Detection of Salmonella in Shellfish Using SYBR Green™ I-Based Real-Time Multiplexed PCR Assay Targeting invA and spvB

Handle URI:
http://hdl.handle.net/10754/562331
Title:
Detection of Salmonella in Shellfish Using SYBR Green™ I-Based Real-Time Multiplexed PCR Assay Targeting invA and spvB
Authors:
Gangwar, Maulshree; Waters, Alicia M.; Bej, Gautam A.; Bej, Asim K.; Mojib, Nazia ( 0000-0003-4924-5538 )
Abstract:
A SYBR Green™ I-based real-time multiplexed PCR assay was developed targeting invA and spvB for the detection of Salmonella strains in shellfish after both hns and invA genes were identified in all Salmonella strains. Simultaneously, the 16S rRNA gene was used as a PCR internal amplification control (IAC). All 89 Salmonella strains tested in this study exhibited amplification of invA, whereas only 21 (23. 6 %) were PCR positive for spvB. The sensitivity of detection of all three targeted genes was 1 ng, which is equivalent to approximately 105 colony-forming unit (CFU) of Salmonella enterica. The analysis showed specific PCR products that were identified by reproducible melt temperature profiles (invA, 84. 27 ± 1. 7 °C; spvB, 88. 76 ± 1. 0 °C; and 16S rRNA gene, 87. 16 ± 0. 8 °C). The sensitivity of detection was 10 pg purified DNA (invA) or 105 CFU in 1 mL pure culture of S. enterica ATCC 14028. The above molecular detection method for Salmonella strains was successfully applied to the oyster homogenates (food matrix). An initial inoculum of 106 and 102 CFU Salmonella in 1 ml seeded oyster tissue homogenate was detected by multiplexed PCR for all three genes after 5 and 24 h of enrichment, respectively. Natural oysters isolated from Gulf of Mexico during the winter months exhibited negative PCR amplification results suggesting the absence of Salmonella. In contrast to conventional PCR, real-time multiplex PCR assay developed in this study is rapid and sensitive and will help Interstate Shellfish Sanitation Conference undertake appropriate measures to monitor Salmonella in oysters, thereby preventing disease outbreaks and consequently protecting consumer health. © 2012 Springer Science+Business Media, LLC.
KAUST Department:
Red Sea Research Center (RSRC); Marine Science Program
Publisher:
Springer Nature
Journal:
Food Analytical Methods
Issue Date:
23-Sep-2012
DOI:
10.1007/s12161-012-9503-6
Type:
Article
ISSN:
19369751
Sponsors:
This work was supported by the funds from the Mississippi Alabama Sea Grant Consortium, The National Oceanic and Atmospheric Administration, and the University of Alabama at Birmingham (grant NA86RG0039-4; project R/SP-8-Bej). We thank Mia Torphdahl of Denmark for providing us the Salmonella strains for this study. We thank John Dindo (Dauphin Island Sea Lab, AL) and Angelo DePaola (FDA, Dauphin Island, AL) for assisting us in collecting oysters from the Gulf of Mexico.
Appears in Collections:
Articles; Red Sea Research Center (RSRC); Marine Science Program

Full metadata record

DC FieldValue Language
dc.contributor.authorGangwar, Maulshreeen
dc.contributor.authorWaters, Alicia M.en
dc.contributor.authorBej, Gautam A.en
dc.contributor.authorBej, Asim K.en
dc.contributor.authorMojib, Naziaen
dc.date.accessioned2015-08-03T10:01:12Zen
dc.date.available2015-08-03T10:01:12Zen
dc.date.issued2012-09-23en
dc.identifier.issn19369751en
dc.identifier.doi10.1007/s12161-012-9503-6en
dc.identifier.urihttp://hdl.handle.net/10754/562331en
dc.description.abstractA SYBR Green™ I-based real-time multiplexed PCR assay was developed targeting invA and spvB for the detection of Salmonella strains in shellfish after both hns and invA genes were identified in all Salmonella strains. Simultaneously, the 16S rRNA gene was used as a PCR internal amplification control (IAC). All 89 Salmonella strains tested in this study exhibited amplification of invA, whereas only 21 (23. 6 %) were PCR positive for spvB. The sensitivity of detection of all three targeted genes was 1 ng, which is equivalent to approximately 105 colony-forming unit (CFU) of Salmonella enterica. The analysis showed specific PCR products that were identified by reproducible melt temperature profiles (invA, 84. 27 ± 1. 7 °C; spvB, 88. 76 ± 1. 0 °C; and 16S rRNA gene, 87. 16 ± 0. 8 °C). The sensitivity of detection was 10 pg purified DNA (invA) or 105 CFU in 1 mL pure culture of S. enterica ATCC 14028. The above molecular detection method for Salmonella strains was successfully applied to the oyster homogenates (food matrix). An initial inoculum of 106 and 102 CFU Salmonella in 1 ml seeded oyster tissue homogenate was detected by multiplexed PCR for all three genes after 5 and 24 h of enrichment, respectively. Natural oysters isolated from Gulf of Mexico during the winter months exhibited negative PCR amplification results suggesting the absence of Salmonella. In contrast to conventional PCR, real-time multiplex PCR assay developed in this study is rapid and sensitive and will help Interstate Shellfish Sanitation Conference undertake appropriate measures to monitor Salmonella in oysters, thereby preventing disease outbreaks and consequently protecting consumer health. © 2012 Springer Science+Business Media, LLC.en
dc.description.sponsorshipThis work was supported by the funds from the Mississippi Alabama Sea Grant Consortium, The National Oceanic and Atmospheric Administration, and the University of Alabama at Birmingham (grant NA86RG0039-4; project R/SP-8-Bej). We thank Mia Torphdahl of Denmark for providing us the Salmonella strains for this study. We thank John Dindo (Dauphin Island Sea Lab, AL) and Angelo DePaola (FDA, Dauphin Island, AL) for assisting us in collecting oysters from the Gulf of Mexico.en
dc.publisherSpringer Natureen
dc.subjectReal-time multiplex PCRen
dc.subjectSalmonellaen
dc.subjectShellfishen
dc.subjectSYBR Green™ Ien
dc.titleDetection of Salmonella in Shellfish Using SYBR Green™ I-Based Real-Time Multiplexed PCR Assay Targeting invA and spvBen
dc.typeArticleen
dc.contributor.departmentRed Sea Research Center (RSRC)en
dc.contributor.departmentMarine Science Programen
dc.identifier.journalFood Analytical Methodsen
dc.contributor.institutionUniversity of Alabama at Birmingham, Birmingham, AL, United Statesen
dc.contributor.institutionUniversity of Alabama, Tuscaloosa, AL, United Statesen
kaust.authorMojib, Naziaen
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