PMA-qPCR法检测冷冻基质中非可培养状态(VBNC)副溶血性弧菌

Propidium monoazide based real-time PCR to detect Vibrio parahaemolyticus in a viable but nonculturable state

摘要:

【背景】副溶血性弧菌为冰鲜产品及肉制品中常见的污染微生物，致病性强，危害严重，出入境运输及加工的肉食品常采取冷冻冷藏的处理手段来防止微生物污染及生长，以保持食物新鲜。而残留的部分副溶血性弧菌会进入活的非可培养状态(Viable but non-culturable state，VBNC)，从而构成潜在的风险隐患。【目的】建立可用于冷冻食品中VBNC副溶血性弧菌的快速检测方法，并探讨其适用性。【方法】将大西洋鲑鱼1:10匀浆，加入终浓度为6.6×105 CFU/mL的副溶血性弧菌，?20 °C分别诱导10、20、30和50 d。建立实时荧光PCR技术(qPCR)方法，测定其特异性、灵敏度及稳定性。利用PMA-qPCR法对不同冷冻时期样品中的副溶血性弧菌进行检测，同时与qPCR、平板培养法进行比较。【结果】建立的qPCR方法特异性好，与其他阴性参考株无交叉反应；灵敏度高，检测限为19.8 CFU/mL；重复性好，Cq值的变异系数(CV)均在1.5%以下；标准曲线为y=?3.272x+45.310，线性回归系数R2为0.996，定量范围为1×102–1×109 CFU/mL。在低温诱导10?50 d后，qPCR法的Cq值在26.32?27.34之间，与诱导前相比几乎没有变化；叠氮溴化丙锭(PMA)-qPCR法的Cq值则从诱导前的26.43逐步上升到38.84，呈现明显的上升趋势，表明死菌的数量在显著上升。经过比较及统计，PMA-qPCR检测的活菌数均高于平板培养法测出的数量，差异显著(P<0.05)。【结论】PMA-qPCR特异性及灵敏度高，能有效抑制对死菌的扩增，同时能克服传统平板培养法对VBNC的漏检缺陷，可方便、快捷地用于冷冻食品中受损致病微生物，尤其是进入VBNC状态的细菌检测。

Abstract:

[Background] Vibrio parahaemolyticus, which is highly pathogenic and harmful to public health, is a common contaminating microorganism in chilled food and meat products. To keep them fresh, imported and exported food are often refrigerated or frozen to prevent microorganism growth during transportation and machining. However, residual V. parahaemolyticus can enter a viable but non-culturable (VBNC) state, which poses potential risks. [Objective] This study aimed to establish a method to detect V. parahaemolyticus in a VBNC state in frozen food and to explore its applicability. [Methods] A solution containing V. parahaemolyticus was added to homogenized Atlantic salmon matrix to a final concentration of 6.6×105 colony forming units (CFU)/mL. Aliquots of the mixture were induced for 10, 20, 30, and 50 days at ?20 °C. A propidium monoazide (PMA)-based quantitative real-time PCR (qPCR) method was established to detect the V. parahaemolyticus in the samples of different frozen periods and compared with the results of qPCR and plate culture methods. [Results] The established PMA-qPCR method showed good specificity and no cross-reaction with other negative reference strains. The sensitivity of the method was also high and the quantitative limit was 19.8 CFU/mL. The variation coefficients (CV) of Cq values were all below 1.5%. The standard curve was y=?3.272x+45.310 and the linear regression coefficient, R2 was 0.996. The quantitative range was 1×102 to 1×109 CFU/mL. After the low temperature induction from 10?50 d, the Cq value of the qPCR method was between 26.32 and 27.34 which showed little change. However, the Cq value of the PMA-qPCR increased from 26.43 to 38.84, showing a significant increase in the number of dead bacteria. After comparison and statistical analyses, the number of live bacteria detected by PMA-qPCR was higher than that of plate culture, and the difference was significant (P<0.05). [Conclusion] The established PMA-qPCR method had high specificity and sensitivity and could effectively inhibit the amplification of dead bacteria. It was also an effective and fast method to detect VBNC bacteria, which could overcome the limitations of the traditional plate culture method.