( canalytics. ...)Two-dimensional Optimization of a Semi-nested PCR,,, for Detecting Listeria monocytogenes

canalytics. Fig. 5. Under optimized conditions (3 mM MgCl2, 58C annealing temperature) the initial PCR (primer pair L1/L4) as well as the subsequent semi-nested PCR (primer pair L2/L4) was repeated with the Listeria monocytogenes DNA dilution series (20 pg to 2 fg). Discussion

In just a few steps, this simple experimental setup enabled the optimization of a PCR system. Varying the MgCl2 concentration while simultaneously testing the annealing temperature proved to be an effective strategy.

Due to the temperature gradient of Mastercycler gradient, it was possible to test both parameters simultaneously, thereby reducing the time required. Increasing the MgCl2 concentration from 1.5 mM to 3 mM brought about an increase in the product yield, with an increase in the annealing temperature from 55C to 58C, providing a boost to the specificity of primer binding.

Overall, the sensitivity of the PCR system showed a tenfold improvement, with 20 fg Listeria monocytogenes DNA detected, which is roughly equivalent to a copy number of five cells. The fact that the hly gene is a single copy gene explains the negative PCR result for a template quantity of 2 fg DNA.

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