Dierick Evelien, Callens Chana, De Spiegelaere Ward, Ducatelle Richard, Van Immerseel Filip, Goossens Evy
Livestock Gut Health Team (LiGHT), Department of Pathobiology, Pharmacology and Zoological Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Appl Microbiol Biotechnol. 2023 Nov;107(22):6973-6983. doi: 10.1007/s00253-023-12779-8. Epub 2023 Sep 14.
The ClosTron mutagenesis system has enabled researchers to efficiently edit the clostridial genome. Since site-specific insertion of the mobile ClosTron insert may cause errors, validation is key. In this paper we describe the use of digital PCR (dPCR) as an alternative tool in selecting clostridial mutant strains. Clostridium perfringens chitinase mutant strains were constructed in which the mobile ClosTron intron was inserted into one of the chitinase genes. On-target insertion of the mobile intron was validated through conventional PCR. In order to confirm the absence of off-target insertions, dPCR was used to determine the amount of the ClosTron intron as well as the amount of a reference gene, located in close proximity to the interrupted gene. Subsequently, mutant strains containing an equivalent amount of both genes were selected as these do not contain additional off-target mobile ClosTron inserts. The outcome of this selection procedure was confirmed through a validated PCR-based approach. In addition to its application in mutant selection, dPCR can be used in other aspects of clostridial research, such as the distinction and easy quantification of different types of strains (wildtype vs. mutant) in complex matrices, such as faecal samples, a process in which other techniques are hampered by bacterial overgrowth (plating) or inhibition by matrix contaminants (qPCR). This research demonstrates that dPCR is indeed a high-throughput method in the selection of clostridial insertion mutants as well as a robust and accurate tool in distinguishing between wildtype and mutant C. perfringens strains, even in a complex matrix such as faeces. KEY POINTS: • Digital PCR as an alternative in ClosTron mutant selection • Digital PCR is an accurate tool in bacterial quantification in a complex matrix • Digital PCR is an alternative tool with great potential to microbiological research.
ClosTron诱变系统使研究人员能够高效地编辑梭菌基因组。由于移动ClosTron插入序列的位点特异性插入可能会导致错误，因此验证是关键。在本文中，我们描述了使用数字PCR（dPCR）作为选择梭菌突变菌株的替代工具。构建了产气荚膜梭菌几丁质酶突变菌株，其中移动ClosTron内含子插入到一个几丁质酶基因中。通过常规PCR验证了移动内含子的靶向插入。为了确认不存在脱靶插入，使用dPCR来确定ClosTron内含子的量以及位于中断基因附近的参考基因的量。随后，选择含有等量两种基因的突变菌株，因为这些菌株不包含额外的脱靶移动ClosTron插入序列。通过一种经过验证的基于PCR的方法证实了这种选择程序的结果。除了其在突变体选择中的应用外，dPCR还可用于梭菌研究的其他方面，例如在复杂基质（如粪便样本）中区分和轻松定量不同类型的菌株（野生型与突变型），在这个过程中，其他技术会受到细菌过度生长（平板培养）或基质污染物抑制（qPCR）的阻碍。这项研究表明，dPCR确实是一种高通量方法，可用于选择梭菌插入突变体，也是一种强大而准确的工具，可用于区分野生型和突变型产气荚膜梭菌菌株，即使在诸如粪便这样的复杂基质中也是如此。要点：• 数字PCR作为ClosTron突变体选择的替代方法 • 数字PCR是复杂基质中细菌定量的准确工具 • 数字PCR是一种对微生物学研究具有巨大潜力的替代工具。
