Races and hosts of Pseudomonas syringae pv. tomato in Serbia

During the past few years, frequent appearance of bacterial speck of tomatoes was recorded in several tomato-growing regions in Serbia. A three-year survey of tomato fields in Serbia (2002-2004) resulted in the isolation of numerous bacterial strains, with 30 representative strains selected for further analyses. Based on the results of pathogenicity, biochemical, and physiological tests, all strains isolated from diseased tomato plants were identified as P. syringae pv. tomato. The identity of strains was confirmed by the polymerase chain reaction (PCR), since PCR products of expected size (650 bp) specific for coronatine-producing strains of P. syringae pv. tomato were amplified from all tested strains. Study of the host range of P. syringae pv. tomato strains originating from Serbia confirmed tomato as the sole host. The reaction of tomato differential cultivar Ontario 7710 showed that the Serbian strains belonged to races 0 and 1 of P. syringae pv. tomato.

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A Comparative Study of 5S rDNA Non-Transcribed Spacers in Elaeagnaceae Species

Plants ◽

10.3390/plants10010004 ◽

2020 ◽

Vol 10 (1) ◽

pp. 4

Author(s):

Oleg S. Alexandrov ◽

Olga V. Razumova ◽

Gennady I. Karlov

Keyword(s):

Polymerase Chain Reaction ◽

Molecular Markers ◽

Dna Markers ◽

5S Rdna ◽

Chain Reaction ◽

Closely Related Species ◽

Pcr Products ◽

Polymerase Chain ◽

Species Specific ◽

Shepherdia Canadensis

5S rDNA is organized as a cluster of tandemly repeated monomers that consist of the conservative 120 bp coding part and non-transcribed spacers (NTSs) with different lengths and sequences among different species. The polymorphism in the 5S rDNA NTSs of closely related species is interesting for phylogenetic and evolutional investigations, as well as for the development of molecular markers. In this study, the 5S rDNA NTSs were amplified with universal 5S1/5S2 primers in some species of the Elaeagnaceae Adans. family. The polymerase chain reaction (PCR) products of five Elaeagnus species had similar lengths near 310 bp and were different from Shepherdia canadensis (L.) Nutt. and Sh. argentea (Pusch.) Nutt. samples (260 bp and 215 bp, respectively). The PCR products were cloned and sequenced. An analysis of the sequences revealed that intraspecific levels of NTS identity are high (approximately 95–96%) and similar in the Elaeagnus L. species. In Sh. argentea, this level was slightly lower due to the differences in the poly-T region. Moreover, the intergeneric and intervarietal NTS identity levels were studied and compared. Significant differences between species (except E. multiflora Thunb. and E. umbellata Thunb.) and genera were found. Herein, a range of the NTS features is discussed. This study is another step in the investigation of the molecular evolution of Elaeagnaceae and may be useful for the development of species-specific DNA markers in this family.

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Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction

Clinical Chemistry ◽

10.1093/clinchem/39.9.1927 ◽

1993 ◽

Vol 39 (9) ◽

pp. 1927-1933 ◽

Cited By ~ 39

Author(s):

J B Findlay ◽

S M Atwood ◽

L Bergmeyer ◽

J Chemelli ◽

K Christy ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Pcr Amplification ◽

Automated System ◽

Closed Vessel ◽

Chain Reaction ◽

Panel Testing ◽

Pcr Product ◽

Pcr Products ◽

Polymerase Chain

Abstract An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by "PCR product carryover." The system uses a single vessel for both PCR amplification and the subsequent detection of PCR products, eliminating the need to handle PCR products in an open environment and risk product carryover. The sample and PCR reagents are introduced into one compartment within the vessel, and amplification occurs as they are thermally cycled. Other compartments contain the reagents for detection of PCR products. Pressure from a roller provides for sequential delivery of the contents of the compartments to a detection area. The PCR products are biotinylated at their 5' ends during amplification through the use of biotinylated primers. After delivery to the detection area, they are specifically captured by hybridization with immobilized oligonucleotide probes. Subsequent reaction with streptavidin-horseradish peroxidase conjugate forms a complex that catalyzes dye formation from dye precursor. Wash steps minimize nonspecific background. This format is amenable to multiplexing, permitting internal controls, speciation of bacteria, typing of viruses, and panel testing. An HIV assay performed with this system demonstrated 100% sensitivity and 95% specificity for 64 patients' samples relative to a conventional PCR assay based on 32P solution hybridization. Similarly, an automated closed-vessel assay of cytomegalovirus exhibited 97.5% sensitivity and 100% specificity.

