Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences/College of Biotechnology, University of Santiago de Compostela, Rúa Carballo Calero s/n, Campus Universitario Norte, E-27002 Lugo, Spain.
Food Microbiol. 2011 May;28(3):605-10. doi: 10.1016/j.fm.2010.10.014. Epub 2010 Oct 27.
A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R(2) values of 0.9969 and 0.9958 respectively. Linear correlations between the log(10) input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10(1) CFU/mL to 1.65 × 10(6) CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.
开发了一种用于实时 PCR（Rti-PCR）检测和定量蜡样芽胞杆菌、地衣芽孢杆菌和枯草芽孢杆菌的新引物-探针集。为此，考虑了属于这些物种的四十八株菌株。这些菌株的 DNA 被分离出来，并扩增了 16S rRNA 基因的片段。扩增子被测序，并将获得的序列与 GenBank 中的参考序列进行比对。为了开发基于 TaqMan 探针的实时 PCR（RTi-PCR）方法，设计了一对针对研究的芽孢杆菌属的特异性引物和探针。为了建立定量方法，构建了两个 RTi-PCR 标准曲线；一个用从连续稀释的蜡样芽胞杆菌培养物中提取的 DNA 构建，另一个用从接种了蜡样芽胞杆菌连续稀释物的无菌食品中提取的 DNA 构建。这两条曲线的 R(2) 值分别为 0.9969 和 0.9958。观察到输入 DNA 浓度的对数（10）与阈值循环（Ct）值之间存在线性关系，两条标准曲线的线性度范围均为 1.65 × 10(1) CFU/mL 至 1.65 × 10(6) CFU/mL。设计的引物和探针的特异性用从蜡样芽胞杆菌、地衣芽孢杆菌和枯草芽孢杆菌菌株中提取的 DNA 进行了测试，得到的 Ct 值在 14 到 15 之间，而对其他食品中感兴趣的微生物物种的 DNA 进行非特异性扩增时，Ct 值超过 28.5。据我们所知，该方法代表了关于食品中腐败和/或致病菌蜡样芽胞杆菌、地衣芽孢杆菌和枯草芽孢杆菌定量的首次研究，旨在防止这些不期望的物种在食物链中存在。
