Prevalence of Emetic Bacillus cereus in Different Ice Creams in Bavaria

2010 ◽  
Vol 73 (2) ◽  
pp. 395-399 ◽  
Author(s):  
U. MESSELHÄUSSER ◽  
P. KÄMPF ◽  
M. FRICKER ◽  
M. EHLING-SCHULZ ◽  
R. ZUCKER ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Genetic Background ◽  
Ice Cream ◽  
Toxin Production ◽  
Pcr Assay ◽  
Cultural Methods ◽  
Different Sources ◽  
Culture Positive

In this study, 809 samples of ice cream from different sources were investigated by using cultural methods for the presence of presumptive Bacillus cereus. Isolates from culture-positive samples were examined with a real-time PCR assay targeting a region of the cereulide synthetase gene (ces) that is highly specific for emetic B. cereus strains. The samples were collected from ice cream parlors and restaurants that produced their own ice cream and from international commercial ice cream companies in different regions of Bavaria during the summer of 2008. Presumptive B. cereus was found in 508 (62.7%) ice cream samples investigated, and 24 (4.7%) of the isolates had the genetic background for cereulide toxin production. The level of emetic B. cereus in the positive samples ranged from 0.1 to 20 CFU/g of ice cream.

Development of Rapid Real-Time PCR and Most-Probable-Number Real-Time PCR Assays To Quantify Enterotoxigenic Strains of the Species in the Bacillus cereus Group

2007 ◽  
Vol 70 (12) ◽  
pp. 2774-2781 ◽  
Author(s):  
I-CHEN YANG ◽  
DANIEL YANG-CHIH SHIH ◽  
JAN-YI WANG ◽  
TZU-MING PAN
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Samples ◽  
Most Probable Number ◽  
Pcr Assay ◽  
Bacillus Cereus Group ◽  
Probable Number ◽  
Cooked Rice ◽  
Group Detection

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 > 0.993), wide dynamic quantification range (102 to 107 CFU/g for cooked rice and chicken, 103 to 107 CFU/ml for milk, and 104 to 107 CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 100 CFU/ml. Both assays were tested with real food samples and shown to be considerably appropriate for B. cereus group detection and quantification.

Detection and Enumeration of Salmonella Enteritidis in Homemade Ice Cream Associated with an Outbreak: Comparison of Conventional and Real-Time PCR Methods

2006 ◽  
Vol 69 (3) ◽  
pp. 639-643 ◽  
Author(s):  
K. H. SEO ◽  
I. E. VALENTIN-BON ◽  
R. E. BRACKETT
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Direct Detection ◽  
Ice Cream ◽  
Plate Count ◽  
Pcr Assay ◽  
The Real ◽  
Real Time Quantitative Pcr

Salmonellosis caused by Salmonella Enteritidis (SE) is a significant cause of foodborne illnesses in the United States. Consumption of undercooked eggs and egg-containing products has been the primary risk factor for the disease. The importance of the bacterial enumeration technique has been enormously stressed because of the quantitative risk analysis of SE in shell eggs. Traditional enumeration methods mainly depend on slow and tedious most-probable-number (MPN) methods. Therefore, specific, sensitive, and rapid methods for SE quantitation are needed to collect sufficient data for risk assessment and food safety policy development. We previously developed a real-time quantitative PCR assay for the direct detection and enumeration of SE and, in this study, applied it to naturally contaminated ice cream samples with and without enrichment. The detection limit of the real-time PCR assay was determined with artificially inoculated ice cream. When applied to the direct detection and quantification of SE in ice cream, the real-time PCR assay was as sensitive as the conventional plate count method in frequency of detection. However, populations of SE derived from real-time quantitative PCR were approximately 1 log higher than provided by MPN and CFU values obtained by conventional culture methods. The detection and enumeration of SE in naturally contaminated ice cream can be completed in 3 h by this real-time PCR method, whereas the cultural enrichment method requires 5 to 7 days. A commercial immunoassay for the specific detection of SE was also included in the study. The real-time PCR assay proved to be a valuable tool that may be useful to the food industry in monitoring its processes to improve product quality and safety.

scholarly journals Detection and Characterization of Diphtheria Toxin Gene-Bearing Corynebacterium Species through a New Real-Time PCR Assay

