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.

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Publisher Full Text

Authors

Ho CC, Wu AK, Tse CW, Yuen KY, Lau SK, Woo PC

Institution

Department of Microbiology, The University of Hong Kong, Hong Kong, China.

Source

Journal of clinical microbiology 50:6 2012 Jun pg 1905-11

MeSH

Bacteriological Techniques
Computational Biology
DNA Primers
Databases, Nucleic Acid
Genome, Bacterial
Humans
Internet
Molecular Diagnostic Techniques
Polymerase Chain Reaction
Salmonella typhi
Sensitivity and Specificity
Typhoid Fever

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Validation Studies

Language

eng

PubMed ID

22442318