| Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples. | |
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MedLine Citation:
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PMID: 23044230 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classicalPCR for detection of Shigella in food and stool specimens to real-time PCR using theSYBR Green format. This method follows a melting curve analysis to be more rapid andprovide both qualitative and quantitative data about the targeted pathogen. A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH-positive and all non-Shigella strains yielded no amplification products. The melting temperature (Tm=81.5±0.5°C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C(q)) versus copy numbers of Shigella showed good linearity (R(2)=0.995; slope=2.952) and the minimum level of detection was 1.5×10(3) CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea. The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples. |
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Authors:
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W Mokhtari; S Nsaibia; A Gharbi; M Aouni |
Publication Detail:
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Type: JOURNAL ARTICLE Date: 2012-10-5 |
Journal Detail:
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Title: Molecular and cellular probes Volume: - ISSN: 1096-1194 ISO Abbreviation: Mol. Cell. Probes Publication Date: 2012 Oct |
Date Detail:
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Created Date: 2012-10-9 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 8709751 Medline TA: Mol Cell Probes Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Copyright Information:
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Copyright © 2012. Published by Elsevier Ltd. |
Affiliation:
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Laboratoire Régional de Santé Publique de Nabeul, Tunisie; Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives (LR99-ES27), Faculté de Pharmacie de Monastir, Tunisie. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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