PMA-qPCR定量检测VBNC副溶血弧菌方法的建立与优化

Development and optimization of PMA-qPCR for the quantitative detection of viable but non-culturable Vibrio parahaemolyticus

副溶血弧菌(Vibrio parahaemolyticus)在某些不利于生长的自然和食品加工条件下能够进入活的非可培养(viable but non-culturable,VBNC)状态,普通检测方式极易漏检,因此VBNC细菌检测技术的开发与优化对副溶血弧菌感染防控具有重要意义。基于叠氮溴化丙锭结合荧光定量PCR技术(propidium monoazide quantitative PCR,PMA-qPCR)能够利用活细胞的膜完整性区分活菌和死菌的原理,以副溶血弧菌ATCC 17802为对象,通过探讨PMA-qPCR方法中各项因素对活菌计数结果的影响,优化并建立了VBNC副溶血弧菌的定量检测方法。实验结果表明,当添加PMA质量浓度为15 mg/L,黑暗孵育10 min,再进行强光照射15 min后,死细胞的DNA扩增得到有效抑制,此时提取DNA模板进行qPCR扩增准确性更高,活菌数与qPCR扩增循环数呈现显著性线性相关(R^(2)=0.99)。PMA-qPCR法测定副溶血弧菌活菌数的标准曲线显示该方法检测范围为2.90×10^(1)~2.90×10^(8) CFU/mL,验证回收率在92.97%~99.57%,相对标准偏差为1.95%。该文建立了更加精准可靠的VBNC副溶血弧菌定量检测方法,为后续进一步探究提供了有效技术手段。

Vibrio parahaemolyticus might turn to a viable but non-culturable(VBNC)state under some natural and food processing conditions where bacteria can hardly grow,which cannot be detected by conventional culture-based methods.Therefore,the development and optimization of the VBNC bacteria detection technology is significant for the prevention and control of V.parahaemolyticus infection.Based on the principle of propidium monoazide quantitative PCR(PMA-qPCR)assay to distinguish the live or death of bacteria according to the membrane integrity,the effect of multiple parameters in PMA-qPCR on the viable count was detected and the quantitative detection method of VBNC V.parahaemolyticus was optimized and established.The results indicated that the optimal treatment method was to incubate in dark with 15 mg/L of PMA for 10 min and followed by 15 min of strong light irradiation.Under the optimal conditions,DNA amplification of dead cells was effectively inhibited,and the accuracy of qPCR amplification was higher.The number of viable bacteria was significantly linear correlated with the Ct values of qPCR(R^(2)=0.99).The detection limit of PMA-qPCR standard was from 29 to 2.90×10^(8) CFU/mL,the recovery rate range was 92.97%-99.57%,and the standard deviation was 1.95%.In conclusion,the more accurate and reliable quantitative detection method for VBNC V.parahaemolyticus was established and provides an effective method for further research.