Digital PCR: a tool in clostridial mutant selection and detection.

ABSTRACT

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.