Cornell University, Department of Food Science, Ithaca, New York, USA.
Appl Environ Microbiol. 2012 Aug;78(16):5855-63. doi: 10.1128/AEM.01361-12. Epub 2012 Jun 8.
Psychrotolerant sporeformers, specifically Paenibacillus spp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. While Paenibacillus spp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number of Paenibacillus spp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detect Paenibacillus spp. in fluid milk and to discriminate between Paenibacillus and other closely related spore-forming bacteria. Specificity was confirmed using 16 Paenibacillus and 17 Bacillus isolates. All 16 Paenibacillus isolates were detected with a mean cycle threshold (C(T)) of 19.14 ± 0.54. While 14/17 Bacillus isolates showed no signal (C(T) > 40), 3 Bacillus isolates showed very weak positive signals (C(T) = 38.66 ± 0.65). The assay provided a detection limit of approximately 3.25 × 10(1) CFU/ml using total genomic DNA extracted from raw milk samples inoculated with Paenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection of Paenibacillus. Heat-treated milk samples where Paenibacillus (≥1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection of Paenibacillus that has the potential to identify raw milk with microbial spoilage potential as a pasteurized product.
耐冷孢子形成菌，特别是类芽孢杆菌属，是巴氏杀菌冷藏食品（如液态奶）中的重要腐败菌。虽然已经从农场环境、生牛乳、加工厂环境和巴氏杀菌液态奶中分离出类芽孢杆菌属，但关于生牛乳中需要存在多少数量的类芽孢杆菌属才能导致巴氏杀菌奶变质，目前尚无信息。设计了一种针对 16S rRNA 基因的实时 PCR 检测方法，用于检测液态奶中的类芽孢杆菌属，并区分类芽孢杆菌属和其他密切相关的孢子形成细菌。使用 16 株类芽孢杆菌属和 17 株芽孢杆菌属分离株证实了特异性。所有 16 株类芽孢杆菌属分离株的平均循环阈值（C(T)）为 19.14±0.54。虽然 17 株芽孢杆菌属分离株中的 14 株未显示信号（C(T)>40），但 3 株芽孢杆菌属分离株显示出非常弱的阳性信号（C(T)=38.66±0.65）。该检测方法使用从接种类芽孢杆菌属的生牛乳样品中提取的总基因组 DNA，提供了约 3.25×10(1)CFU/ml 的检测下限。应用 TaqMan PCR 于从热处理和富集生牛乳中获得的菌落裂解物，可快速准确地检测类芽孢杆菌。用该菌落 TaqMan PCR 检测到热处理牛乳样品中类芽孢杆菌属（≥1 CFU/ml），在 6°C 冷藏储存 21 天后，细菌计数很高（>4.30log CFU/ml）。因此，我们开发了一种快速检测类芽孢杆菌属的工具，该工具有可能识别具有微生物变质潜力的生牛乳作为巴氏杀菌产品。
