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

L1 5-CGG AGG TTC CGC AAA AGA TG-3
L2 5-CAT CGA CGG CAA CCT CGG A-3
L3 5-CCT CCA GAG TGA TCG ATG TT-3

PCR system Primer combination Product size
Initial PCR L1/L4 234 kb
Semi-nested PCR L2/L4 204 kb

All PCR reactions were carried out using the Mastercycler gradient from Eppendorf. Loading buffer (Sambrook et al.) was added to the amplified products of both PCR systems at a ratio of 5:1. 15 l of this solution was separated electrophoretically on a 1.5% agarose gel with 3.5 l ethidium bromide (10 mg/ml) in 1xTBE (Sambrook et al.) at 100 V for 15 minutes and then photographed in UV light by means of a gel-documentation system.

Experimental Procedure and Results

The detection limit of the PCR system was determined using the starting conditions (see materials and method), with DNA quantities of 20 pg 2 fg.

For the subsequent semi-nested reaction, the amplified products of the initial PCR were diluted at 1:100 in distilled water, with 2 l used as a template in the reaction.

Under the starting conditions, it was barely possible to detect 200 fg L. m. DNA with the start PCR (Fig.1). The subsequent semi-nested PCR achieved a sensitivity of 20 fg, although the product yield was not satisfactory.

To enable a positive result to be obtained for the semi-nested reaction, the start PCR was optimized with 20 fg template.

First optimization step: MgCl2 concentration: 1.5 mM 3.5 mM; temperature gradient: 58C 70C (Fig. 2)

20 fg L. m. DNA was used as the te
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