scholarly journals Multiplex PCR Assay for Detection and Identification of Clostridium botulinum Types A, B, E, and F in Food and Fecal Material

2001 ◽  
Vol 67 (12) ◽  
pp. 5694-5699 ◽  
Author(s):  
Miia Lindström ◽  
Riikka Keto ◽  
Annukka Markkula ◽  
Mari Nevas ◽  
Sebastian Hielm ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Bacterial Species ◽  
Type A ◽  
Food Samples ◽  
Type B ◽  
Fecal Material ◽  
Pcr Assay ◽  
Multiplex Pcr Assay

ABSTRACT Botulism is diagnosed by detecting botulinum neurotoxin andClostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 102 cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10−2 spore/g for types A, B, and F to 10−1 spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.

Multiplex PCR Detection of Enterotoxin Genes in Aeromonas spp. from Suspect Food Samples in Northern Taiwan

2008 ◽  
Vol 71 (10) ◽  
pp. 2094-2099 ◽  
Author(s):  
YU-CHANG CHANG ◽  
JAN-YI WANG ◽  
AMMAIYAPPAN SELVAM ◽  
SHU-CHEN KAO ◽  
SHANG-SHYNG YANG ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Diarrheal Disease ◽  
Simultaneous Detection ◽  
Food Poisoning ◽  
Food Samples ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Enterotoxin Genes ◽  
Causative Agents ◽  
Northern Taiwan

Aeromonads possess an array of virulence factors and are causative agents of a number of human infections. Among them, genes of one cytotoxic (Act) and two cytotonic (Alt, Ast) enterotoxins are implicated in a human diarrheal disease. A rapid, specific, simultaneous detection of these enterotoxin genes in suspected food poisoning samples is not yet reported. Hence, a multiplex PCR assay was designed to amplify the cytotoxic (act), heat-labile cytotonic (alt), and heat-stable cytotonic (ast) enterotoxin genes of aeromonads. The PCR assay was tested with 133 Aeromonas spp. isolated from suspect food poisoning samples and retail samples of poultry and fish from wet markets in and around Taipei, Northern Taiwan. The Aeromonas spp. isolates were divided into six genotypes based on absence or presence of one or more enterotoxin genes. Of these 133 isolates, Aeromonas caviae (52.5%) and Aeromonas hydrophila (43.4%) were the most frequently isolated species from food poisoning samples and retail samples, respectively. Among the species, A. hydrophila had a significantly higher proportion for harboring three enterotoxin genes than had the others, whereas Aeromonas encheleia, considered a nonpathogen, was found harboring three enterotoxin genes. The multiplex PCR assays are rapid and specific, and provide a useful tool for the detection and genotyping of enterotoxin genes of aeromonads.

scholarly journals Multiplex PCR for Detection of Botulinum Neurotoxin-Producing Clostridia in Clinical, Food, and Environmental Samples

2009 ◽  
Vol 75 (20) ◽  
pp. 6457-6461 ◽  
Author(s):  
Dario De Medici ◽  
Fabrizio Anniballi ◽  
Gary M. Wyatt ◽  
Miia Lindström ◽  
Ute Messelhäußer ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Environmental Samples ◽  
Botulinum Neurotoxin ◽  
Standard Procedure ◽  
Toxic Substance ◽  
Type A ◽  
Laboratory Animals ◽  
Mouse Bioassay ◽  
Bacterial Strains ◽  
Multiplex Pcr Assay

ABSTRACT Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.

scholarly journals Honey sold directly by producers in the Silesian region of Poland as a source of Clostridium botulinum types A, B, E and F

2017 ◽  
Vol 35 (No. 3) ◽  
pp. 194-199 ◽  
Author(s):  
Fatma Hayıt ◽  
Hülya Gül
Keyword(s):  
Botulinum Toxin ◽  
Multiplex Pcr ◽  
Clostridium Botulinum ◽  
Type A ◽  
Honey Sample ◽  
Type B ◽  
Subsequent Processing ◽  
Pcr Method ◽  
Centrifugation Method ◽  
Cooked Meat

The level of contamination of honey with Clostridium botulinum spores is considered as an indicator of the adequacy of hygienic practices during collection, extraction, and subsequent processing. A total of 39 honey samples purchased directly from beekeepers at outdoor markets and from small amateur apiaries in Silesia were analysed for Clostridium botulinum spores. The samples were prepared using a dilution centrifugation method and cultured in parallel in cooked meat medium (CMM) and tripticase peptone glucose yeast (TPGY) enrichment broths. Identification of C. botulinum toxin types A, B, E, and F was performed with the use of a multiplex PCR method. The analysis showed six (15.4%) samples to be contaminated with C. botulinum spores. The major serotypes detected were type A – in two (5.1%) and type B – in two (5.1%) honey samples, respectively. Types E and F were found in 1 (2.6%) and 1 (2.6%) positive honey sample analysed, respectively.

