Malays. Appl. Biol. (June 2003) 32(1): 35-44
INTRA- AND INTER-SEROVARS RANDOM AMPLIFICATION
POLYMORPHIC DNA TYPING OF SALMONELLA WELTEVREDEN,
SALMONELLA AGONA AND SALMONELLA HADAR
NOORZALEHA, A.S., 3CHEAH, Y.K., 2*SON, R. and 'GULAM, R.R.A.
Department of Food Science, Department of Biotechnology, Faculty of Food Science and Biotechnology,
Department ofBiomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor.
ABSTRACT
Random amplification of polymorphic DNA (RAPD) method was used for intra- and inter-serovars differentiation of Salmonella Weltevreden (44 strains), Salmonella Agona (23 strains) and Salmonella Hadar (16 strains) obtained from poultry and four types of vegetables namely 'selom' (Oenanthe stolonifera), 'pegaga' (Centella asiatica), 'kesum' (Polygonum minus) and 'kangkong' (Ipomoea aquatica). Intra- and inter-serovars discrimination was obtained between all the typable strains. The similarity of RAPD patterns expressed as clusters, based on numerical analysis of RAPD patterns, was found to correlate with product type and location. However, some of the recurrently isolated strains were repeatedly found in products from a particular wet market, suggesting the persistence house-flora strains. Extracts used in the RAPD-PCR reaction were prepared directly from boiled cells of the test strains, which made it possible to complete the analysis in one day. These results demonstrate the usefulness of the RAPD method to trace the distribution and sources for contamination of products in wet markets.
ABSTRAK
Kaedah amplifikasi secara rawak DNA polimorfik (RAPD) digunakan untuk pembezaan secara intra- dan inter-serovar pencilan Salmonella Weltevreden (44 pencilan), Salmonella Agona (23 pencilan) dan Salmonella Hadar (16 pencilan) yang diperolehi dari sampel daging ayam dan empat jenis sayur (selom, pegaga, kesum dan kangkong). Diskriminasi intra- dan inter-serovar dapat diperolehi untuk kesemua pencilan yang boleh dibezakan. Kesamaan corak RAPD yang diperolehi secara kluster, yang berdasarkan analisis berangka terhadap corak RAPD, didapati berkaitan dengan jenis produk dan juga lokasi. Walau bagaimana pun, ada pencilan yang dapat dipencilkan berulangkali dalam produk tertentu dari pasar yang tertentu sahaja, dan ini menunjukkan kehadiran pencilan yang dianggap "house-flora". Ekstrak yang digunakan dalam tindakbalas RAPD-PCR disediakan dari sel-sel pencilan yang direbus, dan ini membolehkan analisis disempurnakan dalam tempoh satu hari. Hasil-hasil kajian ini menunjukkan potensi yang baik penggunaan teknik RAPD dalam menjejak penyebaran dan juga cara-cara kontaminasi hasilan di pasar berlaku.
Key words: RAPD-PCR, Salmonella spp., typing, poultry, vegetables.
REFERENCES
Aarts, H.J.M., van Lith, L.A.J.T. and Keiger, J. 1998. High-resolution genotyping of Salmonella strains by AFLP-fingerprinting. Letters in Applied Microbiology, 26: 131-135.
Bailey, J.S., Chiu, J.Y., Cox, N.A. and Johnston, R.W. 1988. Improved selective procedure for detection of salmonella from poultry and sausage products. Journal of Food Protection, 51: 391-396.
Blivet, D., Salvat, G., Humbert, F. and Collin, P. 1997. Evaluation of new enrichment broth for the isolation of Salmonella spp. from poultry products. International Journal of Food Microbiology, 38: 211-216.
Beuchat, L.R. 1996. Pathogenic microorganisms associated with fresh produce. Journal of Food Protection, 59: 204-216.
Ellsworth, D.L., Rittenhouse, K.D. and Honeycutt, R.L. 1993. Artifactual variation in randomly amplified polymorphic DNA banding patterns. Biotechniques, 14:214.
Heinitz, M.L., Ruble, R.D., Wagner, D.E. and Tatini, S.R. 2000. Incidence of Salmonella in fish and seafood. Journal of Food Protection, 63: 579-592.
Hilton, A.C., Banks, J.G. and Penn, C.W. 1996. Random amplification of polymorphic DNA (RAPD) of Salmonella: strain differentiation and characterization of amplified sequences. Journal of Applied Bacteriology, 81: 575-584.
Landeras, E., Gonzalez-Hevia, M.A. and Mendoza, M.C. 1998. Molecular epidemiology of Salmonella serotype Enteritidis. Relationship between food, water and pathogenic strains. International Journal of Food Microbiology, 43: 81-90.
Lin, A.W., Usera, M.A., Barret, T.J. and Goldsby, R.A. 1996. Application of random amplified polymorphic DNA analysis to differentiate strains of Salmonella enteritidis. Journal of Clinical Microbiology, 34: 870-876.
MacPherson, J.M., Eckstein, P.E., Scoles, GJ. and Gajahar, A.A. 1993. Variability of the randomly amplified polymorphic DNA assay among thermal cyclers and effects of primer and DNA concentration. Molecular and Cellular Probes, 7: 293-299.
Meunier, J.R. and Grimont, P.A.D. 1993. Factors affecting reproducibility of random amplified polymorphic DNA fingerprinting. Research in Microbiology, 144: 373-379.
Son, R., Sahilah, A.M., Rusul, G., Samuel, L., Zuraini, M.I. and Nasreldin, E.H. 2000. Comparison of arbitrarily primed PCR, antibiotic resistance and plasmid profiling for differentiating Salmonella enteritidis isolated from fish. Asian Fisheries Science, 13:13-20.
Soto, S.M., Guerra, B., Gonzalez_hevia, M.A. and Mendoza, M.C. 1999. Potential of three-way randomly amplified polymorphic DNA analysis as a typing method for twelve Salmonella serotypes. Applied and Environmental Microbiology, 65: 4830-4836.
Tauxe, R.V. 1991. Salmonella : a post modern pathogen. Journal of Food Protection, 54: 563-568.
Welsh, J. and McClelland, M. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research, 18: 7213-7218.
Williams, J.G.K., Hanafey, M.K., Rafalski, J.A. and Tingey, S.V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research, 18: 6531-6535.
Yee, C.F. and Ayob, M.K. 1994. Isolation and identification of Salmonella spp. from some local food products. Tropical Biomedicine, 11: 53-60.
