Available at www.veterinaryworld.org/Vol.6/Nov-2013/16.pdf RESEARCH ARTICLE Open Access Detection of OmpA gene by PCR for specific detection of

Aim: The study was carried out to determine the sensitivity and specificity of OmpA gene in Salmonella serovars through PCR. Materials and Methods: A set of primers were designed targeting the OmpA gene specific for the Salmonella and polymerase chain reaction was standardized using Salomonella Typhimurium as a positive control and as a negative control 4 non salmonella cultures such as Campylobacter coli, Arcobacter butzleri, Brucella abortus and E. coli. Sensitivity of the test was determined by serial dilution of genomic DNA of standard S. Typhimurium. The PCR standardized was used for screening 68 strains of different serovars of Salmonella. Results: The PCR developed targeting OmpA specific for Salmonella was highly specific in detection of the salmonella serovar alone and sensitivity was upto 68.8 fg. A total of 68 virulent/ natural strains of different serovars of salmonella taken up for the study were positive by OmpA based PCR. Conclusions: This study reports that, OmpA gene which is conserved among Salmonella serovars can be used for the detection of Salmonella in food or clinical samples in further studies, with high sensitivity and specificity.


Introduction
interfacing bacteria with the mammalian host and its defenses, bacteriophages, and other bacteria or micro-Salmonella is a food-borne pathogen that is typically organisms [6].The outer membrane proteins comprise acquired through consumption of contaminated food almost 50% of the bacterial membranes of Gram and water [1].Salmonellosis continues to be a major negative bacteria [7].public health problem worldwide.It also contributes to OmpA super family is one of the most abundant negative economic impacts due to the cost of surveillouter membrane proteins in prokaryotes and is most ance investigation, treatment and prevention of illness widely studied [8].Its main role is to provide integrity [2].The global burden of human gastroenteritis due to to the membrane by ensuring physical linkages Salmonella has been estimated 93.8 million cases, between the outer membrane and the underlying resulting in 155,000 deaths each year [3].Poultry is peptidoglycan layer as well as having importance in considered a major reservoir for many non-host specific bacterial conjugation [9,10].It also serves as a receptor motile serovars of Salmonella, and often human infection to some of the bacteriophages [11] and colicins [12].Of is attributed to consumption of contaminated poultry all the OMPs, OmpA appears to be a major antigenic products, such as eggs and meats [4].
protein in Salmonella induced ReA/uSpA, as it is The outer membrane protein (OMPs) of Gramcommon to most of the stimulatory fractions [13].negative bacteria plays a major role in the adaptation of Salmonella OmpA is immunostimulatory as the bacterium to its various external environments, by demonstrated by stimulation of IFN-g production and passively and/ or selectively controlling influx and enhanced expression of MHC and costimulatory efflux of important solutes, peptides or proteins, molecules in dendritic cells and/or T cells and may play nucleic acids, and other organic compounds such as a role in modulation of the immune response against lipids and polysaccharides [5].Most OMPs are surface salmonellosis [14,15].exposed and, therefore, are potentially important in Traditional culture-based methods for detecting Salmonella are reliable but labor-intensive and timeconsuming, demanding several days for a definitive result.Immunoassays such as enzyme-linked immunosorbent assay (ELISA) have been developed for

