1. Listeria Experiment Name:PCR nr 114_05_listeria_hly
Detection and identification of Listeria monocytogenes in food. Optimization PCR duplex reaction
Anna Misiewicz, Sylwia Wróblewska, Irena Sikorska, Maria Spera Culture Collection on Industrial Micro-organisms, Department of Microbiology, Institute of Agricultural and Food Biotechnology, Warsaw
900 .
INTRODUCTION
Listeria monocytogenes is a recognized pathogenic bacteria for animals and humans. Listeria are Gram-positive, non sporulating, rod-shaped bacteria occurring ubiquitously in soil and water. Listeriosis, caused by Listeria monocytogenes, is known to be transferred via contaminated foods, because they grow under conditions used for food preservation. A peculiar property of L. monocytogenes that affects its food-borne transmission is to ability to multiply at low temperatures. The bacteria may therefore grow and accumulate in contaminated food stored in the refrigerator. Pathogenity of Listeria is mediated by proteins that specifically interact with host proteins.
The genus Listeria comprises six species: Listeria monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L.welshimeri and L. grayi.
In the IAFB Culture Collection were developed identification method of Listeria monocytogenes using duplex PCR technique. First pair of primers was chosen to amplify iap gene – coding extracellular protein p60, which 660-bp DNA fragment is specific for L. monocytogenes. The second pair of starters were selected to obtain an 750-bp DNA fragment of gen hly, virulence gene, coding protein listeriolysin, infection factor in endocellular invasion of L. monocytogenes.
THE AIM
The objective of the study was to develop the method of detection and identification Listeria monocytogenes occuring in food.
MATERIALS AND METHODS
Genomic DNA was prepared by phenol method from liquid cultures (50 ng/25 ml reaction mixture) as well as directly from bacteria from agar plates. PCR was performed with TaqDNA polymerase (DNA Gdansk) according to the manufacture’s instructions and with genomic DNA from L. ivanovii, L. innocua, L. grayi , L. welcheri, L. seelighieri, L. murayi bacteria collected from food sample, our collection and CCM.  
All isolates were identified to the species level by API Listeria kit (BioMerieux) PCR products were visualized by gel electrophoresis using 1.5% agarose gels containing ethidium bromide. The molecular sizes of the obtained fragments were estimated by comparison with the DNA standard. 2. 3. 1.
~ 660 pz
1000 pz
900 pz
600 pz
1. Listeria monocytogenes 1
2. Listeria monocytogenes 24
3. H2O
Listeria gen iap
Listeria Experiment Name:PCR nr 114_05_listeria_hly
1000
900
800
700
600
~ 730 pz 2 5 21 20 19 18 17 16 15 14 13
8. L. welhieri 22
9. L. monocytogenes 5.
10. L. grayi 21
11. L. ivanovii 12.
12. L. 18.
13. L. monocytogenes 24.
14. L. monocytogenes 1.
1. L. innocua 10
2. L. monocytogenes 6.
3. L. seelighierii 19
4. L innocua 16
5. L. monocytogenes 4.
6. L. monocytogenes 2.
7. L. monocytogenes 12
15. L. monocytogenes 8.
16. L. monocytogenes 23.
17. L. monocytogenes 26.
18. L. monocytogenes 11.
19. L. monocytogenes 27.
20. H2O
21. E. coli B
Listeria żel 117_05_żel nr 1
~ 730
~ 660
CONCLUSIONS
This identification method can be used to pathogen detect directly from agar plate. In the near future, after technique improving, we will detect and identify Listeria monocytogenes directly from food sample.
*Collection is a member of WFCC and ECCO. Collection has a status of IDA.