A PCR Assay Based on a Sequence-Characterized Amplified Region Marker for Detection of Emetic Bacillus cereus

2004 ◽  
Vol 67 (8) ◽  
pp. 1694-1701 ◽  
Author(s):  
SHIGERU NAKANO ◽  
HIDEKI MAESHIMA ◽  
ATSUSHI MATSUMURA ◽  
KATSUTOSHI OHNO ◽  
SHIGEKO UEDA ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Enrichment Culture ◽  
Random Amplified Polymorphic Dna ◽  
Agar Plate ◽  
Brain Heart Infusion ◽  
Pcr Assay ◽  
Bacterial Colony ◽  
Sequence Characterized Amplified Region ◽  
Rapd Fragment ◽  
Bacillus Strains

A PCR assay for the detection of Bacillus cereus strains able to produce an emetic toxin (cereulide) was developed in this study based on a sequence-characterized amplified region (SCAR) derived from a random amplified polymorphic DNA (RAPD) fragment. One of the RAPD fragments generated was selected, cloned, and sequenced. A set of PCR primers was newly designed from the SCAR obtained (the sequence of the cloned RAPD fragment) and used in this assay. To determine the specificity of the assay, 30 different B. cereus strains, 8 other Bacillus strains (of six species), and 16 other non-Bacillus strains (from 16 genera) were tested. Results were positive for every emetic B. cereus strain and for only one nonemetic B. cereus strain. For all other bacterial strains, results were negative. Bacterial DNA for PCR was prepared by a simple procedure using Chelex 100 resin from the bacterial colony on the agar plate or from culture after growth in brain heart infusion medium. This PCR assay enabled us to detect the bacteria of emetic B. cereus grown on agar plates but not the bacteria of nonemetic B. cereus. To test this PCR assay for the monitoring of the emetic bacteria, 10 to 70 CFU of B. cereus DSM 4312 (emetic) per g of food was inoculated into several foods as an indicator, followed by a 7-h enrichment culture step. Because this PCR assay based on the SCAR derived from the RAPD fragment was able to detect bacterial cells, this assay should be useful for rapid and specific detection of emetic B. cereus.

scholarly journals Development of SCAR Primers for PCR Assay to Detect Diplodia seriata

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
M. T. Martín ◽  
M. J. Cuesta ◽  
L. Martín
Keyword(s):  
Random Amplified Polymorphic Dna ◽  
Fungal Species ◽  
Dna Purification ◽  
Pcr Assay ◽  
Diplodia Seriata ◽  
Castilla Y Leon ◽  
Target Dna ◽  
Pcr Products ◽  
Scar Primer ◽  
Rapd Fragment

The aim of this study was to develop primer pairs for Diplodia seriata identification, one of the most common fungal species associated with grapevine decline in Castilla y León (Spain). Genetic variability of selected isolates of D. seriata was estimated. A molecular marker was generated from a random amplified polymorphic DNA (RAPD) fragment. PCR products of around 1200 bp were obtained with OPE20 primer. The PCR products were cloned and sequenced. The sequences were compared and a fragment of 1207 bp was used to design primer pairs. Two primer pairs were selected (DS3.8 S3-DS3.8 R6 and DS3.8 S3-DS3.8 R4) that amplified a single DNA product of 634 bp and 233 bp, respectively, with D. seriata isolates. No amplification was obtained for any of the 57 isolates of other species. The designed SCAR primer pairs allowed a rapid detection of D. seriata, and were able to detect 0.1 pg of the target DNA. Detection was specific and sensitive for D. seriata. The established protocols detected these fungi in naturally infected grapevines after DNA purification. Diplodia seriata was detectable without DNA purification and isolation in 62.5% to 75% of reactions. The detection of this pathogen in wood samples has great potential for use in pathogen-free certification schemes.

scholarly journals Development of PCR-Based Assays for Detecting and Differentiating Three Species of Botrytis Infecting Broad Bean

Plant Disease ◽  
2015 ◽  
Vol 99 (5) ◽  
pp. 691-698 ◽  
Author(s):  
Xuan Fan ◽  
Jing Zhang ◽  
Long Yang ◽  
Mingde Wu ◽  
Weidong Chen ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Broad Bean ◽  
Random Amplified Polymorphic Dna ◽  
Pcr Assay ◽  
Sequence Characterized Amplified Region ◽  
Multiplex Pcr Assay ◽  
Dna Assays ◽  
Chocolate Spot ◽  
Polymerase Chain ◽  
Primer Sets