scholarly journals Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay

2017 ◽  
Vol 83 (18) ◽  
Author(s):  
Charles H. D. Williamson ◽  
Adam J. Vazquez ◽  
Karen Hill ◽  
Theresa J. Smith ◽  
Roxanne Nottingham ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Gene Cluster ◽  
Botulinum Neurotoxin ◽  
Comparative Genomic ◽  
Pcr Assay ◽  
Content Type ◽  
Multiplex Pcr Assay ◽  
Group I ◽  
Pcr Assays ◽  
Group Ii

ABSTRACT Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha +] or orfX +). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters. IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type (ha + or orfX +) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest.

scholarly journals Aetiology of Lobar Pneumonia Determined by Multiplex Molecular Analyses of Lung and Pleural Aspirate Specimens in The Gambia

2021 ◽  
Author(s):  
Grant A Mackenzie ◽  
Jessica McLellan ◽  
Eunice Machuka ◽  
Malick Ndiaye ◽  
Jayani Pathirana ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Pneumococcal Pneumonia ◽  
Blood Cultures ◽  
Haemophilus Influenzae Type ◽  
Haemophilus Influenzae Type B ◽  
Type B ◽  
Pcr Assay ◽  
Lobar Pneumonia ◽  
Multiplex Pcr Assay ◽  
Vaccine Type

Background Pneumonia aetiology generally relies on insensitive blood cultures or an assumption that organisms in the pharynx are causal. We determined the causes of lobar pneumonia in rural Gambia using lung aspiration. Methods Pneumonia surveillance was undertaken among all ages. Blood culture and chest radiographs were performed routinely while lung or pleural aspirates were collected from selected patients. 7-valent pneumococcal conjugate vaccine (PCV7) was introduced in August 2009 and replaced by PCV13 from May 2011. We used conventional microbiology, and from April 8, 2011 to July 17, 2012, utilized a multiplex PCR assay on lung aspirates. We calculated proportions with pathogens, associations between co-infecting pathogens, and PCV effectiveness. Results 2,550 patients were admitted with clinical pneumonia; 741 with lobar pneumonia or pleural effusion. We performed multiplex PCR on 156 lung and 4 pleural aspirates. Pathogens were detected in 116 specimens, Streptococcus pneumoniae (n=68), Staphylococcus aureus (n=26), and Haemophilus influenzae type b (n=11). Bacteria (n=97) were more common than viruses (n=49). Common viruses were bocavirus (n=11) and influenza (n=11). Co-infections were frequent (n=55). M. catarrhalis was detected in eight patients and in every case there was co-infection with S. pneumoniae. The odds ratio of vaccine-type pneumococcal pneumonia in patients with two or three compared to zero doses of PCV was 0.17 (95% CI 0.06, 0.51). Conclusions Lobar pneumonia in rural Gambia was caused primarily by bacteria, particularly S. pneumoniae and S. aureus. Co-infection was common and M. catarrhalis always co-infected with S. pneumoniae. PCV was highly efficacious against vaccine-type pneumococcal pneumonia.

A Collagenase-Targeted Multiplex PCR Assay for Identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus

2005 ◽  
Vol 68 (1) ◽  
pp. 150-153 ◽  
Author(s):  
ANGELA DI PINTO ◽  
GIUSEPPINA CICCARESE ◽  
GIUSEPPINA TANTILLO ◽  
DOMENICO CATALANO ◽  
VITO TONY FORTE
Keyword(s):  
Multiplex Pcr ◽  
Vibrio Cholerae ◽  
Vibrio Parahaemolyticus ◽  
Food Samples ◽  
Vibrio Alginolyticus ◽  
Pcr Assay ◽  
Molecular Approaches ◽  
Multiplex Pcr Assay ◽  
Primer Sets ◽  
Species Specific

A multiplex PCR assay using three collagenase-targeted primer pairs for the species-specific detection of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus was developed. The results highlight the species specificity of the three primer sets designed. Because of the increasing importance of Vibrio spp. in human foodborne diseases, molecular approaches for routine microbial screening and monitoring of clinical, environmental, and food samples also have become more important. The results of this study indicate that the gene coding for collagenase should be used as an alternative molecular target to discriminate among the three Vibrio species.

Evaluation of a Multiplex PCR Assay for the Identification of Salmonella Serovars Enteritidis and Typhimurium Using Retail and Abattoir Samples

2017 ◽  
Vol 80 (2) ◽  
pp. 295-301 ◽  
Author(s):  
Dele Ogunremi ◽  
Susan Nadin-Davis ◽  
Andrée Ann Dupras ◽  
Imelda Gálvan Márquez ◽  
Katayoun Omidi ◽  
...  
Keyword(s):  
Salmonella Typhimurium ◽  
Multiplex Pcr ◽  
Internal Control ◽  
Broiler Chickens ◽  
Salmonella Enteritidis ◽  
Bacterial Species ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Retail Outlets ◽  
Dna Fragment

