PMA－qPCR 定量检测畜禽肉类中沙门菌活菌的研究

摘要

Objective To enumerate Salmonella in meat of livestock and poultry rapidly and accurately by using propidium monoazide( PMA) combined with real-time fluorescence quantitation polymerase chain reaction ( qPCR). Methods The light exposure time and the concentration of PMA were optimized to establish PMA-qPCR.The standard curve was established by standard plasmid.The sensitivity and specificity were investigated.This method was used for the quantitation determination of Salmonella in livestock and poultry meat.Results The amplification of DNA derived from Salmonella dead cells could be inhibited without affecting the viable cells when PMA was at a dose of 15 μg/mL and exposed for 5 min.The cycle threshold values(Ct) and standard plasmid model cell copy number presented the satisfactory linear, and the correlation coefficient r 2 approached 0.997 9.This method could detect as low as 10 copies/reaction.The minimum detection level was 21 copies/μL by PMA-qPCR.In artificial chicken samples, PMA-qPCR could detect as low as 103 CFU/mL.Conclusions It was possible to quantify viable Salmonella in meat of livestock and poultry by PMA-qPCR.%目的：将叠氮溴化丙锭（PMA）与实时荧光定量聚合酶链反应（qPCR）相结合定量检测畜禽肉类中活的沙门菌。方法通过优化光反应时间、PMA 浓度等 PMA 作用条件，建立 PMA－qPCR 方法，构建重组质粒建立标准曲线，考察该方法的灵敏性、特异性，并将该方法用于定量检测未经增菌培养的畜禽肉类样品中的沙门菌。结果在 PMA 浓度为15μg／mL、曝光5 min 的条件下可完全抑制样品中死菌 DNA 的扩增。建立的定量标准曲线循环阈值（Ct 值）与质粒标准品模板的拷贝数呈良好线性关系（r 2＝0．9979），最低可检出10拷贝／反应体系。所建立的 PMA－qPCR 方法最低可检出21拷贝／μL 沙门菌。采用 PMA－qPCR 检测人工染菌鸡肉样品，最低可检出103 CFU／mL沙门菌。结论用 PMA－qPCR 方法可实现定量检测畜禽肉类样品中活的沙门菌，从而达到快速检测的目的。