scholarly journals Real-time Screening of Foods Using Repetitive Element PCR Reveals a DNA Marker Characteristic for Enterotoxigenic Bacillus Species

Fine Focus ◽  
2021 ◽  
Vol 7 (1) ◽  
pp. 36-53
Author(s):  
Breanna R. Brenneman ◽  
Kyla L. Adamson ◽  
Matthew R. Beer ◽  
Yenling Ho ◽  
Kiev S. Gracias ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Dna Marker ◽  
Repetitive Element ◽  
Bacillus Species ◽  
Gram Positive ◽  
Banding Patterns ◽  
Bacillus Spp ◽  
Gram Positive Cocci

Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables, and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic, including not just food-associated gastrointestinal toxicoinfections, but human endophthalmitis as well. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Repetitive element PCR was used to compare the banding patterns of each sample against B. cereus ATCC 14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe and were capable of causing gastroenteritis in consumers.

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.

Diagnosis of intramammary infection in samples yielding negative results or minor pathogens in conventional bacterial culturing

2010 ◽  
Vol 78 (1) ◽  
pp. 49-55 ◽  
Author(s):  
Ricardo Bexiga ◽  
Mikko T Koskinen ◽  
Jani Holopainen ◽  
Carla Carneiro ◽  
Helena Pereira ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Gram Positive ◽  
Milk Samples ◽  
Negative Results ◽  
Streptococcus Uberis ◽  
Cell Counts ◽  
Significant Difference ◽  
Culture Negative

Up to half of quarter milk samples submitted for mastitis diagnosis are culture-negative results or lead to identification of coagulase-negative staphylococci or Corynebacterium bovis in conventional culturing, the so-called minor pathogens. The interpretation and usefulness of these results in terms of udder and animal health management is limited, even though the amount of resources spent is relatively high. This work aimed to test two methods of analysis of milk samples with the goal of increasing detection of intramammary pathogens. In the first study, 783 milk samples were processed in duplicate: before and after freezing at −20°C for 24 h, using standard bacteriological techniques. There was a significant difference between the two methods with samples frozen for 24 h yielding significantly fewer Gram-positive catalase-positive cocci, Gram-negative bacilli, Gram-positive bacilli and significantly more samples leading to no growth, than samples before freezing. The number of samples yielding Gram-positive catalase-negative cocci was not significantly affected by freezing. In the second study, a real-time PCR-based test was performed on milk samples with an individual quarter somatic cell count above 500 000 cells/ml that were either negative (n=51 samples) or that led to the isolation of minor pathogens in culturing: Corynebacterium bovis (n=79 samples) or non-aureus staphylococci (NAS, n=32). A mastitis pathogen, beyond the result obtained with standard bacteriology, was detected on 47% of the no-growth samples, on 35% of the samples from which C. bovis had been isolated and on 25% of the samples from which NAS had been isolated. The most commonly detected major pathogen was Escherichia coli, followed by Streptococcus uberis, Arcanobacterium pyogenes/Peptoniphilus indolicus and Streptococcus dysgalactiae. These results suggest that simply freezing milk samples for 24 h does not increase the detection of intramammary bacteria in milk samples and therefore should not be recommended. However, use of the real-time PCR-based test may be useful in diagnosing intramammary infections when milk samples with high somatic cell counts are culture-negative or when culturing results in the detection of minor pathogens.

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

scholarly journals A Real-time Quantitative Polymerase Chain Reaction for the Specific Detection of Hammondia Hammondi and Its Differentiation from Toxoplasma Gondii

2020 ◽  
Author(s):  
Gereon Schares ◽  
Majda Globokar Vrhovec ◽  
Mareen Tuschy ◽  
Maike Joeres ◽  
Andrea Bärwald ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Real Time ◽  
Real Time Pcr ◽  
Post Infection ◽  
In Silico ◽  
Repetitive Element ◽  
Sequence Data ◽  
Quantitative Polymerase Chain Reaction ◽  
Analytical Sensitivity ◽  
Biological Studies

Abstract Introduction: Hammondia hammondi and Toxoplasma gondii are closely related protozoan parasites, but only T. gondii is zoonotic . Both species use felids as definitive hosts and cannot be differentiated by oocyst morphology. In T. gondii, a 529 bp repetitive element (TgREP-529) is of utmost diagnostic importance for PCR diagnostic tests. We identified a similar repetitive region in the H. hammondi genome (HhamREP-529).Material and Methods: Based on reported sequences, primers and probes were selected in silico and optimal primer probe combinations were explored, also by including previously published primers. The analytical sensitivity was tested using serial dilutions of oocyst DNA. For testing analytical specificity, DNA isolated from several related species were used as controls. The newly established TaqMan PCR (Hham-qPCR1) was applied to tissues collected from H. hammondi-infected gamma-interferon knockout (GKO) mice at varying time points post infection. Results: Ten forward and six reverse primers were tested in varying combinations. Four potentially suitable dual-labelled probes were selected. One set based on the primer pair (Hham275F, Hham81R) and the probe (Hham222P) yielded optimal results. In addition to excellent analytic specificity, the assay revealed an analytical sensitivity of genome equivalents of less than 1 oocyst. Investigation of the tissue distribution in GKO mice revealed the presence of parasite DNA in all examined organs, but to a varying extent suggesting 100- to 10.000-fold differences in parasitic loads between tissues in the chronic state of infection, 42 days post infection. Discussion: The use of the 529 bp repeat of H. hammondi is suitable for establishing a quantitative real-time PCR assay because this repeat probably exists about 200-times in the genome of a single organism, like its counterpart in T. gondii. Although there was enough sequence data available, only few of the primers predicted in silico revealed sufficient amplification; the identification of a suitable probe was also difficult. This is in accord with our previous observations on considerable variability in the 529 bp repetitive element of H. hammondi.Conclusions: The H. hammondi real-time PCR represents an important novel diagnostic tool for epidemiological and cell-biological studies on this and related parasites.

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.

scholarly journals Detection of Emetic Bacillus cereus by Real-Time PCR in Foods

2013 ◽  
Vol 18 (4) ◽  
pp. 227-232 ◽  
Author(s):  
SHIGEKO UEDA ◽  
MANAMI YAMAGUCHI ◽  
MIKI IWASE ◽  
YOSHIHIRO KUWABARA
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr

A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food

2016 ◽  
Vol 79 (5) ◽  
pp. 810-815 ◽  
Author(s):  
FEREIDOUN FORGHANI ◽  
SHUAI WEI ◽  
DEOG-HWAN OH
Keyword(s):  
Staphylococcus Aureus ◽  
Listeria Monocytogenes ◽  
High Resolution ◽  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Cost Savings ◽  
High Resolution Melt ◽  
And Staphylococcus Aureus

ABSTRACTThree important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 103 CFU/g without an enrichment step and 3.7 × 101 CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus.

Real-time-PCR-System zum Nachweis von Bacillus cereus (emetischer Typ) in Lebensmitteln

2007 ◽  
Vol 2 (2) ◽  
pp. 190-193 ◽  
Author(s):  
U. Messelhäusser ◽  
M. Fricker ◽  
M. Ehling-Schulz ◽  
H. Ziegler ◽  
D. Elmer-Englhard ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr
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