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Fluorometric Polymerase Chain Reaction (PCR) Enzyme-Linked Immunosorbent Assay for Quantification of Immuno-PCR Products in Microplates

Analytical Biochemistry ◽

10.1006/abio.1996.9989 ◽

1997 ◽

Vol 246 (1) ◽

pp. 140-145 ◽

Cited By ~ 61

Author(s):

Christof M. Niemeyer ◽

Michael Adler ◽

Dietmar Blohm

Keyword(s):

Polymerase Chain Reaction ◽

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Chain Reaction ◽

Pcr Products ◽

Polymerase Chain

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PCR-induced (ligase-free) subcloning: a rapid reliable method to subclone polymerase chain reaction (PCR) products

Nucleic Acids Research ◽

10.1093/nar/18.7.1920 ◽

1990 ◽

Vol 18 (7) ◽

pp. 1920-1920 ◽

Cited By ~ 36

Author(s):

Alan R. Shuldiner ◽

Laurie A. Scott ◽

Jesse Roth

Keyword(s):

Polymerase Chain Reaction ◽

Reliable Method ◽

Chain Reaction ◽

Pcr Products ◽

Polymerase Chain

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Detection of the chromosomal translocation t(11;14) by polymerase chain reaction in mantle cell lymphomas

Blood ◽

10.1182/blood.v83.7.1871.1871 ◽

1994 ◽

Vol 83 (7) ◽

pp. 1871-1875 ◽

Cited By ~ 150

Author(s):

R Rimokh ◽

F Berger ◽

G Delsol ◽

I Digonnet ◽

JP Rouault ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Chromosomal Translocation ◽

Nucleotide Sequencing ◽

Chromosome 11 ◽

Chromosome 14 ◽

Chain Reaction ◽

Mantle Cell ◽

Pcr Products ◽

Polymerase Chain ◽

Ig Heavy Chain

Abstract The t(11;14)(q13;q32) and its molecular counterpart, BCL1 rearrangement, are consistent features of mantle cell lymphoma (MCL). Rearrangement is thought to deregulate the nearby CCND1 (BCL1/PRAD1) proto-oncogene, a member of the cyclin G1 gene family, and thereby to contribute to tumorigenesis. We and others have previously shown that the BCL1 locus is rearranged in 55% to 60% of MCL patients and that, on chromosome 11, more than 80% of the breakpoints are localized within a 1-kbp DNA segment known as the major translocation cluster (MTC). We have determined the nucleotide sequence for a portion of the MTC region, and constructed chromosome 11-specific oligonucleotides that were in conjunction with a consensus immunoglobulin (Ig) heavy chain joining region (JH) primer used to perform the polymerase chain reaction (PCR) to amplify t(11;14) chromosomal junctional sequences in DNA from 16 MCL patients with breakpoints in the MTC region. 15 of the 16 breakpoints that occurred at the MTC region were amenable to PCR detection. The sizes of the amplified bands, the existence or not of a Sac I site in the PCR products, and nucleotide sequencing of the amplified DNA from four patients showed that the breakpoints share a remarkable tendency to tightly cluster within 300 bp on chromosome 11, some of them occurring at the same nucleotide. On chromosome 14, the breakpoints were localized within the Ig JH. Our findings indicate that a BCL1 rearrangement can be detected using this approach in roughly one half of the MCL patients. This has implications for both the diagnosis and the clinical management of MCL.

