Specific detection of Salmonella by multiplex polymerase chain reaction.

Persistent Link:
http://hdl.handle.net/10150/186207
Title:
Specific detection of Salmonella by multiplex polymerase chain reaction.
Author:
Way, Jaw-Shiow Chu.
Issue Date:
1993
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
This study evaluates the use of multiplex polymerase chain reaction (PCR) technology for detection of Salmonella species in pure cultures and also in environmental samples. Three sets of oligonucleotide primers were used in the PCR assay: PhoP primers specific to a 299 bp region in the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, E. coli and Citrobacter species, served as a presumptive indicator of enteric bacteria. Subsequently Hin and H-1i primers, which targeted a 236 bp region of the hin/H2 gene, and a 173 bp region of the H-1-i flagellar gene in Salmonella typhimurium respectively, were used for specific detection of salmonellae. Specificity and sensitivity of PCR amplified products were evaluated by ethidium bromide (EtBr) staining or gene probe analysis. Under optimal PCR conditions described in this study, Salmonella species can be specifically detected by these 3 primer sets. The sensitivity of detection in terms of whole cells was a 10⁻⁵ dilution (approximately 10³ cells) of boiled late log phase cultured cells (detection by EtBr) after 25 cycles of PCR and 10⁻⁸ dilution (approximately 10° cells) after a 50 cycle 'double PCR' protocol. A similar level of sensitivity was observed using a gene probe analysis (³²P end-labeling) to detect the PCR amplified products. The multiplex PCR products observed from known environmental isolates allowed identification of several isolates as Salmonella species. These results agreed with conventional analyses. The efficacy of this PCR technology for environmental applications was also evaluated by testing the primers on soil and environmental water samples. All unseeded soil samples showed no PCR amplified products when detected by EtBr staining after 25 and 50 cycles PCR. Only one pond surface water sample resulted in a positive PCR result. This water sample was also positive when tested by conventional methodologies. Seeded soil and well water samples all resulted in PCR products being observed from 25 or 50 cycles. These results indicate that the primers have the potential to specifically detect Salmonella species in environmental samples.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology.; Agriculture.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Soil and Water Science; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Pepper, Ian L.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleSpecific detection of Salmonella by multiplex polymerase chain reaction.en_US
dc.creatorWay, Jaw-Shiow Chu.en_US
dc.contributor.authorWay, Jaw-Shiow Chu.en_US
dc.date.issued1993en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThis study evaluates the use of multiplex polymerase chain reaction (PCR) technology for detection of Salmonella species in pure cultures and also in environmental samples. Three sets of oligonucleotide primers were used in the PCR assay: PhoP primers specific to a 299 bp region in the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, E. coli and Citrobacter species, served as a presumptive indicator of enteric bacteria. Subsequently Hin and H-1i primers, which targeted a 236 bp region of the hin/H2 gene, and a 173 bp region of the H-1-i flagellar gene in Salmonella typhimurium respectively, were used for specific detection of salmonellae. Specificity and sensitivity of PCR amplified products were evaluated by ethidium bromide (EtBr) staining or gene probe analysis. Under optimal PCR conditions described in this study, Salmonella species can be specifically detected by these 3 primer sets. The sensitivity of detection in terms of whole cells was a 10⁻⁵ dilution (approximately 10³ cells) of boiled late log phase cultured cells (detection by EtBr) after 25 cycles of PCR and 10⁻⁸ dilution (approximately 10° cells) after a 50 cycle 'double PCR' protocol. A similar level of sensitivity was observed using a gene probe analysis (³²P end-labeling) to detect the PCR amplified products. The multiplex PCR products observed from known environmental isolates allowed identification of several isolates as Salmonella species. These results agreed with conventional analyses. The efficacy of this PCR technology for environmental applications was also evaluated by testing the primers on soil and environmental water samples. All unseeded soil samples showed no PCR amplified products when detected by EtBr staining after 25 and 50 cycles PCR. Only one pond surface water sample resulted in a positive PCR result. This water sample was also positive when tested by conventional methodologies. Seeded soil and well water samples all resulted in PCR products being observed from 25 or 50 cycles. These results indicate that the primers have the potential to specifically detect Salmonella species in environmental samples.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology.en_US
dc.subjectAgriculture.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineSoil and Water Scienceen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairPepper, Ian L.en_US
dc.contributor.committeememberHendricks, David M.en_US
dc.contributor.committeememberMiller, Raina M.en_US
dc.contributor.committeememberSinclair, Norval A.en_US
dc.contributor.committeememberSonger, J. Glennen_US
dc.identifier.proquest9322718en_US
dc.identifier.oclc702669889en_US
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