Inv A gene specific PCR for detection of Salmonella from broilers

Poultry meat has been identified as one of the principal foodborne source of Salmonella. In this preliminary study the prevalence of Salmonella spp. contamination of broiler carcasses, were determined. Sixty samples were collected from poultry carcasses from the commercial broiler slaughtering facility in Namakkal, Tamil Nadu. The presence of Salmonella spp in collected samples was assessed by performing the pre-enrichment and enrichment culture, followed by PCR assay. The primers were selected from the invA gene specific for the detection of Salmonella spp. In this study 8.3% of poultry carcasses were found to be contaminated with Salmonella spp. In order to provide a more accurate profile of the prevalence of Salmonella spp in broiler carcasses, it is pertinent to use inv A gene specific PCR method that could be considered as an appropriate alternative to conventional culture method.


Introduction
methods, it needs to modify and improve them (Chiu and Jonathan, 1996).

Poultry and poultry products have been
In vitro amplification of DNA by the PCR implicated as a major source of salmonella infection in method is a powerful tool in microbiological diagnostics human (Amavisit et al., 2001).Salmonella spp. is the (Malorny et al., 2003).Several genes have been used very important bacterial pathogen of poultry in the to detect Salmonella in natural environ-mental world cause an important economic loss in poultry samples as well as food and faecal samples.Virulence rearing and food industries.It has been reported that in chromosomal genes including; invA, invE, himA addition to mishandling of poultry product and raw phoP are target genes for PCR amplification of poultry carcasses, uncooked poultry meat is also one Salmonella species.(Jamshidi,et.al., 2009).The invA of the most frequent cause of human infection by gene of Salmonella contains sequences unique to this Salmonella species (Panisello et al., 2000).
genus and has been proved as a suitable PCR target, Salmonella enterica serovar Typhimurium and with potential diagnostic applications (Rahn et al., Salmonella enterica serovar Enteritidis are the most 1992).In this research, samples from broiler chickens frequently isolated serovar from food borne outbreaks were tested, for isolation of Salmonella, by culturing throughout the world (Herikstad et al., 2002).and biochemical method and then they were Established conventional methods to detect and confirmed by invA specific PCR methods.identify Salmonella are time consuming and include

Materials and Methods
selective enrichment and plating followed by biochemical tests (Bennasar et al., 2000;Burtscher et al., Samples from broiler carcasses and culture 1999; Chiu and Jonathan, 1996).Several techniques methods: Sixty samples of broilers were collected have been developed for the improvement in the from slaughter houses of Namakkal.Samples were detection of Salmonella serovars, such as the use of aseptically cultured into selenite F broth (Himedia) 0 selective culture medium and enzyme-linked and incubated at 37 C for 24 hours.Subsequently, a immunosorbent assay.loopful of each broth was streaked on surface of However, because of controversy in interpreting MacConkey agar plates (Himedia) and Xylose Lysine of results, low sensitivity and specificity of these Desoxycholate agar (Himedia) for further incubation 0 project, showed high selectivity on 242 Salmonella at 37 C for 24 hrs.The biochemical characters of strains (sensitivity 99.6%) and 122 non-Salmonella bacteria from non-lactose fermenting determined strains (specificity 100%).Amplification of invA gene using triple sugar iron agar (Himedia).Colonies that now has been recognized as an international standard show biochemical reactions like Salmonella were for detection of Salmonella genus (Malorny et  The ability of Salmonella specific primers to Thermocycler (Eppendorf).The cycle conditions were detect Salmonella species rapidly and accurately in the 0 0 present study is primarily due to the primer sequences as follow: An initial incubation at 94 C for 60 sec.Studies in other countries have reported that the gels stained with ethidium bromide and visualized by prevalence of Salmonella in poultry carcasses, with UV illumination.A current of 120 V was applied to contamination percentages ranging from 3% to 66% each gel.Eight micro liter of PCR product mixed with (Zhao et al., 2001;Uyttendaele et al., 1998), although 3 micro liters of 6 X-loading dye (Genei, Bangalore), our results were in this range indicate the need to were loaded on to agarose gel.A 100 bp DNA ladder improve hygiene and sanitary standards in poultry was used as a marker for PCR products.
slaughter lines, besides the information to consumers.

Results and Discussion
Culture techniques are universally recognized as the standard methods for the detection of bacterial In recent years most investigators (Guo et  ).These techniques generally take longer establish a method, which can reduce the periods of time (Malorny et al., 2003) and are less sensitive Salmonella identification procedures from various samples.In an international research project for the compared to PCR based methods (Oliveira et al., validation and standardization of PCR for the 2002).The use of inv A Gene specific PCR method in detection of five major food borne pathogens most diagnostic and research laboratories is possible including Salmonella, the most selective primer set and through the molecular basis Salmonella was found to be 139-141, which targets the invA gene.identification techniques, this method is the simplest This specific PCR assay, which was validated in that and less expensive.

0
Followed by 35 cycles of denaturation at 94 C for 60 that are selected from the gene invA of S. typhimurium 0 as reported earlier by Darwin and Miller, 1999.In sec, annealing at 64 C for 30 sec and elongation at 0 other similar studies, Salmonellae have been 72 C for 30 sec, followed by 7 min final extension 0 recovered from poultry meat (S.Typhimurium,S.Saint period at 72 C. Paul, S. Indiana, S. Stanley, S. Derby and S. Newport) Electrophoresis of PCR products: The amplified and fresh buffalo meat (S.Typhimurium) (Sharma, et.DNA products from Salmonella specific-PCR were al., 1995 & 1989).analyses with electrophoresis on 1.2% agarose w/v Genei, Bangalore), 2 (invA) and were confirmed as Salmonella positive by micro liter of each primer, 19 micro liters of molecular the predicted product a 284-bp DNA fragment.The grade water and 2 micro litres of extraction for each results obtained in the present study were in isolate were used in the reaction.corroborationwithNagappaet al., (2007).Amplification was conducted in Master-gradient al., pathogens, such as Salmonella in food stuffs (White et 1999; Ferretti et al., 2001; Schneder et al., 2002 ) try to al., 2002