ABSTRACT A multiplex PCR was developed to identify the two most common serovars of Salmonella causing foodborne illness in Canada, namely, serovars Enteritidis and Typhimurium. The PCR was designed to amplify DNA fragments from four Salmonella genes, namely, invA gene (211-bp fragment), iroB gene (309-bp fragment), Typhimurium STM 4497 (523-bp fragment), and Enteritidis SE147228 (612-bp fragment). In addition, a 1,026-bp ribosomal DNA (rDNA) fragment universally present in bacterial species was included in the assay as an internal control fragment. The detection rate of the PCR was 100% among Salmonella Enteritidis (n = 92) and Salmonella Typhimurium (n = 33) isolates. All tested Salmonella isolates (n = 194) were successfully identified based on the amplification of at least one Salmonella-specific DNA fragment. None of the four Salmonella DNA amplicons were detected in any of the non-Salmonella isolates (n = 126), indicating an exclusivity rate of 100%. When applied to crude extracts of 2,001 field isolates of Salmonella obtained during the course of a national microbiological baseline study in broiler chickens and chicken products sampled from abattoir and retail outlets, 163 isolates, or 8.1%, tested positive for Salmonella Enteritidis and another 80 isolates, or 4.0%, tested as Salmonella Typhimurium. All isolates identified by serological testing as Salmonella Enteritidis in the microbiological study were also identified by using the multiplex PCR. The new test can be used to identify or confirm pure isolates of the two serovars and is also amenable for integration into existing culture procedures for accurate detection of Salmonella colonies.

scholarly journals “Pathotyping” Multiplex PCR Assay for Haemophilus parasuis: a Tool for Prediction of Virulence

2017 ◽  
Vol 55 (9) ◽  
pp. 2617-2628 ◽  
Author(s):  
Kate J. Howell ◽  
Lucy A. Weinert ◽  
Sarah E. Peters ◽  
Jinhong Wang ◽  
Juan Hernandez-Garcia ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Generalized Linear Model ◽  
Bacterial Species ◽  
Haemophilus Parasuis ◽  
Pcr Assay ◽  
Current Standard ◽  
Content Type ◽  
The United Kingdom ◽  
Multiplex Pcr Assay ◽  
Upper Respiratory

ABSTRACTHaemophilus parasuisis a diverse bacterial species that is found in the upper respiratory tracts of pigs and can also cause Glässer's disease and pneumonia. A previous pangenome study ofH. parasuisidentified 48 genes that were associated with clinical disease. Here, we describe the development of a generalized linear model (termed a pathotyping model) to predict the potential virulence of isolates ofH. parasuisbased on a subset of 10 genes from the pangenome. A multiplex PCR (mPCR) was constructed based on these genes, the results of which were entered into the pathotyping model to yield a prediction of virulence. This new diagnostic mPCR was tested on 143 field isolates ofH. parasuisthat had previously been whole-genome sequenced and a further 84 isolates from the United Kingdom from cases ofH. parasuis-related disease in pigs collected between 2013 and 2014. The combination of the mPCR and the pathotyping model predicted the virulence of an isolate with 78% accuracy for the original isolate collection and 90% for the additional isolate collection, providing an overall accuracy of 83% (81% sensitivity and 93% specificity) compared with that of the “current standard” of detailed clinical metadata. This new pathotyping assay has the potential to aid surveillance and disease control in addition to serotyping data.

scholarly journals A Multiplex PCR Assay to Detect and Differentiate Select Agent Strains of Ralstonia solanacearum

Plant Disease ◽  
2015 ◽  
Vol 99 (3) ◽  
pp. 333-341 ◽  
Author(s):  
Michael J. Stulberg ◽  
Jonathan Shao ◽  
Qi Huang
Keyword(s):  
United States ◽  
Multiplex Pcr ◽  
Ralstonia Solanacearum ◽  
Species Complex ◽  
Bacterial Species ◽  
The United States ◽  
Pcr Assay ◽  
Plant Dna ◽  
Multiplex Pcr Assay ◽  
Verification Methods

Ralstonia solanacearum race 3 biovar 2 strains are considered select agents by the U.S. government because they are not endemic to the United States and have the potential to cause brown rot in our potato production fields. Simple and accurate methods are needed for quick identification prior to more discriminating but time-consuming verification methods. We developed a multiplex PCR assay that identifies R. solanacearum species complex strains, signals whether the strain detected is a select agent, and controls for false negatives associated with PCR inhibition or unsuccessful DNA extractions in one reaction. We identified unique sequences of non-phage-related DNA for the R. solanacearum species complex strains, and for select agent strains, using in silico genome subtraction. We also designed and included an internal plant DNA control assay. Our multiplex PCR assay correctly identified 90 R. solanacearum species complex strains and 34 select agent strains, while not recognizing five out-group bacterial species. Additionally, the multiplex PCR assay facilitated the detection of plant DNA and R. solanacearum from infected tomato, potato, geranium, and tobacco plants. Our rapid, accurate, and reliable detection assay can help government officials make timely and appropriate recommendations to exclude this bacterium from the United States.

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