Botrytis cinerea, B. fabae, and B. fabiopsis are known to cause chocolate spot on broad bean. This study was conducted to develop polymerase chain reaction (PCR)-based assays to detect and differentiate these three species. Two sets of primers, Bc-f/Bc-r for B. cinerea and Bfab-f/Bfab-r for B. fabiopsis, were designed based on two sequence-characterized amplified region markers derived from two random amplified polymorphic DNA assays. The other primer set, Bfa-f/Bfa-r for B. fabae, was designed based on the necrosis and ethylene-inducing protein 1 gene sequence. The three primer sets were highly specific for the corresponding species of Botrytis in both single and multiplex PCR assays. The PCR detection limit was 40, 40, and 400 pg of DNA per 25-μl reaction mixture for B. fabae, B. fabiopsis, and B. cinerea, respectively. Presence of the broad bean DNA in the PCR reactions at 1:1000 (Botrytis DNA/broad bean DNA [wt/wt]) had negligible effects on detection of the targeted Botrytis spp. The multiplex PCR assay was able to detect three Botrytis spp. in artificially infected and naturally infected broad bean leaves. These results suggest that the multiplex PCR assay developed in this study could be used to monitor the epidemics of chocolate spot of broad bean in the field.

Development of a PCR Assay for Detection of Enterobacteriaceae in Foods

2003 ◽  
Vol 66 (10) ◽  
pp. 1798-1804 ◽  
Author(s):  
SHIGERU NAKANO ◽  
TORU KOBAYASHI ◽  
KENICHI FUNABIKI ◽  
ATSUSHI MATSUMURA ◽  
YASUHIRO NAGAO ◽  
...  
Keyword(s):  
Salmonella Enteritidis ◽  
Enrichment Culture ◽  
Simple Procedure ◽  
Bacterial Species ◽  
Brain Heart Infusion ◽  
Rrna Gene ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Pcr Assay ◽  
Chelex 100

A broad-range PCR assay for the detection of bacteria belonging to the Enterobacteriaceae family was developed in this study. Primers targeting the bacterial 16S rRNA gene were newly designed and used in this PCR assay. To determine the specificity of the assay, 72 different bacterial species (of 49 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for every tested Vibrioaceae or Enterobacteriaceae strain except Proteus mirabilis. For all other bacterial strains and eukaryotes tested, results were negative. Bacterial DNA for PCR was prepared by a simple procedure with the use of Chelex 100 resin from culture after growth in brain heart infusion medium. To test this PCR assay for the monitoring of the Enterobacteriaceae family, either Escherichia coli or Salmonella Enteritidis was inoculated into various foods as an indicator. Prior to the PCR, the inoculation of 10 to 40 CFU of bacteria per g of food was followed by a 5-h enrichment culture step, and the PCR assay allowed the detection of bacterial cells. When actual examinations of the contamination of 15 noodle foods with Enterobacteriaceae by this PCR assay were conducted, 33% (5 of 15) of the samples tested positive. These results agreed with those of the Petri film Enterobacteriaceae Count Plate assay. Including the enrichment culture step, the entire PCR detection process can be completed within 7 h.

PCR Detection of Bacillus and Staphylococcus in Various Foods

2004 ◽  
Vol 67 (6) ◽  
pp. 1271-1277 ◽  
Author(s):  
SHIGERU NAKANO ◽  
TORU KOBAYASHI ◽  
KENICHI FUNABIKI ◽  
ATSUSHI MATSUMURA ◽  
YASUHIRO NAGAO ◽  
...  
Keyword(s):  
Enrichment Culture ◽  
Brain Heart Infusion ◽  
Pcr Detection ◽  
Rrna Gene ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Pcr Assay ◽  
Bacterial Dna ◽  
Chelex 100 ◽  
Pcr Product

