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Article: 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

TitleAutomated 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
Authors
Issue Date2012
PublisherAmerican Society for Microbiology.
Citation
Journal of Clinical Microbiology, 2012, v. 50 n. 6, p. 1905-1911 How to Cite?
AbstractWith 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.
Persistent Identifierhttp://hdl.handle.net/10722/157706
ISSN
2015 Impact Factor: 3.631
2015 SCImago Journal Rankings: 2.151
PubMed Central ID
ISI Accession Number ID
Funding AgencyGrant Number
HKSAR
Research Grants Council
University Development Fund
Committee for Conference and Research
University of Hong Kong
Croucher Foundation
Funding Information:

This work was partly supported by the HKSAR Research Fund for the Control of Infectious Diseases of the Health, Welfare and Food Bureau, Research Grants Council Grant, University Development Fund, Committee for Conference and Research Grant, The University of Hong Kong, and the Croucher Foundation.

References

 

DC FieldValueLanguage
dc.contributor.authorHo, CCen_US
dc.contributor.authorWu, AKLen_US
dc.contributor.authorTse, CWSen_US
dc.contributor.authorYuen, KYen_US
dc.contributor.authorLau, SKPen_US
dc.contributor.authorWoo, PCYen_US
dc.date.accessioned2012-08-08T08:52:26Z-
dc.date.available2012-08-08T08:52:26Z-
dc.date.issued2012en_US
dc.identifier.citationJournal of Clinical Microbiology, 2012, v. 50 n. 6, p. 1905-1911en_US
dc.identifier.issn0095-1137en_US
dc.identifier.urihttp://hdl.handle.net/10722/157706-
dc.description.abstractWith 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.en_US
dc.languageengen_US
dc.publisherAmerican Society for Microbiology.-
dc.relation.ispartofJournal of Clinical Microbiologyen_US
dc.rightsJournal of Clinical Microbiology. Copyright © American Society for Microbiology.-
dc.rightsCopyright © American Society for Microbiology, [Journal of Clinical Microbiology, 2012, v. 50 n. 6, p. 1905-1911]-
dc.subject.meshBacteriological Techniques - methods-
dc.subject.meshComputational Biology - methods-
dc.subject.meshDNA Primers - genetics-
dc.subject.meshMolecular Diagnostic Techniques - methods-
dc.subject.meshPolymerase Chain Reaction - methods-
dc.titleAutomated pangenomic analysis in target selection for PCR detection and identification of bacteria by use of ssGeneFinder Webserver and its application to Salmonella enterica serovar Typhien_US
dc.typeArticleen_US
dc.identifier.emailYuen, KY: kyyuen@hkucc.hku.hken_US
dc.identifier.emailLau, SKP: skplau@hkucc.hku.hken_US
dc.identifier.emailWoo, PCY: pcywoo@hkucc.hku.hk-
dc.identifier.authorityLau, SKP=rp00486en_US
dc.identifier.authorityWoo, PCY=rp00430en_US
dc.description.naturelink_to_OA_fulltext-
dc.identifier.doi10.1128/JCM.06843-11en_US
dc.identifier.pmid22442318-
dc.identifier.pmcidPMC3372160-
dc.identifier.scopuseid_2-s2.0-84862101373en_US
dc.identifier.hkuros204336-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-84862101373&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume50en_US
dc.identifier.issue6en_US
dc.identifier.spage1905en_US
dc.identifier.epage1911en_US
dc.identifier.isiWOS:000305119000014-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridWoo, PCY=7201801340en_US
dc.identifier.scopusauthoridLau, SKP=7401596211en_US
dc.identifier.scopusauthoridYuen, KY=55018434500en_US
dc.identifier.scopusauthoridTse, CWS=7103295064en_US
dc.identifier.scopusauthoridWu, AKL=55247482100en_US
dc.identifier.scopusauthoridHo, CC=42061526500en_US

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