Document Detail


Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples.
MedLine Citation:
PMID:  23044230     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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.
Authors:
W Mokhtari; S Nsaibia; A Gharbi; M Aouni
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-10-5
Journal Detail:
Title:  Molecular and cellular probes     Volume:  -     ISSN:  1096-1194     ISO Abbreviation:  Mol. Cell. Probes     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-10-9     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8709751     Medline TA:  Mol Cell Probes     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2012. Published by Elsevier Ltd.
Affiliation:
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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