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

ange, reached a plateau at 57.6C 58.9C and then dropped dramatically. The highest OD values, and thus the highest product yield, was obtained at a temperature of 58C.

Verifying the optima determined (Fig. 5)

Under the optimized conditions (3 mM MgCl2, 58C annealing temperature), the initial experiment was repeated with the DNA amounts from 20 pg to 2 fg.
The optimized system enabled 20 fg DNA to be detected in the initial PCR L1/L4, which is equivalent to a tenfold reduction in the detection limit. The subsequent semi-nested PCR produced an unequivocally positive result based on 20 fg DNA.

Fig. 2: First optimization of the initial PCR (primer pair L1/L4) by varying the MgCl2 concentration (1.5 mM 3.5 mM) and by applying a temperature gradient (58C 70C). 20 fg Listeria monocytogenes DNA was used in each case. Fig. 3. Limiting the annealing temperature for the initial PCR (primer pair L1/L4) by applying a temperature gradient (55C 65C; 3 mM MgCl2). 20 fg Listeria monocytogenes DNA was used in each case. Fig. 4. Densitometric evaluation of the bands of agarose gel from Fig. 3. Evaluation was performed using RFLP Scan from S
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