Abstract: To address the issue of the long detection cycle and difficult quantitative analysis of molds in fermented milk, this study developed a systematic method for the detection of molds in commercial fermented milk by using droplet digital polymerase chain reaction (ddPCR) to absolutely quantify the copy number of target sequences in samples. The optimal ddPCR reaction system and amplification program were determined. The proposed method performed well in terms of specificity, sensitivity and quantitative accuracy. It specifically amplification target molds (Aspergillus niger, Penicillium citrinum, and Penicillium chrysogenum), with no cross-reactivity with yeasts, lactic acid bacteria, or pathogenic bacteria. Sensitivity tests indicated that at the minimum detectable concentration (0.625 ng/μL), the positive copy number was 9 copies/μL. In addition, this method established linear equations between bacterial suspension concentration and DNA concentration, as well as between DNA concentration and gene copy number, with correlation coefficients greater than 0.99, and based on them, a linear equation between bacterial suspension concentration and gene copy number was developed. The results of the ddPCR method for artificially contaminated samples were consistent with those of the national standard method (GB 4789.15-2016). Moreover, the method enables absolute quantification without the need for a standard curve, effectively shortening the detection cycle.

Key words: fermented milk; mold count; yeast; droplet digital polymerase chain reaction; quantitative detection

摘要： 为解决发酵乳中霉菌检测周期长、定量难的问题，以发酵乳中常见霉菌为对象，通过微滴式数字聚合酶链式反应（droplet digital polymerase chain reaction，ddPCR）对样品中的目标序列进行绝对定量的基因拷贝数检测，建立市售发酵乳中霉菌的系统检测方法。确定了适用于发酵乳霉菌检测的最优ddPCR反应体系及扩增程序，所建立的方法在特异性、灵敏度及定量准确性方面均呈现良好性能。特异性检验显示，仅目标霉菌（黑曲霉、橘青霉、产黄青霉）出现阳性扩增，对酵母菌、乳酸菌及致病菌无交叉反应；灵敏度检测表明，最低检测质量浓度（0.625 ng/μL）时，阳性基因拷贝数为9 copies/μL。此外，本方法还确定了菌悬液浓度与DNA质量浓度、DNA质量浓度与基因拷贝数的线性关系式，且相关系数均大于0.99，在此基础上得到菌悬液浓度与基因拷贝数的线性关系式。在人工污染样品检测中，ddPCR方法结果与GB 4789.15—2016《食品安全国家标准 食品微生物学检验 霉菌和酵母计数》方法具有一致性，且无需标准曲线即可实现绝对定量，有效缩短检测周期。

关键词: 发酵乳；霉菌计数；酵母；微滴式数字聚合酶链式反应；定量检测