A broad-range PCR assay for the detection of bacteria belonging to Bacillus and Staphylococcus genera was developed. Primers targeting the bacterial 16S rRNA gene were newly designed and used in a PCR assay. To determine the specificity of the assay, 81 different bacterial strains (of 50 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for every tested Bacillus, Staphylococcus, or Aerococcus strain. In addition, the result for Listeria grayi was positive with lower PCR product. For all other bacterial strains and eukaryotes tested, results were negative. Bacterial DNA was prepared with the use of achromopeptidase and Chelex 100 resin from culture after growth in brain heart infusion medium. To test the sensitivity of this PCR assay for Bacillus or Staphylococcus genus, either Bacillus cereus or Staphylococcus aureus was inoculated into various foods with undetectable levels of endogenous microbial contamination as an indicator. Inoculation of bacteria at 10 to 30 CFU/g of food was followed by a 5-h enrichment culture step after which the PCR assay allowed the detection of bacterial cells. When the inoculation ( B. cereus or S. aureus) of 10 to 90 CFU/g into noodle foods containing endogenous microflora (103 to 105 CFU/g) was followed by a 6-h enrichment culture step, the PCR assay detected the bacteria. Including the enrichment culture step, the entire PCR detection process can be completed within 8.5 h.

Prevalence and Genetic Diversity of Bacillus cereus in Dried Red Pepper in Korea

2007 ◽  
Vol 70 (4) ◽  
pp. 917-922 ◽  
Author(s):  
EUIYOUNG CHOO ◽  
SUNG SIK JANG ◽  
KYUMSON KIM ◽  
KWANG-GEUN LEE ◽  
SUNGGI HEU ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Bacillus Cereus ◽  
Multiplex Pcr ◽  
Random Amplified Polymorphic Dna ◽  
Bacterial Pathogen ◽  
Culture Method ◽  
Average Concentration ◽  
Red Pepper ◽  
Pcr Assay ◽  
Multiplex Pcr Assay

Bacillus cereus is a foodborne spore-forming bacterial pathogen that is ubiquitous in the natural environment. Infections with this pathogen manifest as diarrheal or emetic types of food poisoning. In this study, 140 samples of dried red pepper purchased in Korea were assayed for the presence of B. cereus according to the U.S. Food and Drug Administration standard culture method. A multiplex PCR assay was developed for the rapid confirmation of B. cereus as an alternative to conventional biochemical confirmation tests. The genetic diversity of B. cereus isolates was investigated using a random amplified polymorphic DNA (RAPD) assay. B. cereus was found in 84.3% of the dried red pepper samples, with an average concentration of 1.9 × 104 CFU/g. B. cereus could be detected and distinguished from B. thuringiensis in the multiplex PCR assay by using the BCFW1 plus BCrevnew and the K3 plus K5 primer sets designed to detect the gyrB gene of B. cereus and B. thuringiensis and the cry gene of B. thuringiensis. A RAPD assay using the OPG 16 and MUP 3 primers was used to successfully distinguish among isolates, thus elucidating the genetic diversity of B. cereus isolates. The discriminating ability of the OPG 16 primer (142 types) was about threefold higher than that of MUP 3 (52 types) in the RAPD assay.

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.

scholarly journals A Statistical Model for Assessing Sample Size for Bacterial Colony Selection: A Case Study of Escherichia Coli and Avian Cellulitis

2000 ◽  
Vol 12 (2) ◽  
pp. 118-125 ◽  
Author(s):  
Randall S. Singer ◽  
Wesley O. Johnson ◽  
Joan S. Jeffrey ◽  
Richard P. Chin ◽  
Tim E. Carpenter ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Statistical Model ◽  
Clinical Sample ◽  
Agar Plate ◽  
Sample Sizes ◽  
Simple Task ◽  
Single Strain ◽  
Bacterial Colony ◽  
E Coli

A general problem for microbiologists is determining the number of phenotypically similar colonies growing on an agar plate that must be analyzed in order to be confident of identifying all of the different strains present in the sample. If a specified number of colonies is picked from a plate on which the number of unique strains of bacteria is unknown, assigning a probability of correctly identifying all of the strains present on the plate is not a simple task. With Escherichia coli of avian cellulitis origin as a case study, a statistical model was designed that would delineate sample sizes for efficient and consistent identification of all the strains of phenotypically similar bacteria in a clinical sample. This model enables the microbiologist to calculate the probability that all of the strains contained within the sample are correctly identified and to generate probability-based sample sizes for colony identification. The probability of cellulitis lesions containing a single strain of E. coli was 95.4%. If one E. coli strain is observed out of three colonies randomly selected from a future agar plate, the probability is 98.8% that only one strain is on the plate. These results are specific for this cellulitis E. coli scenario. For systems in which the number of bacterial strains per sample is variable, this model provides a quantitative means by which sample sizes can be determined.

scholarly journals Marcadores moleculares na predição do sexo em plantas de mamoeiro

