Visual Detection of Clostridium perfringens Alpha Toxin by Combining Nanometer Microspheres with Smart Phones

Clostridium perfringens α toxin (CPA) is an important virulence factor that causes livestock hemorrhagic enteritis and food poisoning by contaminated meat products. In this study, the nano-silica microspheres combined with smartphone image processing technology was developed to realize real-time CPA detection. First, the N-terminal and C-terminal domain of the CPA toxin (CPAC3 and CPAN) and their anti-sera were prepared. The silica microspheres coupled with the antibody of CPAC3 was prepared to capture the toxin that existed in the detection sample and the fluorescent-labeled antibody of CPAN was incubated. Moreover, the fluorescent pictures of gray value were performed in a cell phone app, corresponding to toxin concentration. The new assay takes 90 min to perform and can detect CPA as little as 32.8 ng/mL. Our results showed a sensitive, stable, and convenient CPA detection system, which provides a novel detection method of native CPA in foods.

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Influence of Several Methodological Factors on the Growth of Clostridium perfringens in Cooling Rate Challenge Studies

Journal of Food Protection ◽

10.4315/0362-028x-67.6.1128 ◽

2004 ◽

Vol 67 (6) ◽

pp. 1128-1132 ◽

Cited By ~ 9

Author(s):

SARAH SMITH ◽

VIJAY JUNEJA ◽

DONALD W. SCHAFFNER

Keyword(s):

Clostridium Perfringens ◽

Food Poisoning ◽

Meat Products ◽

Ground Beef ◽

Inoculum Size ◽

Growth Environment ◽

Significant Difference ◽

Plastic Bag ◽

Major Factors ◽

Inspection Service

Proper temperature control is essential in preventing Clostridium perfringens food poisoning. The U.S. Department of Agriculture Food Safety and Inspection Service cooling guidelines offer two options for the cooling of meat products: follow a standard time-temperature schedule or validate that alternative cooling regimens result in no more than a 1-log CFU/g increase of C. perfringens and no growth of Clostridium botulinum. The latter option requires laboratory challenge studies to validate the efficacy of a given cooling process. Accordingly, the objective of this study was to investigate the role of several methodological variables that might be encountered during typical C. perfringens challenge studies. Variables studied included plastic bag type (Whirlpak or Spiral Biotech), sealing method (Multivac or FoodSaver), initial spore inoculum size (1 to approximately 3 log CFU/g), and growth environment (ground beef or Trypticase–peptone–glucose–yeast extract [TPGY] broth). The major factors that affected growth were sample bag type and growth environment. Samples incubated in Whirlpak bags showed significantly less growth than those incubated in Spiral Biotech bags, which was likely due to the former bag's greater oxygen permeability. C. perfringens spores showed shorter germination, outgrowth, and lag times and C. perfringens cells showed faster growth rates in ground beef compared with TPGY broth. No significant difference was observed between two different sealing methods. Initial spore inoculum levels in the range studied had no significant effect on final C. perfringens cell concentration.

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Inorganic Phosphate Induces Spore Morphogenesis and Enterotoxin Production in the Intestinal Pathogen Clostridium perfringens

Infection and Immunity ◽

10.1128/iai.02090-05 ◽

2006 ◽

Vol 74 (6) ◽

pp. 3651-3656 ◽

Cited By ~ 28

Author(s):

Valeria A. Philippe ◽

Marcelo B. Méndez ◽

I-Hsiu Huang ◽

Lelia M. Orsaria ◽

Mahfuzur R. Sarker ◽

...