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Detection of Subclinical Systemic Disease in Primary CNS Lymphoma by Polymerase Chain Reaction of the Rearranged Immunoglobulin Heavy-Chain Genes

Journal of Clinical Oncology ◽

10.1200/jco.2006.06.7165 ◽

2006 ◽

Vol 24 (29) ◽

pp. 4754-4757 ◽

Cited By ~ 55

Author(s):

Kristoph Jahnke ◽

Michael Hummel ◽

Agnieszka Korfel ◽

Thomas Burmeister ◽

Philipp Kiewe ◽

...

Keyword(s):

Bone Marrow ◽

Polymerase Chain Reaction ◽

Systemic Disease ◽

Primary Cns Lymphoma ◽

Cns Lymphoma ◽

Chain Reaction ◽

Blood Samples ◽

Pcr Products ◽

Polymerase Chain

Purpose To search for subclinical systemic disease in bone marrow and peripheral blood in patients with primary CNS lymphoma (PCNSL) to elucidate whether extracerebral relapse may represent a sequel of initial occult systemic disease rather than true extracerebral spread. Patients and Methods Bone marrow and peripheral-blood specimens of 24 PCNSL patients were examined using polymerase chain reaction (PCR) for analysis of clonally rearranged immunoglobulin heavy-chain (IgH) genes. Results Identical dominant PCR products were found in bone marrow aspirates, blood samples, and tumor biopsy specimens of two patients, indicating that the same tumor cell population is present in the CNS and in extracerebral sites. Follow-up IgH PCR performed in one of these patients in complete remission 24 months after diagnosis yielded a persistent monoclonal product in the blood. An oligoclonal IgH rearrangement pattern was found in the tumor specimen of two other patients, whereas bone marrow and blood samples demonstrated the same dominant PCR products. Follow-up PCR showed a persistent monoclonal amplificate in blood in one of these patients 27 months after diagnosis. Conclusion It could be demonstrated for the first time that subclinical systemic disease can be present in PCNSL patients at initial diagnosis. Our findings may have an impact on the understanding of PCNSL pathogenesis and the extent of staging and treatment.

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Comparative study of five polycyclic aromatic hydrocarbon degrading bacterial strains isolated from contaminated soils

Canadian Journal of Microbiology ◽

10.1139/m97-051 ◽

1997 ◽

Vol 43 (4) ◽

pp. 368-377 ◽

Cited By ~ 58

Author(s):

Fadi Dagher ◽

Eric Déziel ◽

Patricia Lirette ◽

Gilles Paquette ◽

Jean-Guy Bisaillon ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Polycyclic Aromatic Hydrocarbon ◽

Aromatic Hydrocarbon ◽

Contaminated Soils ◽

Bacterial Strains ◽

Chain Reaction ◽

Pah Degradation ◽

Catabolic Genes ◽

Polycyclic Aromatic ◽

Polymerase Chain

Five polycyclic aromatic hydrocarbon (PAH) degrading bacterial strains, Pseudomonas putida 34, Pseudomonas fluorescens 62, Pseudomonas aeruginosa 57, Sphingomonas sp. strain 107, and the unidentified strain PL1, were isolated from two contaminated soils and characterized for specific features regarding PAH degradation. Degradation efficiency was determined by the rapidity to form clearing zones around colonies when sprayed with different PAH solutions and the growth in liquid medium with different PAHs as sole source of carbon and energy. The presence of plasmids, the production of biosurfactants, the effect of salicylate on PAH degradation, the transformation of indole to indigo indicating the presence of an aromatic ring dioxygenase activity, and the hybridization with the SphAb probe representing a sequence highly homologous to the naphthalene dioxygenase ferredoxin gene nahAb were examined. The most efficient strain in terms of substrate specificity and rapidity to degrade different PAHs was Sphingomonas sp. strain 107, followed by strain PL1 and P. aeruginosa 57. The less efficient strains were P. putida 34 and P. fluorescens 62. Each strain transformed indole to indigo, except strain PL1. Biosurfactants were produced by P. aeruginosa 57 and P. putida 34, and a bioemulsifier was produced by Sphingomonas sp. strain 107. The presence of salicylate in solid medium has accelerated the formation of clearing zones and the transformation of indole by Sphingomonas sp. strain 107 and P. aeruginosa 57 colonies. Plasmids were found in Sphingomonas sp. strain 107 and strain PL1. The SphAb probe hybridized with DNA extracted from each strain. However, hybridization signals were detected only in the plasmidic fraction of Sphingomonas sp. strain 107 and strain PL1. Using a polymerase chain reaction (PCR) approach, we determined that several genes encoding enzymes involved in the upper catabolic pathway of naphthalene were present in each strain. Sequencing of PCR DNA fragments revealed that, for all the five strains, these genes are highly homologous with respective genes found in the pah, dox, and nah opérons, and are arranged in a polycistronic operon. Results suggest that these genes are ordered in the five selected strains like the pah, nah, and dox opérons.Key words: polycyclic aromatic hydrocarbons, biodegradation, polymerase chain reaction, naphthalene catabolic genes.

