Detection of Shiga toxin genes stx1, stx2, and the +93 uidA mutation of E. coli O157:H7/H-using SYBR® Green I in a real-time multiplex PCR

Yoshitomi, Ken J.; Jinneman, Karen C.; Weagant, Stephen D.

doi:10.1016/j.mcp.2005.09.002

Cited by 39 publications

(21 citation statements)

References 44 publications

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“…However, EIAs can have relatively high false-positive rates and have been reported to miss E. coli O157 (7,10,14). A number of nucleic acid-based methods, including multiplex, realtime PCR assays, have also been described (10,(15)(16)(17)(18)(19)(20). Additionally, a Luminex-based molecular assay targeting 15 stool pathogens including STEC was recently approved by the FDA (21).…”

mentioning

confidence: 99%

A Sensitive Multiplex, Real-Time PCR Assay for Prospective Detection of Shiga Toxin-Producing Escherichia coli from Stool Samples Reveals Similar Incidences but Variable Severities of Non-O157 and O157 Infections in Northern California

et al. 2013

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dRapid and accurate detection of Shiga toxin-producing Escherichia coli (STEC) of all serotypes from patients with diarrhea is critical for medical management and for the prevention of ongoing transmission. In this prospective study, we assessed the performance of a multiplex, real-time PCR assay targeting stx 1 and stx 2 for the detection of O157 and non-O157 STEC in diarrheal stool samples enriched in Gram-negative broth. We show that the assay is 100% sensitive (95% confidence interval [CI], 89.1% to 100%) and 98.5% specific (95% CI, 90.6% to 99.9%) based on a panel of 40 known STEC-positive specimens and 65 known negative specimens. During a 2-year postvalidation period, the assay detected more positive samples from patients in northern California than did culture and PCR testing performed at a public health reference laboratory, with a positive predictive value of 95.6% (95% CI, 87.6% to 99.1%). Serotyping data showed an incidence rate of 51.2% for non-O157 STEC strains, with 5.8% of patients (1/17) with non-O157 strains and 42.9% (6/14) with O157 strains (P ‫؍‬ 0.03) developing hemolytic-uremic syndrome. The findings from this study underscore the recommendations of the CDC for laboratories to test all diarrheal stool samples from patients with acute community-acquired diarrhea for non-O157 STEC in addition to the O157 serotype by using a sensitive assay. Additionally, a survey of 17 clinical laboratories in northern California demonstrated that nearly 50% did not screen all stool specimens for the presence of Shiga toxins, indicating that many clinical microbiology laboratories still do not routinely screen all stool specimens for the presence of Shiga toxins as recommended in the 2009 CDC guidelines. Shiga toxin-producing Escherichia coli (STEC) infections are a major cause of bacterial gastroenteritis throughout the world and can be complicated by hemorrhagic colitis and hemolyticuremic syndrome (HUS) (1, 2). Although previously the majority of reported cases were attributed to E. coli O157:H7 and many recent studies indicate higher incidence rates of HUS associated with E. coli O157:H7 (3, 4), non-O157 STEC serotypes are emerging as important etiological agents of both sporadic cases and community outbreaks of diarrhea (3-8). Most notably, in May and June 2011, a large outbreak of E. coli O104:H4-associated diarrheal illness in Germany led to HUS in over 800 patients, many of whom were adults, and ultimately resulted in 54 deaths (5, 9). This outbreak and others have underscored the fact that severe disease and HUS can occur in cases related to non-O157 or O157 E. coli strains and that adults as well as children are susceptible to these complications (8-10). Timely laboratory identification of STEC has important implications for outbreak containment and patient management, including prompt parenteral hydration, monitoring for development of HUS, and avoidance of antibiotics and antidiarrheal agents, which can exacerbate disease (1). Importantly, a number of studies have shown that STECrelated illnesses...

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confidence: 99%

A Sensitive Multiplex, Real-Time PCR Assay for Prospective Detection of Shiga Toxin-Producing Escherichia coli from Stool Samples Reveals Similar Incidences but Variable Severities of Non-O157 and O157 Infections in Northern California

et al. 2013

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“…While the development of rapid test methods will facilitate more timely and cost effective testing [37]; a more profound reduction of foodborne shigellosis may depend on the education of employees and consumers as to proper personal hygiene.…”

Section: Discussionmentioning

confidence: 99%

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

Mokhtari¹,

Nsaibia²,

Gharbi³

et al. 2013

Molecular and Cellular Probes

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“…DNA was eluted in 100 ml of ultra pure water and 5 ml were used for PCR amplification. The oligonucleotides used in this work target the b-glucuronidase gene (uidA) of E. coli O157:H7 (Yoshitomi et al, 2006) and the listeriolisin gene (hly) of L. monocytogenes (Rodríguez-Lázaro et al, 2003). They were synthesized by Metabion (Martinsried, Germany).…”

Section: Conventional Pcrmentioning

confidence: 99%

“…In addition, it allows quantification of the target microorganism based on previously generated standard curves, which determine the validity of the qPCR quantification method. In recent years, various real-time PCR procedures have been developed to detect E. coli O157:H7 (Stefan et al, 2007;Yoshitomi, Jinneman, & Weagant, 2006;McKillip & Drake, 2004), L. monocytogenes (O'Grady, SedanoBalbás, Maher, Smiyh, & Barry, 2007;Oravcová, Kuchta, & Kaclíková, 2007;Rodríguez-Lázaro et al, 2003;Rossmanith, Krassnig, Wagner, & Hein, 2006) and Salmonella (Malorny, Löfström, Wagner, Krämer, & Hoorfar, 2008;Seo, Valentin-Bon, Brackett, & Holt, 2004) in a variety of food products. However, the quantitative detection of bacterial pathogens in non-spiked food products by qPCR has scarcely been approached and further research is required for the implementation of this method in routine food laboratories.…”

Section: Introductionmentioning

confidence: 99%

Quantification of Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 in non-spiked food products and evaluation of real-time PCR as a diagnostic tool in routine food analysis

2011

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Detection of Shiga toxin genes stx1, stx2, and the +93 uidA mutation of E. coli O157:H7/H-using SYBR® Green I in a real-time multiplex PCR

Cited by 39 publications

References 44 publications

A Sensitive Multiplex, Real-Time PCR Assay for Prospective Detection of Shiga Toxin-Producing Escherichia coli from Stool Samples Reveals Similar Incidences but Variable Severities of Non-O157 and O157 Infections in Northern California

A Sensitive Multiplex, Real-Time PCR Assay for Prospective Detection of Shiga Toxin-Producing Escherichia coli from Stool Samples Reveals Similar Incidences but Variable Severities of Non-O157 and O157 Infections in Northern California

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

Quantification of Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 in non-spiked food products and evaluation of real-time PCR as a diagnostic tool in routine food analysis

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