乳品中的酵母活菌数PMA-qPCR检测方法的建立及初步应用

李畅; 赵柯; 常江; 张嵩; 李萌; 关玉婷; 孟宪梅; 柳增善; 胡盼; 任洪林; 卢士英; 李岩松; 孙宇; 刘熙

doi:10.13386/j.issn1002-0306.2018.22.023

乳品中的酵母活菌数PMA-qPCR检测方法的建立及初步应用

Development and Application of PMA-qPCR Method for Detecting Viable Yeasts in Dairy Products

摘要

摘要: 本实验旨在对乳品中酵母菌活菌总数进行准确的定量检测,深入研究PMA处理乳品中酵母热损伤菌的最佳工作条件,去除热损伤死菌DNA的影响。针对从乳品中分离的三株酵母菌26S rDNA D1/D2区域序列设计引物Y3和Y4,运用实时荧定定量PCR进行序列扩增,以重组质粒pMD-18T-26S拷贝数对数与扩增循环数(Ct)值为坐标轴建立标准曲线,建立PMA-qPCR检测方法,并对实际样品中的酵母菌活菌富集后进行数量检测。PMA-qPCR法可达到国标规定的食品中酵母菌最高限量值1×10² CFU/mL的检测标准,对应Ct值为36.67±0.43,重复性良好,总耗时6~7 h,可对酵母菌数量进行准确和快速的定量分析,为乳品中腐败微生物的鉴定与监控提供有价值的借鉴。

Abstract: This experiment was aimed to accurately determine the total number of viable yeast strains in dairy. The best working conditions of PMA for heat-damaged yeast in dairy products was investigated to removal of heat-damaged DNA. Primers Y3 and Y4 were designed for the 26S rDNA D1/D2 region sequences of three yeasts isolated from dairy and real-time quantitative PCR was used to perform sequence amplification. The copy number and Ct value of recombinant plasmid pMD-18T-26S were calculated,then the standard curve was established on the coordinate axis,and the PMA-qPCR detection method was established. The amount of live yeast in the actual sample was enriched and quantitatively detected. The PMA-qPCR method could meet the standards of the yeast maximum limit value of 1×10² CFU/mL in foods specified by the national standard with good repeatability,corresponding to a Ct value of 36.67±0.43,and the total time taken was 6~7 h. The amount of yeast could be determined. Accurate and rapid quantitative analysis could provide valuable reference for the identification and monitoring of spoilage microorganisms in dairy products.

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