2007 ◽  
Vol 42 (12) ◽  
pp. 1747-1754 ◽  
Author(s):  
Eder Jorge de Oliveira ◽  
Jorge Luiz Loyola Dantas ◽  
Milene da Silva Castellen ◽  
Diego Souza de Lima ◽  
Helder de Souza Barbosa ◽  
...  
Keyword(s):  
Random Amplified Polymorphic Dna ◽  
Sequence Characterized Amplified Region

O objetivo deste trabalho foi validar marcadores moleculares, previamente identificados como ligados ao sexo do mamoeiro, para utilização na seleção indireta em genótipos comerciais. Foram analisadas duas variedades do grupo Solo e dois híbridos do grupo Formosa, com utilização de 20 plantas por genótipo, quatro marcadores do tipo SCAR (Sequence Characterized Amplified Region) e um RAPD (Random Amplified Polymorphic DNA). O RAPD BC210 permitiu a identificação de todas as plantas femininas e hermafroditas, o que revela grande potencial para ser usado na seleção assistida em alguns dos genótipos mais cultivados no Brasil. Os marcadores do tipo SCAR não permitiram a identificação correta do sexo dos genótipos, pois detectou-se a presença de falso-positivos e falso-negativos nas análises.

Toxin Production by Psychrotrophic Bacillus spp. Present in Milk

1990 ◽  
Vol 53 (9) ◽  
pp. 790-792 ◽  
Author(s):  
M. W. GRIFFITHS
Keyword(s):  
Bacillus Cereus ◽  
Brain Heart Infusion ◽  
Bacterial Count ◽  
Toxin Production ◽  
Latex Agglutination ◽  
Brain Heart Infusion Broth ◽  
Agglutination Assay ◽  
Bacillus Spp ◽  
Latex Agglutination Assay

Using a reversed passive latex agglutination assay, about 85% of psychrotrophic Bacillus spp. tested were shown to produce diarrhoegenic toxin during growth on brain heart infusion broth at 25°C. The majority of these strains were identified as Bacillus cereus or cereus-related strains. However, a number of other species was capable of synthesizing the toxin. Further investigation of four psychrotrophic Bacilli showed that the toxin was produced during growth in milk at temperatures ranging from 6 to 21°C. Toxin production increased with increasing temperatures and was not synthesized in appreciable quantities until the bacterial count exceeded 1 × 107 cfu/ml.

scholarly journals Expression of Bacillus cereus Virulence-Related Genes in an Ocular Infection-Related Environment

2020 ◽  
Vol 8 (4) ◽  
pp. 607 ◽  
Author(s):  
Phillip S. Coburn ◽  
Frederick C. Miller ◽  
Morgan A. Enty ◽  
Craig Land ◽  
Austin L. LaGrow ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Virulence Genes ◽  
Ex Vivo ◽  
Brain Heart Infusion ◽  
Luria Bertani ◽  
Ocular Infection ◽  
Rna Seq ◽  
Specific Factors ◽  
Low Levels

Bacillus cereus produces many factors linked to pathogenesis and is recognized for causing gastrointestinal toxemia and infections. B. cereus also causes a fulminant and often blinding intraocular infection called endophthalmitis. We reported that the PlcR/PapR system regulates intraocular virulence, but the specific factors that contribute to B. cereus virulence in the eye remain elusive. Here, we compared gene expression in ex vivo vitreous humor with expression in Luria Bertani (LB) and Brain Heart Infusion (BHI) broth by RNA-Seq. The expression of several cytolytic toxins in vitreous was less than or similar to levels observed in BHI or LB. Regulators of virulence genes, including PlcR/PapR, were expressed in vitreous. PlcR/PapR was expressed at low levels, though we reported that PlcR-deficient B. cereus was attenuated in the eye. Chemotaxis and motility genes were expressed at similar levels in LB and BHI, but at low to undetectable levels in vitreous, although motility is an important phenotype for B. cereus in the eye. Superoxide dismutase, a potential inhibitor of neutrophil activity in the eye during infection, was the most highly expressed gene in vitreous. Genes previously reported to be important to intraocular virulence were expressed at low levels in vitreous under these conditions, possibly because in vivo cues are required for higher level expression. Genes expressed in vitreous may contribute to the unique virulence of B. cereus endophthalmitis, and future analysis of the B. cereus virulome in the eye will identify those expressed in vivo, which could potentially be targeted to arrest virulence.

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