Department of Food Science and Biotechnology, Kangwon National University, Chuncheon, 24341, Republic of Korea.
Department of Agricultural Biotechnology, National Academy of Agricultural Science, Rural Development Administration, Jeonju, 54874, Republic of Korea.
Microb Pathog. 2018 Feb;115:312-320. doi: 10.1016/j.micpath.2017.12.079. Epub 2018 Jan 3.
The aim of the study was to identify and evaluate specific biomarkers to differentiate within Bacillus cereus group species from contaminated food samples with the use of real-time PCR. A total of 120 strains, comprising of 28 reference, 2 type, 78 wild strains of B. cereus and B. thuringiensis along with 12 strains representing 2 bacterial groups - B. mycoides, B. pseudomycoides, B. weihenstephanensis (B. cereus group); B. amyloliquefaciens, B. subtilis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Micrococcus luteus, Salmonella enterica, Staphylococcus aureus, Streptococcus pyogenes (non-Bacillus sp.) were identified by applying valid biomarkers (groEL and gyrB). In addition, the presence of B. cereus group was determined in three different artificially contaminated vegetable samples (lettuce, spinach, and kimbap), using prominent biomarkers targeting on chaperonin protein (GroEL) and topoisomerase enzyme protein (gyrB). Direct analysis of samples revealed the specificity towards identification and characterization of the B. cereus group among wild, reference and type strains and the type strain inoculated in vegetables. Our results demonstrated two existing biomarkers groEL and gyrB with a high specificity of 98% and 96% respectively to analyze the total B. cereus group. Further, we also reported the detection limit of groEL and gyrB in food samples was 3.5 and 3.7 log CFU/g respectively. Thus, the developed real-time PCR approach can be a reliable and effective tool for the identification of B. cereus group strains present in environment and food samples. This does not require band isolation, re-amplification, sequencing or sequence identification, thus reducing the time and cost of analysis.
本研究旨在利用实时 PCR 技术从污染食物样本中识别和评估特定生物标志物，以区分蜡样芽胞杆菌组内的物种。共检测了 120 株菌，包括 28 株参考菌株、2 株模式菌株、78 株野生蜡样芽胞杆菌和苏云金芽胞杆菌以及 12 株代表 2 个细菌群的菌株——芽胞杆菌属(mycoides)、芽胞杆菌属(pseudomycoides)、芽胞杆菌属(weihenstephanensis)(蜡样芽胞杆菌组)；解淀粉芽胞杆菌、枯草芽孢杆菌、粪肠球菌、大肠杆菌、藤黄微球菌、单核细胞增生李斯特菌、肠炎沙门氏菌、金黄色葡萄球菌、化脓性链球菌(非芽孢杆菌属)。通过应用有效生物标志物（groEL 和 gyrB）对这些菌株进行了鉴定。此外，还使用针对伴侣蛋白（GroEL）和拓扑异构酶酶蛋白（gyrB）的显著生物标志物，在三种不同的人工污染蔬菜样本（生菜、菠菜和紫菜包饭）中检测到了蜡样芽胞杆菌组的存在。直接分析样本显示，这些生物标志物对野生、参考和模式菌株以及接种在蔬菜中的模式菌株的蜡样芽胞杆菌组具有特异性。我们的研究结果表明，现有的两个生物标志物 groEL 和 gyrB 具有高度特异性，分别为 98%和 96%，可用于分析总蜡样芽胞杆菌组。此外，我们还报告了 groEL 和 gyrB 在食品样本中的检测限分别为 3.5 和 3.7 log CFU/g。因此，所开发的实时 PCR 方法可以成为一种可靠且有效的工具，用于鉴定环境和食品样本中存在的蜡样芽胞杆菌组菌株。该方法不需要进行带分离、再扩增、测序或序列鉴定，从而减少了分析的时间和成本。