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Detection and identification of Clavibacter michiganensis subsp. sepedonicus and Clavibacter michiganensis subsp. michiganensis by nonradioactive hybridization, polymerase chain reaction, and restriction enzyme analysis

Canadian Journal of Microbiology ◽

10.1139/m94-161 ◽

1994 ◽

Vol 40 (12) ◽

pp. 1007-1018 ◽

Cited By ~ 18

Author(s):

J. L. W. Rademaker ◽

J. D. Janse

Keyword(s):

Polymerase Chain Reaction ◽

Polymerase Chain Reaction Product ◽

Enzyme Analysis ◽

Restriction Enzyme Analysis ◽

Bacterial Strains ◽

Clavibacter Michiganensis ◽

Chain Reaction ◽

Clavibacter Michiganensis Subsp ◽

Detection And Identification ◽

Polymerase Chain

To develop a rapid and reliable detection and identification method for Clavibacter michiganensis subsp. sepedonicus and C. michiganensis subsp. michiganensis, two biotinylated probes and derived primer sets were evaluated for specificity using a large number of bacterial strains. Detection in dot blot analysis using the Diagen probe against C. michiganensis subsp. sepedonicus was possible with all 32 C. michiganensis subsp. sepedonicus strains tested. Cross-hybridization occurred with all nine C. michiganensis subsp. insidiosus strains tested. No hybridization occurred with any of 54 other related and unrelated bacterial strains including C. michiganensis subsp. michiganensis, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, C. iranicus, C. rathayi, and C. tritici and potato saprophytes. Hybridization of the MIC 1 probe against C. michiganensis subsp. michiganensis was obtained with 22 out of 24 C. michiganensis subsp. michiganensis strains. A weak hybridization signal occurred only with two strains of C. michiganensis subsp. insidiosns. No hybridization occurred with any of the 71 other related and unrelated bacterial strains tested including tomato saprophytes. Restriction fragment length polymorphisms detected with the Diagen probe allowed differentiation between C. michiganensis subsp. sepedonicus and the related C. michiganensis subsp. insidiosus. Restriction fragment length polymorphism analysis using the MIC 1 probe and BamH1 showed at least two groups of patterns within C. michiganensis subsp. michiganensis. By using a primer set derived from the Diagen probe, a DNA sequence could be amplified with all C. michiganensis subsp. sepedonicus strains tested. Only the nontarget organism C. michiganensis subsp. insidiosus yielded a similar polymerase chain reaction product. Restriction enzyme analysis of the polymerase chain reaction product enabled rapid distinction between the subspecies. With a CMM primer set derived from the MIC 1 probe a DNA sequence was amplified from the same 22 out of 24 C. michiganensis subsp. michiganensis strains that showed hybridization with the MIC 1 probe. The polymerase chain reaction product could be verified by restriction enzyme analysis. The Diagen and MIC 1 probes and derived primer sets were shown to be useful for the detection and identification of C. michiganensis subsp. sepedonicus and C. michiganensis subsp. michiganensis. The MIC 1 probe, however, failed to detect two strains of the latter subspecies.Key words: biotin, PCR, REA, potato bacterial ring rot, bacterial canker of tomato, RFLP, Clavibacter michiganensis subsp. insidiosus.

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