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.

Evaluation of a Real-Time PCR Assay for the Detection and Quantification of Bacillus cereus Group Spores in Food

2010 ◽  
Vol 73 (8) ◽  
pp. 1480-1485 ◽  
Author(s):  
JUAN FRANCISCO MARTÍNEZ-BLANCH ◽  
GLORIA SÁNCHEZ ◽  
ESPERANZA GARAY ◽  
ROSA AZNAR
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Dna Isolation ◽  
Food Samples ◽  
Maximum Amount ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Bacillus Cereus Group ◽  
Detection And Quantification

A procedure based on quantitative real-time PCR was evaluated for the detection and quantification of Bacillus cereus spores. Several methods for DNA isolation, such as various heat treatments and germination solutions, were evaluated on spore suspensions of representative strains of the B. cereus group. Overall, the commercially available DNeasy tissue kit yielded the maximum amount of DNA. The procedure also was used to construct calibration curves for different food matrices, with a wide spore quantification range of 5 log units using serial dilutions of spore suspensions of B. cereus CECT 148T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 4 spores per reaction or 60 spores per ml. The newly developed methodology based on the DNeasy tissue kit and an SYBR Green quantitative real-time PCR assay is very suitable for the rapid and accurate detection and quantification of B. cereus group strains and their spores in food samples.

scholarly journals Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters

2007 ◽  
Vol 73 (18) ◽  
pp. 5840-5847 ◽  
Author(s):  
Jessica L. Nordstrom ◽  
Michael C. L. Vickery ◽  
George M. Blackstone ◽  
Shelley L. Murray ◽  
Angelo DePaola
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
The United States ◽  
Internal Amplification Control ◽  
Most Probable Number ◽  
Specific Marker ◽  
Pcr Assay ◽  
Probable Number ◽  
The Real

ABSTRACT Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >104 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh + and trh + strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.

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

scholarly journals Detection and quantification of viable Bacillus cereus group species in milk by propidium monoazide quantitative real-time PCR

2016 ◽  
Vol 99 (4) ◽  
pp. 2617-2624 ◽  
Author(s):  
Fernanda Cattani ◽  
Valdir C. Barth ◽  
Jéssica S.R. Nasário ◽  
Carlos A.S. Ferreira ◽  
Sílvia D. Oliveira
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Propidium Monoazide ◽  
Quantitative Real Time Pcr ◽  
Bacillus Cereus Group ◽  
Group Species ◽  
Detection And Quantification

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.

Assessment of food safety using a new real-time PCR assay for detection and quantification of virulence factors of enterococci in food samples

2016 ◽  
Vol 121 (6) ◽  
pp. 1745-1754
Author(s):  
M. Abouelnaga ◽  
A. Lamas ◽  
M. Guarddon ◽  
M. Osman ◽  
J.M. Miranda ◽  
...  
Keyword(s):  
Food Safety ◽  
Real Time ◽  
Virulence Factors ◽  
Real Time Pcr ◽  
Food Samples ◽  
Pcr Assay ◽  
Detection And Quantification

Detection and Quantification of Campylobacter in Poultry Slaughterhouses Using Conventional Microbiological Technique, Most Probable Number, and Real-Time PCR

2021 ◽  
Author(s):  
Gustavo Perdoncini ◽  
Yuli Melisa Sierra Arguello ◽  
Leonardo Moreira Lima ◽  
Thales Quedi Furian ◽  
Karen Apellanis Borges ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Most Probable Number ◽  
Probable Number ◽  
Detection And Quantification

scholarly journals Development of a Fluorogenic Probe-Based PCR Assay for Detection of Bacillus cereus in Nonfat Dry Milk

2000 ◽  
Vol 66 (4) ◽  
pp. 1453-1459 ◽  
Author(s):  
Young-Rok Kim ◽  
John Czajka ◽  
Carl A. Batt
Keyword(s):  
Bacillus Cereus ◽  
Positive Sequence ◽  
Most Probable Number ◽  
Pcr Assay ◽  
Probable Number ◽  
Fluorogenic Probe ◽  
Hemolysin Gene ◽  
Dry Milk ◽  
Fluorogenic Probes ◽  
Nonfat Dry Milk

ABSTRACT A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5′-to-3′ exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereusstrains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negativeB. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated withB. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.

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