In the present study, for the first time, high sensitive quantitative polymerase chain reaction (qPCR) and digital droplet polymerase chain reaction (ddPCR) assays were developed to detect and quantify total eumycetes with potential application in several food matrices and to specifically determine the level of contamination by Saccharomycopsis fibuligera and Wickerhamomyces anomalus cells directly in bread. Among the candidate target genes used to develop the assays, car1 gene was chosen to detect the two spoilage yeasts S. fibuligera and W. anomalus. The specificity of the PCR assays was tested using purified genomic DNA from 36 bacterial and fungal strains. The sensitivity of the assays was defined by using tenfold serial dilutions of genomic DNA starting from 10⁶ cfu/mL to 1 cfu/mL of S. fibuligera and W. anomalus. Validation of the assays was achieved by enumeration of S. fibuligera and W. anomalus DNA copies from samples of artificially contaminated bread homogenates detecting up to 10 cfu/mL (0.06 ± 0.01 copies/μL) of W. anomalus by using ddPCR. In conclusion, the developed qPCR and ddPCR assays demonstrate strong performance in the early detection of S. fibuligera and W. anomalus in bread, representing promising tools for applying high-throughput approaches to regularly monitor bread quality.

在本研究中，首次开发了高灵敏度定量聚合酶链反应 (qPCR) 和数字液滴聚合酶链反应 (ddPCR) 测定法来检测和量化在几种食品基质中具有潜在应用的总真菌，并具体确定水平直接在面包中受到Saccharomycopsis fibuligera和Wickerhamomyces anomalus细胞的污染。在用于开发检测的候选靶基因中，选择car1基因来检测两种腐败酵母S. fibuligera和W. anomalus. 使用来自 36 种细菌和真菌菌株的纯化基因组 DNA 测试 PCR 测定的特异性。通过使用从 10 ⁶  cfu/mL 到 1 cfu/mL 的S. fibuligera和W. anomalus的基因组 DNA 的十倍系列稀释来定义测定的灵敏度。通过使用 ddPCR检测到 10 cfu/mL (0.06 ± 0.01 拷贝/μL) 的W. anomalus，从人工污染的面包匀浆样品中枚举S. fibuligera和W. anomalus DNA 拷贝来验证检测方法。总之，开发的 qPCR 和 ddPCR 检测在S. fibuligera和W. anomalus的早期检测中表现出强大的性能 在面包中，代表了应用高通量方法定期监测面包质量的有前途的工具。