Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples

Mokhtari, W.; Nsaibia, S.; Gharbi, Amal; Aouni, Mahjoub

doi:10.1016/j.mcp.2012.09.002

Cited by 35 publications

(13 citation statements)

References 32 publications

(39 reference statements)

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“…We kept this region and solved the problem of overlapping with the "CG tails" strategy (17), adding 3 nucleotides (CGC) in front of the forward primer for Shigella spp. This multiplex approach showed higher analytical sensitivity than other methods described in previous studies (18)(19)(20) for detection of these three enteropathogens in stool samples. The efficiencies (90% to 110%) and the regression coefficient (almost 1.00) obtained for all the targets were in agreement with the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines (21).…”

Section: Discussionmentioning

confidence: 74%

Multiplex Real-Time PCR for Detection of Campylobacter, Salmonella, and Shigella

et al. 2013

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Infectious diarrhea can be classified based on its clinical presentation as noninflammatory or inflammatory disease. In developing countries, among inflammatory diarrhea cases, Shigella is the most common cause, followed by Campylobacter and Salmonella. Because the time frame in which treatment choices must be made is short and conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. The purpose of our study was to develop a multiplex real-time PCR procedure to simultaneously identify Campylobacter spp., Salmonella spp., and Shigella spp. Primers were designed to amplify the invA, ipaH, and 16S rRNA genes simultaneously in a single reaction to detect Salmonella, Shigella, and Campylobacter, respectively. Using this approach, we correctly identified 102 of 103 strains of the targeted enteropathogens and 34 of 34 other pathogens. The melting temperatures were 82.96 ؎ 0.05°C for invA, 85.56 ؎ 0.28°C for ipaH, and 89.21 ؎ 0.24°C for 16S rRNA. The limit of accurate quantification for the assay in stool samples was 10 4 CFU g ؊1 ; however, the limit of detection was 10 3 CFU g ؊1 . This assay is a simple, rapid, inexpensive, and reliable system for the practical detection of these three enteropathogens in clinical specimens. Infectious diarrhea is a global health problem that is still responsible for thousands of deaths worldwide, especially in children (1). It can be classified based on its clinical presentation as one of two syndromes-noninflammatory or inflammatory diarrhea (2). Among cases of inflammatory diarrhea, Shigella is the most common cause, followed by Campylobacter and Salmonella (3). These invasive organisms primarily target the lower bowel; they invade the intestinal mucosa to induce an acute inflammatory reaction and activate cytokines and inflammatory mediators (4).Because the time frame in which treatment choices must be made is short and the conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. We searched for DNA sequences that were highly conserved between the different species of each genera, and we selected the following genes as targets, invA (invasion A gene) for Salmonella spp., ipaH (invasion plasmid antigen H) for Shigella spp., and 16S rRNA for Campylobacter spp., to develop a fluorescence-based real-time PCR procedure to simultaneously identify these enteropathogens. In this method the post-PCR products are identified based on melting-point curve analysis. We have also standardized the technique to quantify these bacteria directly from stool samples. MATERIALS AND METHODSBacterial strains. A total of 147 enteropathogenic strains (Table 1) were analyzed, including clinical isolates representative of Salmonella spp., Shigella spp., and Campylobacter spp., as well as other enteropathogens. These clinical strains had previously been identified based on serology, biochemical assays, and real-time PCR for the diarrheagenic Escherichia coli (5). In add...

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Section: Discussionmentioning

confidence: 74%

Multiplex Real-Time PCR for Detection of Campylobacter, Salmonella, and Shigella

et al. 2013

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“…Most PCR assays for the detection of Shigella in food are based on the sole detection of ipaH and cannot differentiate between natural isolates or cross-contamination with the genetically engineered S. flexneri 2457M FDA positive control strain (Mokhtari et al, 2013;Garrido et al, 2013;Lin et al, 2010;Wang et al, 2007;Warren et al, 2006). Accidental in-laboratory sample contamination with 2457M could generate a false-positive result that could lead to regulatory action and/or the destruction of the tested food.…”

Section: Discussionmentioning

confidence: 99%

Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay

2014

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a b s t r a c tFaster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 Â 10 3 Shigella CFU/ml post-enrichment, requiring w18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen.Published by Elsevier Ltd.

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“…During the fermentation, LAB utilise carbohydrate substrates available in the fermentation system and produce organic acids, especially lactic acid, which not only play an important role in the taste and aroma of the product but also lower the product's pH to ensure quality and safety . As a complementary method, qPCR technology which has been used to quantitatively determine microbe in various environments (Mokhtari et al, 2013;Kao et al, 2007) was used to quantitate the quantity of LAB in this study.…”

Section: Introductionmentioning

confidence: 99%

Microbial Community Characteristics in Industrial Matured Chinese paocai, a Fermented Vegetable Food, from Different Factories

Liang

Zhang

et al. 2016

FSTR

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Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples

Cited by 35 publications

References 32 publications

Multiplex Real-Time PCR for Detection of Campylobacter, Salmonella, and Shigella

Multiplex Real-Time PCR for Detection of Campylobacter, Salmonella, and Shigella

Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay

Microbial Community Characteristics in Industrial Matured Chinese paocai, a Fermented Vegetable Food, from Different Factories

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