2020 ◽  
Vol 58 (10) ◽  
Author(s):  
Margaret M. Williams ◽  
Jessica L. Waller ◽  
Janessa S. Aneke ◽  
Michael R. Weigand ◽  
Maureen H. Diaz ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Toxin Production ◽  
Toxin Gene ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Culture Methods ◽  
Content Type ◽  
Corynebacterium Ulcerans

ABSTRACT Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of Corynebacterium diphtheriae, with similar illness produced occasionally by toxigenic Corynebacterium ulcerans or, rarely, Corynebacterium pseudotuberculosis. While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene (tox). Nontoxigenic tox-bearing (NTTB) Corynebacterium isolates have been described, but impact of these isolates on the accuracy of molecular diagnostics is not well characterized. Here, we describe a new triplex RT-PCR assay to detect tox and distinguish C. diphtheriae from the closely related species C. ulcerans and C. pseudotuberculosis. Analytical sensitivity and specificity of the assay were assessed in comparison to culture using 690 previously characterized microbial isolates. The new triplex assay characterized Corynebacterium isolates accurately, with 100% analytical sensitivity for all targets. Analytical specificity with isolates was 94.1%, 100%, and 99.5% for tox, Diph_rpoB, and CUP_rpoB targets, respectively. Twenty-nine NTTB Corynebacterium isolates, representing 5.9% of 494 nontoxigenic isolates tested, were detected by RT-PCR. Whole-genome sequencing of NTTB isolates revealed varied mutations putatively underlying their lack of toxin production, as well as eight isolates with no mutation in tox or the promoter region. This new Corynebacterium RT-PCR method provides a rapid tool to screen isolates and identify probable diphtheria cases directly from specimens. However, the sporadic occurrence of NTTB isolates reinforces the viewpoint that diphtheria culture diagnostics continue to provide the most accurate case confirmation.

A novel diagnostic real-time PCR assay for quantification and differentiation of emetic and non-emetic Bacillus cereus

Food Control ◽  
2013 ◽  
Vol 32 (1) ◽  
pp. 176-185 ◽  
Author(s):  
Monika Dzieciol ◽  
Martina Fricker ◽  
Martin Wagner ◽  
Ingeborg Hein ◽  
Monika Ehling-Schulz
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Assay

scholarly journals Diagnostic Real-Time PCR Assays for the Detection of Emetic Bacillus cereus Strains in Foods and Recent Food-Borne Outbreaks

2007 ◽  
Vol 73 (6) ◽  
pp. 1892-1898 ◽  
Author(s):  
Martina Fricker ◽  
Ute Messelhäußer ◽  
Ulrich Busch ◽  
Siegfried Scherer ◽  
Monika Ehling-Schulz
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Care Center ◽  
Food Poisoning ◽  
Sybr Green ◽  
Sybr Green I ◽  
Taqman Assay ◽  
Pcr Assay ◽  
Food Borne

ABSTRACT Cereulide-producing Bacillus cereus can cause an emetic type of food-borne disease that mimics the symptoms provoked by Staphylococcus aureus. Based on the recently discovered genetic background for cereulide formation, a novel 5′ nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic B. cereus in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesis-causing food pathogens B. cereus and S. aureus. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic B. cereus and 14 S. aureus strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic B. cereus by using the real-time PCR assay.

scholarly journals Development of a Real-Time PCR for Detection of Mycoplasma Bovis in Bovine Milk and Lung Samples

2005 ◽  
Vol 17 (6) ◽  
pp. 537-545 ◽  
Author(s):  
Hugh Y. Cai ◽  
Patricia Bell-Rogers ◽  
Lois Parker ◽  
John F. Prescott
Keyword(s):  
Real Time ◽  
Lung Tissue ◽  
Real Time Pcr ◽  
Bovine Milk ◽  
Melting Peak ◽  
Clinical Samples ◽  
Mycoplasma Bovis ◽  
Pcr Assay ◽  
Milk Samples ◽  
Culture Positive

A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6°C. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1°C. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 × 104 and 7.7 × 108 cfu/ml and the M. bovis culture-positive lungs between 1 × 103 and 1 × 109 cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs.

Evaluation of a real-time PCR assay for the differentiation of Bacillus cereus group species

Food Control ◽  
2021 ◽  
Vol 120 ◽  
pp. 107530
Author(s):  
Hendrik Frentzel ◽  
Ylanna Kelner-Burgos ◽  
Carlus Deneke
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
Bacillus Cereus Group ◽  
Group Species
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