Rapid identification using a chromogenic medium and molecular chraracterization of hly positive listeria monocytogene

• Main Author: Broooke., Pearlycia
• Format: Final Year Project Report
• Language: English; English
• Published: Universiti Malaysia Sarawak (UNIMAS) 2005
• Subjects: QD Chemistry; SF Animal culture
• Online Access: http://ir.unimas.my/id/eprint/20086/1/Pearlycia%20Brooke%2024pgs.pdf; http://ir.unimas.my/id/eprint/20086/4/Pearlycia%20Brooke%20ft.pdf; http://ir.unimas.my/id/eprint/20086/

A total of 87 raw meat samples (chicken, beef, and pork) marketed in markets in Kuching were investigated for the presence of hly positive Listeria monocytogenes. The identification of the bacterial species were based on morphological appearance on a chromogenic medium called CHROMagar Listeria agar (CHROMagar Microbiology, France), biochemical tests, followed by a specific PCR to detect the presence of hly gene in all the strains isolated. Using a pair of primer to amplify a product of 730 bp in size, 15 strains of L. monocytogenes were successfully isolated from the 87 samples. Along with 20 isolates from the laboratory collection, these isolates were subjected to RAPD-PCR to investigate their genetic relatedness. Four random primers were screened for their discriminatory abilities by visualizing the amplification products electrophoretically. GENI-50-09 which amplified useful banding patterns was selected and tested against all the isolates. The primer successfully produced 19 distinctive RAPD patterns with bands having molecular izes ranging from 500 bp to 1 kbp. The genetic relationships between the isolates were represented in the form of a dendogram based on the proportion of identical bands in the RAPD profiles. Clustering analysis allowed the differentiation of 2 major clonal clusters, each containing 8 and 27 isolates respectively, with different animal hosts and geographical origins. Cluster 1 was found to be highly heterogeneous with the average genetic distance of D = 0.69. Cluster 2 on the other hand, was more homogeneous with the average genetic distance of D = 0.18, showing that the isolates in this cluster have 82% of genetic similarity. In addition, there were 17 identical strains in these clusters. The different RAPD patterns demonstrated in this tudy may have implications to our understanding of the dissemination and evolution ofL. monocytogenes.