Identification of Salmonella Strains of Phyllosphere Food Poisoning by Melt Curve Analysis : In Silico approach

Article history: Received Oct 15 th ,2014 Revised Nov 17 th ,2014 Accepted Dec 24 th , 2014 During last few decades, there have been increased incidences of outbreak of diseases due to consumption of fresh vegetables and fruits contaminated with human pathogens. Such threats warrant rapid detection test. The standard method of diagnosis relies on culture-plate and serological methods which lack discrimination and are time consuming having several drawbacks, inconsistency and are less efficient. We applied bioinformatics approaches to develop of a realtime PCR simulation for detecting Salmonella serovars which are involved in most disease outbreaks associated with phyllosphere. Salmonella enterica subspecies enterica (designated as S. enterica) are common in plants, on surface as well as present internally in tissues. Though more than 2500 serovars of Salmonella enterica are known but the reports of serovars colonizing in plants are limited. Nucleotide sequence variation in target genes, viz. PurE, SucA, hisD, hivA and fliC were used in in silico to differentiate Salmonella serovars. A large number of reference sequences of target genes were retrieved from NCBI, and common conserved region were used for development of multiplex primer design using muPlex. Primer thermodynamic properties and secondary structure were assessed using Beacon designer. Sequences were truncated to remove sequences outside of the region bounded by the primers. We performed in silico DNA melting simulations with several Salmonella serovars using the programs umelt, and tested the utility of the programs for assay design, which will save time and cost of in vitro testing several multiple primers in RT PCR. Keyword:


INTRODUCTION
Salmonella is a major foodborne pathogenic bacterium.Salmonellosis is responsible for large numbers of infections in both humans and animals [1].Salmonella strains are not detectable in certain clinical samples that contain small numbers of organisms [2].The Salmonella genus consists of 2 species: S. bongori and S. enterica.The latter includes 6 subspecies: S. enterica ssp.houtenae, arizonae, diarizonae, enterica, salamae and indica.S. enterica spp.enterica includes the human pathogenic Salmonella and consists of more than 2000 serovars including Typhimurium (of which more than 500 phage types are recognized), Typhi, Dublin, Enteritides, Montevideo, Newport etc.Salmonella spp.are resilient bacteria and can adapt to extreme environmental conditions [3].Salmonellae have been isolated from many types of raw fruits and vegetables.The inspection of http://www.ijcb.infood for the presence of Salmonella has become routine all over the world.Due to the low infective dose of Salmonella, methods for its detection are required to prove the presence of one cell in a defined food sample.Cultural methods for Salmonella detection involve a no selective pre-enrichment, followed by selective enrichment and plating on selective and diagnostic agars.Suspect colonies are confirmed biochemically and serologically; the complete test requires three to four days to obtain a negative result and up to seven days to get a confirmed positive result [4].
A number of rapid methods for the detection of Salmonella in foods have been developed, including electrical techniques, immunoassays and nucleic acid probe analyses [5,6,7,8].However, there are still problems with their sensitivity and specificity.Several PCR assays have been developed by targeting various Salmonella genes, such as invA [9,10], 16S rRNA [11], agfA [12], and viaB [13], and virulenceassociated plasmids [14,15].Guo et al. (2000) developed a PCR to detect S. Montevideo in artificially infected tomatoes after enrichment, using two pairs of primers to amplify the hilA gene [11].Thus, rapid and sensitive methods for detecting salmonellae are in great demand in order to assure produce safety.High Resolution Melting (HRM) is a simple, rapid and low cost genotyping method [16].Its advantage is the fact that PCR amplification and melting curve analysis are performed within the same tube or plate, without any post-PCR processing.This feature is particularly important for a routine diagnostic setting.
Since HRM is based on thermodynamic differences between DNA fragments, it has been used in particular for scanning of heterozygous sequences.However, in its original form, discrimination between homozygous genotypes is more difficult, because the difference between homozygous sequence melting profiles is usually merely represented by a slight shift in the melting temperature (Tm), but not by a change of the melting curve profile [17].Therefore, HRM has been adapted for analysis of polymorphic SNPs via PCR amplification of small amplicons.Such a reduction of the amplicon length results in a broader divergence between melting profiles and increases the sensitivity of the technique, which then could be used not only for scanning, but also for accurate genotyping.Moreover, differences between homozygous wild type and homozygous mutant DNA fragments are thus more apparent [18].
Although reviews and reports on the use of HRM for mutation scanning and genotyping were published previously [16], there is no report on diagnostic validation of this technique as required by OECD and/or ISO guidelines .In this manuscript, we have evaluated in silico DNA melting simulations with several Salmonella serovars using the POLAND program and umelt program, and tested the utility of the programs for assay design, which will save time and cost of in vitro testing several multiple primers in RT-PCR.