Salmonella detection, however, low specificity has
The genomic DNA from the reference strain S. limited their use [16].
Typhimurium was extracted by phenol chloroform PCR technology represents a rapid procedure method [19] and checked for quality and quantity with high sensitivity and high specificity to detect spectrophotometerically, while, the genomic DNA Salmonella in a wide variety of food.Several PCR from the other salmonella and non-salmonella isolates assays have been developed by targeting various were extracted by snap chill method.Briefly, 1 mL of Salmonella genes, such as 16S rRNA , agfA , and viaB the overnight incubated cultures was boiled in boiling and virulence-associated plasmids [17].
water for 10 minutes and immediately transferred onto The study was thus, focused on polymerase chain ice.For PCR assay, 2 µL of this heated broth was taken reaction based amplification of OmpA gene to prove its as template.conserved nature in Salmonella serovars and to finally documented using gel documentation system (UVP).PCR amplification and detection: The sequence of the primers used is as follows: Forward: 5'-AGT CGA GC PCR specificity and sensitivity: PCR specificity and TCATGAAAAAGACAGCTATCGC-3' Reverse: 5'sensitivity was determined using all the pure cultures AGTCAAGCTTTTAAG CCTGCG GCTGAGTT A-3'.lysate (Table -1).The specificity of OmpA primers was determined by PCR and observed for any product DNA extraction and quantification: Oligonucleotide generated from other bacteria.For determination of primers were designed targeting the OmpA gene of sensitivity of the PCR assay, the DNA was diluted ten Salmonella serovar based on the nucleotide sequence fold and the ability of this PCR assay to detect the available in the Genbank and the primers were got minimum concentration of DNA was determined and synthesized commercially.assay was repeated thrice to confirm its repeatability.been developed, and different targets DNAs for amplification have been applied [21].

Results
A total of 68 isolates were screened for the presence of OmpA gene and it was found that all the PCR was standardised for the amplification of Salmonella isolates were positive for the presence of OmpA gene by optimizing reaction mixture and cycling OmpA gene.Our results were in confirmation with conditions using the genomic DNA from standard S.
Freudl and Cole [22] who reported that OmpA Typhimurium extracted and purified by phenol: regulatory region of S. Typhimurium is highly conserved chloroform method.Standardized PCR yielded the and has an overlapping twin-promoter arrangement.In expected single band of 1053 bp on agarose gel addition, Okamura et al [23] based on bioinformatic electrophoresis.The size for PCR product was as per analysis also reported that OmpA is well conserved the expected size of the primers which were designed to among the various Salmonella serovars.amplify the OmpA gene.
A total of 68 isolates were screened for the Conclusion presence of OmpA gene and it was found that all the From the overall study, it can be concluded that Salmonella isolates were positive for the presence of OmpA gene is conserved among Salmonella serovars.OmpA gene and non of the non Salmonella cultures Since our preliminary study proved the conserved were positive for OmpA gene ( Table -1 ). nature of gene in different Salmonella serovars, this The concentration of the genomic DNA from the may be used for the detection of Salmonella in food or reference S. Typhimurium was 3240 ng/µL and the clinical samples in further studies.minimum detection limit (sensitivity) of the PCR was found that upto 64.8 fg DNA template which produced physiology, and adaptation to environmental stresses, Motile Serovars of Salmonella enterica from Breeder and whereas in disease they can serve as virulence factors Commercial Broiler Poultry Farms in Bangladesh.PLoS causing adhesion, invasion and damage of host tissue One 8(3): e57811.or evasion of host defences resulting in clinical disease 5. Confer, A.W. and Ayalew, S. (2013) The OmpA family of or death [5].proteins: Roles in bacterial pathogenesis and immunity.Vet.Microbiol.163: 207-222.The development of molecular methods for 6.Krishnan, S. and Prasadarao, N.V. (2012) Outer membrane diagnosis of infectious diseases has improved the protein A and OprF-versatile roles in Gram-negative sensitivity, specificity, quality and availability of bacterial infections.FEBS J. 279:919-930.diagnosis and treatment.Several polymerase chain 7. Koebnik, R., Locher, K.P. and Van, Gelder P. (2000) reaction (PCR) assays for detection of Salmonella have Structure and function of bacterial outer membrane proteins:

Figure- 1 .
Figure-1.Sensitivity of OmpA PCR after tenfold dilution of genomic DNA of S.Typhimurium.

Detection of OmpA gene by PCR:
The PCR mixture of evaluate its specificity and sensitivity in detection.25µl contained 2.5 µL of Dream taq buffer, 2.5 µL of

Table - 1
. PCR results for different Salmonella serotypes targeting OmpA gene