Keyword(s):

Clostridium Perfringens ◽

Inorganic Phosphate ◽

Food Poisoning ◽

Inhibitory Effect ◽

Physiological Signal ◽

Food Borne ◽

Intestinal Pathogen ◽

Important Virulence Factor ◽

Dna Partitioning ◽

Effect Of Glucose

ABSTRACT Clostridium perfringens enterotoxin (CPE) is an important virulence factor for food poisoning and non-food borne gastrointestinal (GI) diseases. Although CPE production is strongly regulated by sporulation, the nature of the signal(s) triggering sporulation remains unknown. Here, we demonstrated that inorganic phosphate (Pi), and not pH, constitutes an environmental signal inducing sporulation and CPE synthesis. In the absence of Pi-supplementation, C. perfringens displayed a spo0A phenotype, i.e., absence of polar septation and DNA partitioning in cells that reached the stationary phase of growth. These results received support from our Northern blot analyses which demonstrated that Pi was able to counteract the inhibitory effect of glucose at the onset of sporulation and induced spo0A expression, indicating that Pi acts as a key signal triggering spore morphogenesis. In addition to being the first study reporting the nature of a physiological signal triggering sporulation in clostridia, these findings have relevance for the development of antisporulation drugs to prevent or treat CPE-mediated GI diseases in humans.

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Comparative Experiments To Examine the Effects of Heating on Vegetative Cells and Spores of Clostridium perfringens Isolates Carrying Plasmid Genes versus Chromosomal Enterotoxin Genes

Applied and Environmental Microbiology ◽

10.1128/aem.66.8.3234-3240.2000 ◽

2000 ◽

Vol 66 (8) ◽

pp. 3234-3240 ◽

Cited By ~ 133

Author(s):

Mahfuzur R. Sarker ◽

Robert P. Shivers ◽

Shauna G. Sparks ◽

Vijay K. Juneja ◽

Bruce A. McClane

Keyword(s):

Clostridium Perfringens ◽

Gastrointestinal Disease ◽

Food Poisoning ◽

Meat Products ◽

Gastrointestinal Diseases ◽

Vegetative Cells ◽

Food Borne ◽

Enterotoxin Genes ◽

Plasmid Genes ◽

High Degree

ABSTRACT Clostridium perfringens enterotoxin (CPE) is an important virulence factor for both C. perfringens type A food poisoning and several non-food-borne human gastrointestinal diseases. Recent studies have indicated that C. perfringensisolates associated with food poisoning carry a chromosomalcpe gene, while non-food-borne human gastrointestinal disease isolates carry a plasmid cpe gene. However, no explanation has been provided for the strong associations between certain cpe genotypes and particular CPE-associated diseases. Since C. perfringens food poisoning usually involves cooked meat products, we hypothesized that chromosomalcpe isolates are so strongly associated with food poisoning because (i) they are more heat resistant than plasmid cpeisolates, (ii) heating induces loss of the cpe plasmid, or (iii) heating induces migration of the plasmid cpe gene to the chromosome. When we tested these hypotheses, vegetative cells of chromosomal cpe isolates were found to exhibit, on average approximately twofold-higher decimal reduction values (Dvalues) at 55°C than vegetative cells of plasmid cpeisolates exhibited. Furthermore, the spores of chromosomalcpe isolates had, on average, approximately 60-fold-higherD values at 100°C than the spores of plasmidcpe isolates had. Southern hybridization and CPE Western blot analyses demonstrated that all survivors of heating retained theircpe gene in its original plasmid or chromosomal location and could still express CPE. These results suggest that chromosomalcpe isolates are strongly associated with food poisoning, at least in part, because their cells and spores possess a high degree of heat resistance, which should enhance their survival in incompletely cooked or inadequately warmed foods.

