Detection of Salmonella typhimurium in chicken meat imported in the local markets of Diwaniya city By using PCR technique

The aim of this study to detected contamination with Salmonella spp. In imported chicken meat in the local markets of AlDiwaniyia city . to protect health of consumer and Determintion the most contaminated origin with salmonella spp. A toatl of 100 chicken meat samples collected from different origin. The bacteria cultured and isolated in enrichment and selective media . Salmonella isolates were subjected to some biochemical tests show positive productive results H2S .TSI . SIM And its give negative for indole , vo-gs Proskauer and ureas , Biochemical identification was carried out using API 20-E test ..the result showed isolation sample (33\55)60% on bismuth sulphate agar and the results of isolation on chromogenic agar were 87.8 |%(29\33) .according to reading Api20-E system the results of confirmation of isolates 92%(25\26) In this study,( 23) Salmonella isolates were detected by polymerase chain reaction (PCR) by using 16s rRNA and invA gene these primers were selected specifically for the detection of Salmonella to amplify a 406bp and 558 bp DNA fragments, respectively. Only 7 isolates out of 23 were identified as S. typhimurium the results of this study showed the highest percent of s.typhimurim isolates was ( 50%) ( 3/6) for India origin and the lowest was Turkish origin Introduction Food borne diseases caused typhoid Salmonellosis represent an important public health problem worldwide. It is estimated that approximately 70%–80% of food borne bacterial outbreaks were caused by Salmonella spp. in China (1). In the United States Salmonella infections (approximately 32,000 annually) were reported during 1998–2002 (2). , beef and poultry /chicken meat have been recognized as significant sources of human salmonellosis (3). Salmonella serotypes, (S.) Typhimurium is one of the most important agents of food borne Salmonellosis in humans . [4] It was estimated that approximately 75% of human salmonellosis cases were due to contaminated food products, such as beef, pork, poultry and Chicken products are recognized as an important source molecular methods such as polemear chain reaction (PCR), have shown high sensitivity and specificity for detecting target pathogens, including Salmonella, in different types of foods, and the time required to obtain results can be as short as 12 h (5 ) The use of 16s r RNAgene or invA gene specific PCR method in most diagnostic and research laboratories is possible and through the molecular basis of Salmonella identification techniques, this method is the simplest and less expensive ( 6) the 16S rRNA genes are highly conserved among isolates belonging to the same bacterial species ,( 7) invA is located on the pathogenicity island 1 of Salmonella spp. encoding proteins of a type (T3SS) III secretion system this gene is highly conserved among the Salmonella spp. and is associated with the adherence and invasion of mammalian cell. AL-Qadisiya Journal of Vet.Med.Sci. Vol./12 No./2 2013 ______________________________________________________________________________ 134 Material and Methods 1-Collection of samples Chicken samples were collected from different market in al-diwaniyia city with different origin include different trademark ( al-kafeel ,al murad , thighs U.S.A, Turkish Chicken, Chicken JD) the sample transport by ice box about 25 g from meat sample were placed in 225 ml of enrichment medium tetrathionate broth in microbiological laboratory in veterinary collage for 18-24 h at 37°C.. this study took place during the period from December 2011 and carry on June2012. 2Isolation and identification of salmonella spp.:The samples were cultivated on to selective medium such as bismuth sulphate agar and chromogenic agar For identification of salmonella colonies, incubation at 37c ̊ for 18-24 hr samples were subjected to biochemical tests such as (TSI), SulfideIndole(SIM), (MRVP), Urea, and finally confirmed by using Api20-E system, Colonies that showed biochemical characteristics similar to that of Salmonella spp. were tested by API20-E system and the confirmation was identified by PCR with 16s rRNA and invA genes primers for the detection ofsalmonellaspp .3-Specific Primers Sequence Used for PCR Amplification: The primers used for the detection specific sequence of 16s rRNA gene ribosomal genes of Salmonella spp [8]. And invA gene encoding proteins of a type (T3SS) III secretion system [9]These primers are specific for designed in this study by using NCBI Gene Bank and Primer: online and provided by (Bioneer company, Korea) as following Table(1): Table(1):Specific primers used for the detection specific sequence of 16s rRNA gene and invA gene Size of PCR product( bp) Position Orientation Sequence 406 16s rRNA Forward CGG.,ACG,GGT,GAG,TAA,TGT ,CT Reverse GTT,AGC,CGG,TGC,TTC,TTC,. TG 558 Avni Forward ATG,CCC,GGT,AAA,CAG.ATG, ATG,AG Reverse CTC,GCC,TTT,GTC,GGT,TTT,A