Development of a quantitative polymerase chain reaction method using a live bacterium as internal control for the detection of Listeria monocytogenes

A novel real-time quantitative polymerase chain reaction (Q-PCR) assay was developed for rapid and accurate detection of Listeria monocytogenes. In this Q-PCR assay, a computational DNA random shuffling method was used to design an internal amplification control (IAC) sequence, which was the same in length and G + C content to the hly amplicon. This IAC sequence was inserted into the genome of L. monocytogenes to create a mutant strain named L. monocytogenes-IAC. The LM-IAC was used as an internal control during the PCR assay and produced accurate quantification of L. monocytogenes due to similar DNA extraction and amplification efficiencies between LM-IAC strain and wild-type L. monocytogenes. Quantification by this method was over a 5-log linearity range of initial L. monocytogenes with an R2 value of 0.9997. This PCR method will provide accurate quantification of L. monocytogenes and can be used in the clinic and food assays for diagnostic purposes.