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Prevalence and Characterization of Enterotoxin Gene-Carrying Clostridium perfringens Isolates from Retail Meat Products in Japan

Applied and Environmental Microbiology ◽

10.1128/aem.00783-08 ◽

2008 ◽

Vol 74 (17) ◽

pp. 5366-5372 ◽

Cited By ~ 42

Author(s):

Yasuhiro Miki ◽

Kazuaki Miyamoto ◽

Ikuko Kaneko-Hirano ◽

Kanako Fujiuchi ◽

Shigeru Akimoto

Keyword(s):

Clostridium Perfringens ◽

Food Poisoning ◽

Meat Products ◽

Disease Outbreaks ◽

Enterotoxin Gene ◽

Food Borne ◽

Causative Agents ◽

Retail Meat ◽

Meat Samples

ABSTRACT Clostridium perfringens is an important anaerobic pathogen causing food-borne gastrointestinal (GI) diseases in humans and animals. It is thought that C. perfringens food poisoning isolates typically carry the enterotoxin gene (cpe) on their chromosome, while isolates from other GI diseases, such as antibiotic-associated diarrhea, carry cpe on a transferable plasmid. However, food-borne GI disease outbreaks associated with C. perfringens isolates carrying plasmid-borne cpe (plasmid cpe isolates) were recently reported in Japan and Europe. To investigate whether retail food can be a reservoir for food poisoning generally, we evaluated Japanese retail meat products for the presence of two genotypes of enterotoxigenic C. perfringens. Our results demonstrated that approximately 70% of the Japanese retail raw meat samples tested were contaminated with low numbers of C. perfringens bacteria and 4% were contaminated with cpe-positive C. perfringens. Most of the cpe-positive C. perfringens isolates obtained from Japanese retail meat carried cpe on a plasmid. The plasmid cpe isolates exhibited lower spore heat resistance than did chromosomal cpe isolates. Collectively, these plasmid cpe isolates might be causative agents of food poisoning when foods are contaminated with these isolates from equipment and/or the environment after cooking, or they may survive in food that has not been cooked at a high enough temperature.

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Proposal of Multiple Detection Method in Human Detection System using Terrestrial Digital TV Waves

IEEJ Transactions on Electronics Information and Systems ◽

10.1541/ieejeiss.132.500 ◽

2012 ◽

Vol 132 (4) ◽

pp. 500-508

Author(s):

Masahiro Nishi ◽

Koichi Shin ◽

Teruaki Yoshida

Keyword(s):

Detection Method ◽

Detection System ◽

Digital Tv ◽

Human Detection ◽

Multiple Detection

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A Putative Amidase Endolysin Encoded by Clostridium perfringens St13 Exhibits Specific Lytic Activity and Synergizes with the Muramidase Endolysin Psm

Antibiotics ◽

10.3390/antibiotics10030245 ◽

2021 ◽

Vol 10 (3) ◽

pp. 245

Author(s):

Hiroshi Sekiya ◽

Maho Okada ◽

Eiji Tamai ◽

Toshi Shimamoto ◽

Tadashi Shimamoto ◽

...

Keyword(s):

Cell Wall ◽

Clostridium Perfringens ◽

Antimicrobial Agents ◽

Catalytic Domain ◽

Food Poisoning ◽

Binding Activity ◽

Lytic Activity ◽

Intestinal Bacterium ◽

Terminal Domain ◽

Cell Wall Binding

Clostridium perfringens is an often-harmful intestinal bacterium that causes various diseases ranging from food poisoning to life-threatening fulminant disease. Potential treatments include phage-derived endolysins, a promising family of alternative antimicrobial agents. We surveyed the genome of the C. perfringens st13 strain and identified an endolysin gene, psa, in the phage remnant region. Psa has an N-terminal catalytic domain that is homologous to the amidase_2 domain, and a C-terminal domain of unknown function. psa and gene derivatives encoding various Psa subdomains were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. Purified His-tagged full-length Psa protein (Psa-his) showed C. perfringens-specific lytic activity in turbidity reduction assays. In addition, we demonstrated that the uncharacterized C-terminal domain has cell wall-binding activity. Furthermore, cell wall-binding measurements showed that Psa binding was highly specific to C. perfringens. These results indicated that Psa is an amidase endolysin that specifically lyses C. perfringens; the enzyme’s specificity is highly dependent on the binding of the C-terminal domain. Moreover, Psa was shown to have a synergistic effect with another C. perfringens-specific endolysin, Psm, which is a muramidase that cleaves peptidoglycan at a site distinct from that targeted by Psa. The combination of Psa and Psm may be effective in the treatment and prevention of C. perfringens infections.

