Detection of Virulence invA Gene in the Salmonella Isolated from Fecal Samples of Poultry by Pcr Method

| The presence of Salmonella spp in collected faecal samples was assessed by performing the pre-enrichment and enrichment culture, followed by PCR assay. The primer was selected from the invA gene, specific for the detection of Salmonella spp. It was observed that 28% (10/35) of Salmonella were isolated by the conventional method. All Salmonella isolates thus obtained were subjected to PCR for the invA gene and all were found positive. In order to provide a more accurate profile of the prevalence of Salmonella spp in faecal samples, it was pertinent to use invA gene specific PCR method that could be considered as rapid technique for identification of Salmonella spp.

lates by the boiling method, and for this approx.1.5ml of the cultured broth was centrifuged at 10,000rpm for 5 minutes.The supernatant was discarded and the pellets were washed twice with sterile water.After this, 200µl of sterile water was added to the pellets, the pellets were vortexed to homogenize and boiled in a dry bath at 100°C for 10 minutes.This was followed by vortexing and centrifugation at 12,000rpm for 5 minutes.The supernatant containing the DNA was transfer to another tube.The purity of the extracted DNA was estimated using a Nanodrop spectrophotometer (Smith et al., 2015).
Primers set and PCR amplification: PCR Salmonellaspecific primers, S139 and S141 (Rahn et.al., 1992) have respectively the following nucleotide sequence based on the invA gene of Salmonella 5´ -GTG AAA TTA TCG CCA CGT TCG GGC AA -3´and 5´ -TCA TCG CAC CGT CAA AGG AAC C -3´. Reaction with these primers were carried out in a 20µl amplification mixture consisting of 10µl of PCR Master mix (Qiagen), 20 pmol of each primer, 3µl of molecular grade water and 5µl of DNA were used in the reaction.Amplification was conducted in Master-gradient Thermocycler (Eppendorf ).The cycle conditions were as follow: An initial incubation at 94 0 C for 60 sec.Followed by 35 cycles of denaturation at 94 o C for 60 sec, annealing at 64 o C for 30 sec and elongation at 72 o C for 30 sec, followed by 7 min final extension period at 72 o C.

Electrophoresis of PCR products:
The amplified DNA products from Salmonella specific-PCR were analyzed with electrophoresis on 1.2% agarose w/v gels stained with ethidium bromide and visualized by UV illumination.A current of 120 V was applied to each gel.Eight µl of PCR product mixed with two µl of 6X loading dye were loaded on to agarose gel.A 100 bp DNA ladder (Himedia make-MBT049-50LN) was used as a marker for PCR products.

RESULTS AND DISCUSSION
The present study supports the ability of these specific primer sets to confirm the isolates as Salmonella.From the 35 fecal samples, 10 isolates were found Salmonella positive as per conventional methods.On Triple sugar Iron agar, the salmonella showed alkaline reaction of the slant with red coloration of the medium, butt showed the acidic reaction with yellowing of the medium with slight gas production.All the isolates were able to utilize citrate as a sole carbon source and showed the change in colour from green to blue.All the 10 Salmonella positive isolates were screened by PCR for invA gene, and all of them resulted in 284 bp amplified fragment of invA gene (Karmi, 2013).Detection of invA in all the isolates proved that they were Salmonella species (Kocabiyik, 2006) (Figure 1).The invA gene contains sequences unique to the genus Salmonella and is considered the international standard for its identification (Malorny et al., 2003).All Salmonella isolates, positive for the presence of invA gene which is responsible for invasion of cells, have the capacity to invade and survive in macrophages (Gole et al., 2013).The ability of Salmonella specific primers to detect Salmonella species rapidly and accurately is primarily due to the primer sequences that are selected from the gene invA.The invA gene codes for protein in inner membrane of bacteria, which is necessary for invasion to epithelial cells (Darwin and Miller, 1999).Rapid detection of Salmonella in poultry farms has an effective role in this study.PCR based methods with genus-specific primers belong to invA, according to its quick, specificity and sensitivity is a reliable technique for this proposes.In other hand this technique provides a tool, in confirmation of isolates as Salmonella.Although techniques which in recent years proposed for rapid and reliable detection and confirmation of Salmonella are very progress, but the PCR method which we use in our study, yet is a certain technique in identification and confirmation of Salmonella.The use of this method in most diagnostic and research laboratories is possible and through the molecular basis Salmonella identification techniques, this method is the simplest and a less expensive.
The amplification of the invA gene has been proposed as an international standard for genus of Salmonella detection (Malorny et al., 2003b).In the present study we used S138 and S141 primers for specific detection of Salmonella and the results of this study highlight the usefulness of the PCR for confirm detection of Salmonella spp from poultry faecal samples.In conclusion, with attention to high level of Salmonella infections in poultry farms, it is necessary to consider control programs to prevention of economically loss resulting from mortality and spreading of infection.