Cloning and Characterisation of a Δ-prfA Listeria monocytogenes Strain Containing an Artificial Single Copy Genomic Internal Amplification Control (IAC) for Use as Internal Sample Process Control (ISPC)

摘要

Conventional internal amplification controls (IAC) are DNA-based controls which monitor the amplification reaction of real-time PCR in food pathogen detection. Food pathogen detection using real-time PCR, however, includes necessarily sample preparation and DNA isolation/purification. This modular structure leads to an analytical chain. To cover the whole analytical chain, the concept of the IAC has to be extended to internal sample process controls (ISPCs) which include supporting pre-analytical steps. One concept for such ISPCs is the use of recombinant bacterial cells comprising a deleted target and an artificial competitive target instead, which are derived from the actual target strain. In this work, we present an ISPC for the molecular detection of Listeria monocytogenes. A Δ-prfA L. monocytogenes EGDe strain was cloned with a pPL2 phage insertion vector to include a single copy artificial DNA target, resulting in a fluorescence signal not interfering with the respective signal of the L. monocytogenes EGDe wild-type strain during real-time PCR. The recombinant strain was confirmed and characterized with conventional and real-time PCR including sequencing. Microbiological examination revealed a distinct phenotype pattern on selective plate media which enables discrimination of Δ-prfA L. monocytogenes EGDe from wild-type L. monocytogenes EGDe and Listeria innocua. The ISPC was applied in an examination of artificially contaminated ultra high temperature-treated milk to demonstrate its analytical suitability. The resulting corrected recovery values of the ISPC as obtained by the whole molecular quantification procedure correspond to the respective values determined for the actual target strain (P ≤ 0.05).