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Two-dimensional Optimization of a Semi-nested PCR,,, for Detecting Listeria monocytogenes

In the case of the PCR-based detection for L. m., the hly gene was selected as a target sequence, which codes for listeriolysin O, one of the pathogenicity factors.

The PCR system consists of an initial PCR, primer combination L1/L4 (Furrer et al., 1991) with a subsequent semi-nested reaction, primer combination L2/L4 (Allmann et al., 1995) for confirmation/signal re-amplification of the results.

Objective

The aim of the investigations was to boost the sensitivity of this PCR system while, at the same time, maintaining the selectivity. The following questions were dealt with:

What detection limit does the existing system have under the starting conditions?
Can the detection limit be reduced by varying the above-mentioned temperatures?
What effect does increasing the MgCl2 concentration have on the product yield?
What is the relation between the MgCl2 concentration and annealing temperature with regard to the sensitivity limit?

Materials and Method

The Listeria monocytogenes strain EGD was used as test material. An overnight culture in TSB+Y was incubated at 37C, and DNA purification was carried out using the High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals). Following fluorimetric measurement, a 10-fold dilution series of 10 pg/l to 1 fg/l in distilled water was produced. The starting conditions for initial PCR as well as for subsequent semi-nested PCR were as follows:

The following primers were used:

Primer Primer sequence

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