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

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    Type
    Article
    Authors
    Gangwar, Maulshree
    Waters, Alicia M.
    Bej, Gautam A.
    Bej, Asim K.
    Mojib, Nazia cc
    KAUST Department
    Red Sea Research Center (RSRC)
    Marine Science Program
    Date
    2012-09-23
    Online Publication Date
    2012-09-23
    Print Publication Date
    2013-06
    Permanent link to this record
    http://hdl.handle.net/10754/562331
    
    Metadata
    Show full item record
    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.
    Citation
    Gangwar, M., Waters, A. M., Bej, G. A., Bej, A. K., & Mojib, N. (2012). Detection of Salmonella in Shellfish Using SYBR Green™ I-Based Real-Time Multiplexed PCR Assay Targeting invA and spvB. Food Analytical Methods, 6(3), 922–932. doi:10.1007/s12161-012-9503-6
    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.
    Publisher
    Springer Nature
    Journal
    Food Analytical Methods
    DOI
    10.1007/s12161-012-9503-6
    ae974a485f413a2113503eed53cd6c53
    10.1007/s12161-012-9503-6
    Scopus Count
    Collections
    Articles; Red Sea Research Center (RSRC); Marine Science Program

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