Abstract
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 real- time 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. Keywords - In Silico ; Salmonella ; Phyllosphere;Real-time PCR (RT-PCR)
Highlights
Salmonella is a major foodborne pathogenic bacterium
The Salmonella genus consists of 2 species: S. bongori and S. enterica
Reviews and reports on the use of High Resolution Melting (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
Summary
Salmonella is a major foodborne pathogenic bacterium. Salmonellosis is responsible for large numbers of infections in both humans and animals [1]. 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 could be used for scanning, and for accurate genotyping. 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. Role in the citric acid cycle hisD (histidinol dehydrogenase) hivA(invasion protein regulator)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