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Image detection and parameterization for different components in cross-sections of radial tires

Proceedings of the Institution of Mechanical Engineers Part D Journal of Automobile Engineering ◽

10.1177/09544070211023628 ◽

2021 ◽

pp. 095440702110236

Author(s):

Chen Liu ◽

Yude Dong ◽

Yanli Wei ◽

Jiangtao Wang ◽

Hongling Li

Keyword(s):

Image Processing ◽

Digital Image Processing ◽

Cross Sections ◽

Digital Image ◽

Detection Method ◽

Detection System ◽

Practical Significance ◽

Vehicle Safety ◽

Wire Steel ◽

Good Detection

The internal structure analysis of radial tires is of great significance to improve vehicle safety and during tire research. In order to perform the digital analysis and detection of the internal composition in radial tire cross-sections, a detection method based on digital image processing was proposed. The research was carried out as follows: (a) the distribution detection and parametric analysis of the bead wire, steel belt, and carcass in the tire section were performed by means of digital image processing, connected domain extraction, and Hough transform; (b) using the angle of location distribution and area relationship, the detection data were optimized through coordinate and quantity relationship constraints; (c) a detection system for tire cross-section components was designed using the MATLAB platform. Our experimental results showed that this method displayed a good detection performance, and important practical significance for the research and manufacture of tires.

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Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies

Toxins ◽

10.3390/toxins13040266 ◽

2021 ◽

Vol 13 (4) ◽

pp. 266

Author(s):

Thea Neumann ◽

Maren Krüger ◽

Jasmin Weisemann ◽

Stefan Mahrhold ◽

Daniel Stern ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Clostridium Perfringens ◽

Food Poisoning ◽

Receptor Interaction ◽

Clostridium Perfringens Enterotoxin ◽

Fast Detection ◽

Highly Sensitive ◽

Basic And Applied Research ◽

Highly Sensitive Detection

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.

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A Multiplex PCR Assay Mediated by Universal Primers for the Detection of Adulterated Meat in Mutton

Journal of Food Protection ◽

10.4315/0362-028x.jfp-18-302 ◽

2019 ◽

Vol 82 (2) ◽

pp. 325-330 ◽

Cited By ~ 1

Author(s):

WANWAN LIU ◽

XIAONAN WANG ◽

JING TAO ◽

BANGSHENG XI ◽

MAN XUE ◽

...

Keyword(s):

Multiplex Pcr ◽

Detection System ◽

Meat Products ◽

Pcr Primers ◽

Rapid Identification ◽

Food Samples ◽

Detection Sensitivity ◽

Universal Primers ◽

Multiplex Pcr Assay ◽

Multiple Pcr

ABSTRACT This study aimed to establish a multiplex PCR detection system mediated by “universal primers,” which would be able to determine whether mutton meat contained nonmutton ingredients from rats, foxes, and ducks. Based on the sequence variation of specific mitochondrial genes, nine different multiplex PCR primers were designed, and four kinds of meat products were rapidly identified by electrophoresis using an optimized multiplex PCR system based on the molecular weight differences of the amplified products. Multiplex PCR applications optimized for meat food source from food samples for testing was used to verify the accuracy of the identification method. The results showed that the primers in multiple PCR system mediated by universal primers could be used for the rapid identification of rat, fox, duck, and sheep meat in mutton products, and the detection sensitivity could reach 0.05 ng/μL. The identification of food samples validated the practical value of this method. Therefore, a multiplex PCR system mediated by universal primers was established, which can be used to quickly identify the origin of animal ingredients from rats, foxes, and ducks in mutton products.

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