ANSES, Food Safety Laboratory, Bacteriology and Parasitology of Fishery and Aquaculture products Unit, F-62200 Boulogne-Sur-Mer, France.
UMR UMET, INRA, CNRS, Univ. Lille 1, 59650 Villeneuve d'Ascq, France.
Int J Food Microbiol. 2018 Feb 2;266:289-294. doi: 10.1016/j.ijfoodmicro.2017.12.012. Epub 2017 Dec 16.
This study was designed to assess the efficiency of eight extraction methods regarding their ability to release superficial (exogenous) and intracellular (endogenous) DNA from B. cereus spores for subsequent analysis by quantitative PCR (qPCR). B. cereus spore suspensions were subjected to both commercial DNA extraction kits and mechanical DNA extraction methods. The spores were observed by transmission electron microscopy to evaluate any damage caused during extraction. The efficiency of both extraction and purification were assessed using a qPCR assay targeting the bclA gene. Most of the extraction methods assessed, except the passage through the French press or the use of the QIAamp DNA Blood Mini kit without 95°C treatment, allowed the amplification of significant amounts of DNA. By using propidium monoazide, which is a photoreactive DNA-binding dye, the presence of non-negligible amounts of amplifiable DNA at the spore surface was highlighted. A further set of extraction assays was then performed on spores previously treated with PMA. The results of this study show that both superficial and intracellular spore DNA can be released by extraction methods to a greater or lesser extent and then further amplified by qPCR. The Precellys extraction allowed the detection of both intracellular and superficial DNA, the DNeasy Blood & Tissue kit the specific detection of intracellular DNA, while the Instagene kit detected only superficial DNA. Of the methods tested in this study, the Precellys extraction was the most efficient in terms of further DNA detection.
In order to verify the presence or absence of B. cereus spores in food or on surfaces in the food environment, the use of an efficient extraction method is required, followed by a qPCR analysis on the DNA released. Conversely, in order to quantify the population of Bacillus spores, any superficial DNA must be blocked, e.g. with PMA, prior to intracellular DNA extraction and further amplification.
本研究旨在评估八种提取方法从 B. cereus 孢子中释放表面（外源性）和细胞内（内源性）DNA 的效率，以便随后通过定量 PCR（qPCR）进行分析。B. cereus 孢子悬浮液经过商业 DNA 提取试剂盒和机械 DNA 提取方法处理。通过透射电子显微镜观察孢子，以评估提取过程中造成的任何损伤。使用针对 bclA 基因的 qPCR 检测评估提取和纯化的效率。评估的大多数提取方法（除了通过法国压榨机或不使用 QIAamp DNA Blood Mini 试剂盒在 95°C 下处理的方法）都允许扩增大量的 DNA。通过使用光反应性 DNA 结合染料原卟啉单甲醚，可以突出孢子表面存在相当数量的可扩增 DNA。然后对先前用 PMA 处理的孢子进行了另一组提取检测。本研究的结果表明，表面和细胞内孢子 DNA 可以通过提取方法在不同程度上释放出来，然后通过 qPCR 进一步扩增。Precellys 提取允许检测到细胞内和表面 DNA，DNeasy Blood & Tissue 试剂盒特异性检测细胞内 DNA，而 Instagene 试剂盒仅检测表面 DNA。在本研究中测试的方法中，Precellys 提取在进一步检测 DNA 方面效率最高。
为了验证食品或食品环境表面是否存在 B. cereus 孢子，需要使用有效的提取方法，然后对释放的 DNA 进行 qPCR 分析。相反，为了定量检测芽孢杆菌孢子的数量，必须在提取细胞内 DNA 并进一步扩增之前，用 PMA 等方法阻断任何表面 DNA。