METHODOLOGY 2.1 Target Genes
Five different housekeeping genes were selected as a target for identification of different strains of salmonella from the genome of salmonella serovars .The genes used are purE (phosphoribosylaminoimidazole carboxylase), sucA (alpha ketoglutarate dehydrogenase), hisD (histidinol dehydrogenase), hivA (invasion protein regulator) and fliC (flagellin) (Table 1) and their Corresponding sequence were downloaded from GenBank (http://www.ncbi.nlm.nih.gov).

RESULT AND DISCUSSION
Differentiation of PCR products using DNA melting curve analysis was first demonstrated by Ririe et al with the double-stranded DNA-specific dye SYBR Green I and has since seen widespread adoption in real-time PCR applications [19].Melting curve analysis provides immediate practical benefits in real-time PCR, obviating the need for gel electrophoresis by providing a reproducible signature of the amplified DNA sequence that may be used for typing PCR products.Typing is typically achieved by examining the first derivative of the melting curve and identifying the characteristic "melt peak" (Tm), which is the temperature at which the rate of fluorescence change (DNA denaturation) is highest and is observed in the raw data as a sudden decrease in fluorescence [20].Genotyping by HRM of small amplicons is a technique associated with high sensitivity and specificity [20].We were able to prove these observations by discriminating different strains of salmonella serovars.Complex melting profiles resulting in multiple melting peaks provide a superior tool for identification of different strains of salmonella serovars compared with simple profiles that result in only a single peak.In addition to Tm, both the number of peaks and relative peak heights can be used as additional diagnostic characters to facilitate identification of different strains salmonella serovars.Until this study, a systematic method for developing such assays had not been reported.The ability to simply submit a DNA sequence to melting simulation software and generate a useful prediction of the likely real time PCR melting curve profile in silico was an interesting and it also obviates the need of gel electrophoresis.

CONCLUSION
The phyllosphere is both scientifically and economically an important habitat in which to study microbial ecology.Because of the importance of many phyllosphere microbial inhabitants to plant health, there will likely be many practical applications that result from a better understanding of the interactions of microbes with plants and among themselves.This enhanced knowledge may contribute also to our understanding of the ecology of humanpathogenic bacteria on plant surfaces and provide new insights for the development of prevention or control strategies to manage preharvest contamination of crops with enteric pathogens.Computer aided melting simulation provides a gateway to the exploitation of this information.The demonstration that melting curve assays can be designed reliably in silico may stimulate more widespread use of whole product melting curve analysis and ultimately lead to dedicated melting curve assay design software.
A number of programs are available to help design primers pairs and pick target sequence.Primer-Blast, a freeware option for desiging oligonucleotides, is a program developed by NCBI that uses the algorithm primer3.Primer sequences are compared users selected database to ensure they are unique and specific for the gene of interest.Melt peak temperature (T) comparison in 14 serovars of S.entrica sub sp.Entrica (Fig 1) and the melt map for each sequence/amplicon (base position versus predicted Tm) shows/gives a clear result demonstrate that the melting curves we designed has high specificity in the identification of different strains of salmonella serovars.Multiple melting domains in a melting curve provide a rich source of information for typing samples-like salmonella (Fig 2).

Table 2 .
Primer designing and properties (Designing of Primers using muPlex software and properties obtained from free web version of Becon Designer).