PCR detection of Listeria monocytogenes in milk and milk products and differentiation of suspect isolates

Abstract

The main goal of our work was to solve the problems of normative diagnostic method for the detection of Listeria monocytogenes in foods by STN ISO 10560 (1999), i.e. to shorten the long time of analysis (5-10 days) and differentiate the non-haemolysed strains of Listeria monocytogenes from non-pathogenic Listeria innocua yielding the same results of biochemical and serological typing (serotype 4ab). PCR was employed to confirm the method for testing 60 suspect isolates from other laboratories of the Slovak Republic. We verified the detection of Listeria monocytogenes in milk and dairy products by nested PCR. The objective of this experiment was to analyze 100 various samples by traditional cultivation method and compare the results with those yielded by PCR. The results were in good agreement; all 18 positive and 72 negative samples were detected by both methods. However, the PCR method yields results within 2 days. This makes diagnosis in food control laboratories much more efficient. For PCR detection, two pairs of primers (PRFA 1 and 2, LIP 1 and 2) were used with affinity to prfA gene involved in the regulation of listeriolysin synthesis. The size of the PCR product was 1060 bp fragment in a first step of PCR and 273 bp fragment in nested PCR. The sensitivity and reliability of PCR was comparable with conventional methods. The PCR method solved the problem of interpretation of classical biochemical and serological typing in the only one step without the necessity of using additional examinations. Furthermore, we found that in most of the strains isolated from foods with a biochemical profile of non-haemolytic Listeria monocytogenes on blood agar serotype 4ab were not Listeria monocytogenes but a non-pathogenic Listeria innocua. This finding is very important from the point of view of food evaluation according to the STN ISO 10560 (1999).