采用叠氮溴化丙锭(propidium monoazide, PMA)结合实时荧光定量PCR对乳制品中活菌DNA进行定量分析,建立一种快速而准确检测发酵乳品中植物乳杆菌P-8(Lactobacillus plantarum P-8)活菌数的新方法。通过对影响PMA作用的浓度、暗孵育和曝光时间等因素进行试验,确定最佳PMA处理方案。结果表明:L. plantarum P-8经80 ℃处理60 s,即为膜损伤菌;当PMA质量浓度为40 μg/mL,暗孵育时间10 min,曝光时间为20 min时,PMA既不影响活菌DNA的PCR扩增,又能渗透进入细胞膜受损的死菌并抑制其PCR扩增;通过制备L. plantarum P-8质粒标准品并建立标准曲线,其表现出良好的线性关系,相关系数(R2)为0.992 9,最低检测限为103 CFU/mL,特异性良好。该方法为完善发酵乳产品益生菌活菌数的检测奠定了基础。
Propidium monoazide (PMA) was combined with real-time fluorescent quantitative PCR to detect DNA of living bacteria in milk products. A new, fast, and accurate method to detect Lactobacillus plantarum P-8 in fermented dairy products was developed. Influencing factors, such as concentrations, dark incubation, duration of illumination were determined to establish the optimal PMA treatment scheme. The results showed that after treating L. plantarum P-8 at 80℃ for 60 s, they became membrane-damaging. When 40 ug/mL PMA was used for 10 min dark incubation, followed by espousing for 20 min, PMA did not influence the living bacteria's DNA amplification. In addition, it inhibited DNA amplification of dead cells. A standard curve with a good linear relationship was set up based on L. plantarum P-8 plasmid standards. The relative coefficient of the curve was 0.992 9, and the lowest detection limit was 103 CFU/mL with good specificity. This method laid a foundation for improving the detection of viable probiotics in fermented milk products.
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