was developed and evaluated for the reliable quantification of the VBNC cells of V. parahaemolyticus. The samples of different ratios of viable and dead cells were detected by PMA-qPCR and qPCR to assess this method. Then the method was used to monitor the VBNC induction process of V. parahaemolyticus and quantify the viable cells along with confirmation of cell integrity and respiratory activity by fluorescence microscopy and confocal laser scanning microscope (CLSM) observation, flow cytometry (FCM) analysis. In addition, the applicability of the method was verified to detect artificially contaminated seafood samples with different states of V. parahaemolyticus. The results showed that the optimal PMA treatment condition was 5 min-PMA incubation in dark followed by 15 min-light exposure. This method can significantly distinguish viable from dead cells and specifically enumerate viable cells. By PMA-qPCR method, the number change of cells during VBNC induction was monitored successfully. The cells incubated at 4 °C in sterile 3% NaCl entered into VBNC state, and 106 cell/mL of VBNC cells (10% of the original viable cells) were detected on the fifth day of induction, then the density of VBNC cells was 5.8 × 104 cell/mL on the 40th day when no culturable cells were observed. FCM analysis showed the viability of the VBNC cells which were also observed red revealing respiratory activity by CTC/DAPI staining under CLSM. For the detection of the spiked seafood samples (shrimp, pomfret and scallop) with different states of V. parahaemolyticus, it could detect as low as 1.2 × 102 cell/g of the viable or VBNC state of V. parahaemolyticus without any interference from food matrices and dead cells. In summary, the PMA-qPCR method developed in this study is rapid and reliable to quantify VBNC cells of V. parahaemolyticus.

中文翻译：

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通过 PMA 结合实时定量 PCR 并确认呼吸活动，对 VBNC 状态下的副溶血性弧菌进行计数

摘要 副溶血性弧菌是一种世界性病原体，已被证明能够进入可存活但不可培养（VBNC）状态并在压力条件下存活。在这项研究中，开发了一种实时定量 PCR 与单叠氮化丙啶 (PMA-qPCR) 结合的方法，并评估了对副溶血性弧菌 VBNC 细胞的可靠定量。通过PMA-qPCR和qPCR检测不同比例的活细胞和死细胞的样品以评估该方法。然后将该方法用于监测副溶血性弧菌的 VBNC 诱导过程，并通过荧光显微镜和共聚焦激光扫描显微镜 (CLSM) 观察、流式细胞术 (FCM) 分析对活细胞进行量化，同时确认细胞完整性和呼吸活性。此外，验证了该方法的适用性，以检测具有不同副溶血性弧菌状态的人为污染的海鲜样品。结果表明，最佳的 PMA 处理条件是在黑暗中进行 5 分钟的 PMA 孵育，然后是 15 分钟的光照。这种方法可以显着区分活细胞和死细胞，并具体列举活细胞。通过PMA-qPCR方法，成功监测了VBNC诱导过程中细胞数量的变化。4 ℃培养的 3% NaCl 无菌细胞进入 VBNC 状态，诱导第 5 天检测到 106 cell/mL VBNC 细胞（原活细胞的 10%），则 VBNC 细胞密度为在第 40 天未观察到可培养细胞时为 5.8 × 104 cell/mL。FCM 分析显示 VBNC 细胞的活力，在 CLSM 下通过 CTC/DAPI 染色也观察到红色显示呼吸活动。对于检测不同状态的副溶血性弧菌的加标海鲜样品（虾、鲳鱼和扇贝），它可以检测低至 1.2 × 102 cell/g 的副溶血性弧菌的存活或 VBNC 状态，而不受食物的干扰基质和死细胞。总之，本研究中开发的 PMA-qPCR 方法可快速可靠地量化副溶血性弧菌的 VBNC 细胞。副溶血性，不受食物基质和死细胞的干扰。总之，本研究中开发的 PMA-qPCR 方法可快速可靠地量化副溶血性弧菌的 VBNC 细胞。副溶血性，不受食物基质和死细胞的干扰。总之，本研究中开发的 PMA-qPCR 方法可快速可靠地量化副溶血性弧菌的 VBNC 细胞。