Vet. World, 2012, Vol.5(3):169-172 RESEARCH Screening of poultry samples for Salmonella Typhimurium by PCR assay

Poultry samples viz., cloacal swabs, egg swabs, poultry faeces and feed were screened for Salmonella Typhimurium. A set of primers derived from fli C gene were employed to standardize PCR for detection of Salmonella Typhimurium from poultry samples, which gave specific amplification of a 620 bp fragment. Boiling and snap chilling method used for template preparation. Screening of 112 samples revealed that 12 samples positive for Salmonella Typhimurium by PCR assay.


2000)
. Poultry products can be major vehicles of food borne salmonellosis because the raw Food safety hazards caused by food-borne products are initially contaminated with salmonella pathogens such as Salmonella Typhimurium cells (Bryan and Doyle 1995).remain a major problem for the food industry, Salmonella enterica is mainly transmitted to particularly poultry processors (USDA-2003).It humans following consumption of contaminated is the major causes of food-borne disease eggs and poultry products.Hence, a rapid throughout the world (Wang et al., 2008).
detection and identification method of this Salmonella infected chickens represent a source serovar is necessary in the food industry (Lim of pathogens for humans, causing severe illness et.al. 2003).The rapid, cost effective and and even death.Salmonella Typhimurium is also automated diagnosis of food borne pathogens the most frequently isolated serovar from global throughout the food chain continues to be a major food-borne outbreaks.Poultry are one of the most concern for the industry and public health.The important reservoirs of Salmonellae that can be test supplies the growing demand for validated transmitted to humans through the food-chain.diagnostic PCR methods for screening of The commonest serotypes causing disease in samples in meat production chain to assure safe humans are S. enteritidis and S. Typhimurium food Lofstrom et. al. (2010).(Aktas et al., 2007).Kotova et. al. (1988) observed

Materials and Methods
that humans develop the salmonella carrier state after acute salmonellosis and it is due to result of Sample collection: 20 each cloacal swabs and egg occupational exposure to poultry (6.1% -8.8%).swabs, 12 poultry faecal samples and 10 feed There has long been an association between the samples from poultry farm were aseptically contamination of eggs and egg products with collected from All India Coordinated Research salmonella and human infection (Humphrey, (AICRP) on poultry for eggs, Hyderabad.Fifty chicken samples (50g) were collected aseptically annealing temperatures and cycling conditions.from local shops of Rajendranagar.
The reaction mixture consisted of 5ul of the template, 2.5 µl of 10x assay buffer for Taq Enrichment of samples: Cloacal and egg swabs, polymerase containing 1.5 mM MgCl , 1 µl of 25 2 faecal samples and feed samples were inoculated µM each dNTP mix, 1 µl each of forward and in buffered peptone water in test tubes (50ml) and o reverse primer (4 pmol) and 0.9 U/ µl of Taq DNA incubated at 37 C, 16h.About 10g of each chicken polymerase made up to 25 µl using molecular samples were inoculated into 90ml buffered grade water.Routinely, master mix was prepared peptone water (BPW) in individual sterile polythene and 20 µl each was distributed to the PCR tubes, bags homogenized thoroughly in a stomacher for to which 5 µl of the template was added.The bacterial strains Salmonella 72 C at 7 min.The amplification products were Typhimurium and Salmonella Enteritidis were analyzed by agarose gel electrophoresis using obtained from Department of Veterinary 1.5% agarose gel containing 0.5 µg/ml ethidium Microbiology, College of Veterinary Science, bromide at constant voltage 5 V/cm in 1x TAE.Rajendranagar

Results and Discussion
DNA isolation: The genomic DNA isolation was In India the Salmonella has been isolated carried out by phenol: chloroform: iso amyl alcohol from variety of livestock foods including milk method from the bacterial strain Salmonella and milk products, eggs, fish, crustaceans, meat Typhimurium to standardize PCR assay for and meat products and also from various detection of Salmonella Typhimurium.DNA environmental samples collected from slaughter templates were prepared from samples by boiling houses and fish processing plants (Nagappa et al and snap chilling method.In this method, about 2007; Sharma et.al.,1995).There is wide 1000 µl of the 24h inoculums from the selective difference in the isolation of Salmonella spp.by enrichment was centrifuged at 6000rpm for 5 min various scientists from various foods ranging and resuspended in 50 µl of molecular grade from 0 (Ramasastry et.al.1999) to 100% (Sharma water.The suspension was then kept in a boiling et.al. 1989;Kamat et.al. 1991).These differences water bath for 10 min and immediately transferred might be due to geographic, seasonal variations onto ice, later it was centrifuged at 13000rpm for and also due to procedures adopted for isolation.5min.for PCR technique, five µl of supernatant Therefore, there is a significant need for was used as template.16): 5'-ACT GGT the application of such methods, and there is still AAA GAT GGC T-3'.The PCR protocol was a need for a rapid, sensitive, specific and userinitially standardized by optimizing the friendly method.One possible approach involves concentration of the components of the reaction polymerase chain reaction based assay, which has mixture in the PCR assay and by varying become a powerful and increasingly popular tool in other areas of microbial detection and analysis and pulsed-field gel electroidentification.As PCR relies on the detection of phoresis.Inter.J. Antimicrob.Agent, 30: specific gene fragments, it can be applied in mixed 541-545.microbial cultures, avoiding problems which 2. Bennett, A.R., Greenwood, D,.Tennant, C., may arise using other biochemical and morpho-Banks J.G. and Betts, R.P. (1998 o 3 to 5 min and incubated at 37 C for 16h.Amplification was carried out with initial Selective enrichment was done in Tetra thionate o denaturation at 94 C for 5 min, followed by 35 (TT) broth for which 1ml of pre-enrichment o cycles each of denaturation at 94 C for 1 min, inoculum was transferred to TT (10ml) and were o o annealing at 45.1 C for 30 sec and extension at incubated at 42 C for 18h.o 72 C for 38 sec with a final extension period of o Bacterial strains: more rapid and/or sensitive methods for the Standardization of PCR: The primers derived identification of Salmonella.Several techniques from fli C gene for detection of Salmonella have been developed to address this need, Typhimurium were custom synthesized by integrated including DNA hybridization (Chevrier et.al.DNA technologies.The nucleotide sequence of 1995), fluorescent antibodies and enzyme-linked the primers (Olivera et.al.2002) used in this study immunosorbent assay (Fadeel et.al.2004).However, was Fli 15 (22): 5'-CGG TGT TGC CCA GGT sensitivity and specificity problems have limited TGG TAA T-3' and Typ 04 ( Bennett et.al.1998; Chao et.al.1998; Tsen and and Campylobacter jejuni in raw poultry.J. Chen, 2001).Of these, inv A gene and fli C gene Food Protect.58 326-344.have been the most frequently targeted genes for 4. Catarame, T.M.G., O'Hanlon, K.A., primer selection in PCR based Salmonella spp.Salmonella and simultaneous Out of 20 swabs from eggs, 4 swabs were detection of Salmonella and Shiga-like positive.These observations were in accordance et.al.toxin producing Escherichia coli using the with the findings of Moussa (2010).One sample was found positive out of 10 feed samples magnetic capture hybridization polymerase analyzed.Out of 12 faecal samples and 20 cloacal chain reaction.Lett Appl Microbiol., 32: 7swabs, one and 3 samples were positive.These 11. findings are more or less similar to the results of 7. Chevrier, D., Popoff, M.Y., Dion, M.P., Gonclaves et.al.(1998).Hermant, D. and Guesdon, J.L. (1995).Rapid detection of Salmonella subspecies I