title: Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples.
abstract: 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 classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide 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 (T(m) = 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 (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ 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.affiliation: Laboratoire Régional de Santé Publique de Nabeul, 8, Rue Bab Ezzaouia, Nabeul 8000, Tunisia.
authors.forename: W
authors.initials: W
authors.lastname: Mokhtari
authors.forename: S
authors.initials: S
authors.lastname: Nsaibia
authors.forename: A
authors.initials: A
authors.lastname: Gharbi
authors.forename: M
authors.initials: M
authors.lastname: Aouni
mesh_terms: D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D004405:Dysentery, Bacillary / Q000175:diagnosis*; D005243:Feces / Q000382:microbiology*; D005260:Female; D005516:Food Microbiology; D006801:Humans; D008297:Male; D009930:Organic Chemicals; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012760:Shigella / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I