Automated Pangenomic Analysis in Target Selection for PCR Detection and Identification of Bacteria by Use of ssGeneFinder Webserver and Its Application to Salmonella enterica Serovar Typhi

Abstract

With the advent of high-throughput DNA sequencing, more than 4,000 bacterial genomes have been sequenced and are publicly available. We report a user-friendly web platform, ssGeneFinder Webserver ( http://147.8.74.24/ssGeneFinder/ ), which is updated weekly for the automated pangenomic selection of specific targets for direct PCR detection and the identification of clinically important bacteria without the need of gene sequencing. To apply the ssGeneFinder Webserver for identifying specific targets for Salmonella enterica serovar Typhi, we analyzed 11 S . Typhi genomes, generated two specific targets, and validated them using 40 S . Typhi, 110 non-Typhi Salmonella serovars (serovar Paratyphi A, n = 4; Paratyphi B, n = 1; Typhimurium, n = 5; Enteritidis, n = 12; non-Paratyphi group A, n = 6; non-Paratyphi group B, n = 29; non-Paratyphi group C, n = 12; non-Typhi group D, n = 35; group E and others, n = 6), 115 Enterobacteriaceae isolates ( Escherichia , n = 78; Shigella , n = 2; Klebsiella , n = 13; Enterobacter , n = 9; others, n = 13), and 66 human stool samples that were culture negative for S . Typhi. Both targets successfully detected all typical and atypical S . Typhi isolates, including an H1-j flagellin gene mutant, an aflagellated mutant which reacted with 2O Salmonella antiserum, and the Vi-negative attenuated vaccine strain Ty21a. No false positive was detected from any of the bacterial isolates and stool samples. DNA sequencing confirmed the identity of all positive amplicons. The PCR assays have detection limits as low as 100 CFU per reaction and were tested using spiked stool samples. Using a pangenomic approach, ssGeneFinder Webserver generated targets specific to S . Typhi. These and other validated targets should be applicable to the identification and direct PCR detection of bacterial pathogens from uncultured, mixed, and environmental samples.

Gene Amplification and Sequencing for Bacterial Identification

“…This provides coding probes with phenotypic correlation (Bielaszewska et al, 2011), as long as the phenotypic or proteomic survey is comprehensive. The bottom-up approach, conversely, starts with obtaining the genetic information of target and non-target bacteria and uses in vitro (Akopyants et al, 1998;Coudeyras, Marchandin, Fajon, & Forestier, 2008;Marenda, Sagne, Poumarat, & Citti, 2005) or in silico Ho et al, 2012;Ho, Yuen, Lau, & Woo, 2011;Ou, Ju, et al, 2007;Qin et al, 2011;Yu et al, 2010) methods to select genomic regions that are only present in the targeted species but not in other ones. This allows the discovery process to reach into intergenic spacers, non-coding regions and genes that are not expressed in experimental conditions, resulting in a theoretically larger repertoire of potential probes.…”

“…The GenoList webserver (Lechat, Hummel, Rousseau, & Moszer, 2008) is an integrated platform with genome browsing and subtractive proteome analysis capabilities. The mGenomeSubtractor (Shao et al, 2010) and ssGeneFinder webserver (Ho et al, 2012) are similar and offer BLAST-based comparison of multiple prokaryotic genomes to allow species-specific gene discovery; ssGeneFinder additionally provides a standalone version (Ho, Yuen, et al, 2011) for users who may need to perform their analysis using proprietary or pre-publication data.…”

“…Using DNA probes generated from subtractive genome comparison, a number of assays have been designed, with applications ranging from environmental microbiology (Price et al, 2013), industrial quality control (Yu et al, 2010), clinical diagnostics (Ho et al, 2012;Ho, Yuen, et al, 2011;Wang, Sun, & Lu, 2012) and rapid detection and identification of bacterial pathogens with bioterrorism potential Janse, Hamidjaja, Hendriks, & van Rotterdam, 2013;. As the probes generated from the in silico analysis can be selected based on their genomic characteristics, e.g., number of copies, highly sensitive assays and quantitative assays may be designed using multi-copy and single-copy probes, respectively.…”

Rapid and SensitiveSalmonellaTyphi Detection in Blood and Fecal Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification

“…The main nucleic acid amplification diagnosis technologies, such as polymerase chain reaction (PCR) (Ho et al, 2012), reverse transcription (RT)-PCR (Nga et al, 2010), and nested PCR (Kumar et al, 2012) assays, have been used in Salmonella Typhi detection. However, each of these methods has displayed certain disadvantages, such as high susceptibility to inhibition factors (Fredricks et al, 1998) or false positives, and they may require skilled laboratory personnel and expensive equipment (Anthony et al, 2000;Ortu et al, 2006).…”

Pan-PCR, a Computational Method for Designing Bacterium-Typing Assays Based on Whole-Genome Sequence Data

“…Essential to the design of an effective assay is the ability to identify a set of genes whose variable presence is capable of distinguishing strains of interest. One approach that has been taken to automate this process is to computationally find signatures that exist within a group of target genomes but that do not exist in other background genomes (18,19). A limitation of this approach is that it can determine only whether a given isolate is a member of a particular strain and cannot discern relationships among multiple strains.…”