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

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

Andrea Tanzer and Christian Rohrer
Andrea Tanzer, Institute of Milk Hygiene, Milk Technology and Food Science, Austria
Christian Rohrer, Eppendorf AG, Germany Introduction

Bacterial enteritides, which are triggered by contaminated foodstuffs, pose a significant health risk for consumers. The legally prescribed food tests are intended to prevent such products from finding their way into retail outlets. However, microbiological tests usually take several days since the pathogenic bacteria are often present in very low numbers in relation to the overall number of bacteria, which means that selective enhancement steps must be carried out prior to the actual detection reactions.

Among the problem bacteria is Listeria monocytogenes, a gram-positive, rod-shaped, facultative anaerobic bacterium. L. m. is ubiquitous and normally enters foodstuffs during processing and packaging. Milk and milk products are commonly affected by this bacteria.

The Austrian milk hygiene decree stipulates that L. m. must not be present in untreated cow's milk that is intended for human consumption, cheese (with the exception of hard cheeses), pasteurized milk or any other dairy products.

The standard method for detecting L. m. is the IDF (International Dairy Federation) method, although the test requires up to seven days. Modern molecular biology methods enable results to be obtained rapidly. The publication by Allmann et al. (1995) presents various, PCR*-based systems for detecting pathogenic microorganisms in dairy products.

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.


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

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 mplate DNA in all optimization reactions. The MgCl2 concentration as well as the annealing temperature were varied systematically. Amplified products were detectable on the agarose gel up to a temperature of 62.8C. The optimal annealing temperature should be below this temperature. At a MgCl2 concentration of 3 mM, the product yields were at their maximum; no increase in yield was ascertained when the concentration was increased to 3.5 mM.

Second optimization step: MgCl2 concentration 3 mM; temperature gradient: 55C 65C (Fig. 3)

In order to further determine the exact annealing temperature, a temperature window of 55C 65C was applied at the optimal MgCl2 concentration of 3 mM. A visual assessment of the band intensity was sufficient for assuming a temperature optimum of 58C.

To quantify the product yield, a densitometric measurement of the electrophoretically separated amplification products was carried out.

Fig.1. Determining the detection limit for Listeria monocytogenes DNA based on the initial PCR (primer pair L1/L4), as well as on the subsequent semi-nested PCR (primer pair L2/L4). Starting conditions: 20 pg 2 fg DNA per reaction; 1.5 mM MgCl2; 55C annealing temperature. Densitometric measurement (Fig. 4)

Using an OD measurement, the relative product yield is determined on the basis of the intensity of the bands in the gel (in Fig. 3). With rising temperature, the OD also increased continuously in the initial r 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 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.


Allmann M. et al. Polymerase chain reaction (PCR) for detection of pathogenic microorganisms in bacteriological monitoring of dairy products. Res Microbiol. 1995:146; 85-97.

Furrer B, et al. Detection and identification of Listeria monocytogenes in cooked sausage products and in milk by in vitro amplification of haemolysin gene fragments. J Appl Bacteriol. 1991:70; 327-379.

Sambrook S, Fritsch EF, Maniatis T, eds. Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.

Andrea Tanzer
Institute of Milk Hygiene, Milk Technology and Food Science, Austria,
Veterinrmedizinische Universitt Wien,
Veterinrplatz 1, A-1210 Wien, Austria

*This product is sold under licensing arrangements with F. Hoffman-La Roche Ltd., Roche Molecular Systems, Inc. and Applied Biosystems.



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