Microarray for molecular typing of Salmonella enterica serovars

Scaria, Joy; Palaniappan, Raghavan U.M.; Chiu, Dorothy T.; Phan, Julie; Ponnala, Lalit; McDonough, Patrick L.; Gröhn, Yrjö T.; Porwollik, Steffen; McClelland, Michael; Chiou, Chien-Shun; Chu, Chishih; Chang, Yung-Fu

doi:10.1016/j.mcp.2008.04.002

Cited by 46 publications

(40 citation statements)

References 23 publications

(38 reference statements)

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“…While 16S rRNA gene sequences have been used to type isolates to the subspecies level (29), molecular serogrouping required the simultaneous interrogation of gene clusters involved in Salmonella antigen biosynthesis, such as the rfb genes for various O antigens (9). DNA microarray-based typing has revealed the presence of groups of serovar-specific genes (20,22) and multiplex PCR assays (12,16), and panels (1) intending to differentiate among the different serovars have been published. Although a commercial low-density DNA microarray for the identification of Salmonella serovars has been marketed, it failed to distinguish between some serovars and was not tested using S. Typhi (28).…”

Section: Discussionmentioning

confidence: 99%

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

Tse

et al. 2012

J Clin Microbiol

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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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Section: Discussionmentioning

confidence: 99%

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

Tse

et al. 2012

J Clin Microbiol

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“…A number of recent strategies have employed PCR-based approaches to determine different O and H antigens as a means to replace serologic identification of these antigens (14,20,25,32). Others have proposed alternative strategies examining genetic differences as a means of identifying serovars, including ribotyping (17), pulsed-field gel electrophoresis (PFGE) (28), multiplex PCR (3,4,29), IS200 analysis (18,46), random amplification of DNA polymorphisms (45), and DNA microarray analysis (38,42).…”

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confidence: 99%

High-Throughput Molecular Determination of Salmonella enterica Serovars by Use of Multiplex PCR and Capillary Electrophoresis Analysis

Leader

Frye²,

et al. 2009

J Clin Microbiol

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Salmonella enterica is a leading cause of food-borne illness worldwide and is also a major cause of morbidity and mortality in domestic and wild animals. In the current study, a high-throughput molecular assay was developed to determine the most common clinical and nonhuman serovars of S. enterica in the United States. Sixteen genomic targets were identified based on their differential distribution among common serovars. Primers were designed to amplify regions of each of these targets in a single multiplex PCR while incorporating a 6-carboxyfluorescein-labeled universal primer to fluorescently label all amplicons. The fluorescently labeled PCR products were separated using capillary electrophoresis, and a Salmonella multiplex assay for rapid typing (SMART) code was generated for each isolate, based upon the presence or absence of PCR products generated from each target gene. Seven hundred fifty-one blind clinical isolates of Salmonella from Washington State, collected in 2007 and previously serotyped via antisera, were screened with the assay. A total of 89.6% of the isolates were correctly identified based on comparison to a panel of representative SMART codes previously determined for the top 50 most common serovars in the United States. Of the remaining isolates, 6.2% represented isolates that produced a new SMART code for a previously determined serotype, while the final 8.8% were from serotypes not screened in the original panel used to score isolates in the blinded study. This high-throughput multiplex PCR assay allowed simple and accurate typing of the most prevalent clinical serovars of Salmonella enterica at a level comparable to that of conventional serotyping, but at a fraction of both the cost and time required per test.

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“…11 This technology has been used in highthroughput detection of pathogens, such as methicillin-resistant Staphylococcus in hospitals, 44 and in the identification of antimicrobial resistance genes. 16,36 More recently, 3 studies 29,42,50 have focused on molecular serotyping of Salmonella using microarray protocols. These microarrays could identify only a limited number of serovars and required multiple probe sets for each serovar.…”

Section: Introductionmentioning

confidence: 99%

Development of Microarray and Multiplex Polymerase Chain Reaction Assays for Identification of Serovars and Virulence Genes in Salmonella Enterica of Human or Animal Origin

et al. 2010

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Abstract. Salmonella enterica is an important enteric pathogen consisting of many serovars that can cause severe clinical diseases in animals and humans. Rapid identification of Salmonella isolates is especially important for epidemiologic monitoring and controlling outbreaks of disease. Although immunologic and DNA-based serovar identification methods are available for rapid identification of isolates, they are time consuming or costly or both. In the current study, 2 molecular methods for identification of Salmonella serovars were developed and validated. A 70-mer oligonucleotide spotted microarray was developed that consisted of probes that detected genes responsible for genetic variation among isolates of Salmonella that can be used for serotyping. A multiplex polymerase chain reaction (PCR) assay was also developed, which is capable of identifying 42 serovars, thus providing a valuable prediction of the pathogenicity of the isolates by detecting the presence of virulence genes sseL, invA, and spvC. The gene spvC was the best predictor of pathogenicity. In a blind study, traditional serologic methods were correlated at 93.3% with the microarraybased method and 100% with the multiplex PCR-based serovar determination.

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Microarray for molecular typing of Salmonella enterica serovars

Cited by 46 publications

References 23 publications

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

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

High-Throughput Molecular Determination of Salmonella enterica Serovars by Use of Multiplex PCR and Capillary Electrophoresis Analysis

Development of Microarray and Multiplex Polymerase Chain Reaction Assays for Identification of Serovars and Virulence Genes in Salmonella Enterica of Human or Animal Origin

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