scholarly journals Specific and Sensitive Detection of Ralstonia solanacearum in Soil on the Basis of PCR Amplification of fliC Fragments

2003 ◽  
Vol 69 (12) ◽  
pp. 7248-7256 ◽  
Author(s):  
J. Schönfeld ◽  
H. Heuer ◽  
J. D. van Elsas ◽  
K. Smalla
Keyword(s):  
Ralstonia Solanacearum ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Environmental Isolates ◽  
System A ◽  
Gene Coding ◽  
Pcr Product ◽  
Geographic Origins ◽  
Pcr Products

ABSTRACT Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60 available at GenBank, the Ral_fliC PCR primer system was designed; this system yielded a single 724-bp product with the DNAs of all of the R. solanacearum strains tested. However, R. pickettii and four environmental Ralstonia isolates also yielded amplicons. The Ral_fliC PCR products obtained with 12 strains (R. solanacearum, R. pickettii, and environmental isolates) were sequenced. By sequence alignment, Rsol_fliC primers specific for R. solanacearum were designed. With this primer system, a specific 400-bp PCR product was obtained from all 82 strains of R. solanacearum tested. Six strains of R. pickettii and several closely related environmental isolates yielded no PCR product; however, a product was obtained with one Pseudomonas syzygii strain. A GC-clamped 400-bp fliC product could be separated in denaturing gradient gels and allowed us to distinguish P. syzygii from R. solanacearum. The Rsol_fliC PCR system was applied to detect R. solanacearum in soil. PCR amplification, followed by Southern blot hybridization, allowed us to detect about one target DNA molecule per PCR, which is equivalent to 103 CFU g of bulk soil−1. The system was applied to survey soils from different geographic origins for the presence of R. solanacearum.

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

1993 ◽  
Vol 39 (9) ◽  
pp. 1927-1933 ◽  
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.

scholarly journals Shifts in Abundance and Diversity of Mobile Genetic Elements after the Introduction of Diverse Pesticides into an On-Farm Biopurification System over the Course of a Year

2014 ◽  
Vol 80 (13) ◽  
pp. 4012-4020 ◽  
Author(s):  
Simone Dealtry ◽  
Peter N. Holmsgaard ◽  
Vincent Dunon ◽  
Sven Jechalke ◽  
Guo-Chun Ding ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Mobile Genetic Elements ◽  
Replication Initiation ◽  
Genetic Elements ◽  
Class 1 ◽  
Genes Encoding ◽  
Abundance And Diversity ◽  
And Shifts

ABSTRACTBiopurification systems (BPS) are used on farms to control pollution by treating pesticide-contaminated water. It is assumed that mobile genetic elements (MGEs) carrying genes coding for enzymes involved in degradation might contribute to the degradation of pesticides. Therefore, the composition and shifts of MGEs, in particular, of IncP-1 plasmids carried by BPS bacterial communities exposed to various pesticides, were monitored over the course of an agricultural season. PCR amplification of total community DNA using primers targeting genes specific to different plasmid groups combined with Southern blot hybridization indicated a high abundance of plasmids belonging to IncP-1, IncP-7, IncP-9, IncQ, and IncW, while IncU and IncN plasmids were less abundant or not detected. Furthermore, the integrase genes of class 1 and 2 integrons (intI1,intI2) and genes encoding resistance to sulfonamides (sul1,sul2) and streptomycin (aadA) were detected and seasonality was revealed. Amplicon pyrosequencing of the IncP-1trfAgene coding for the replication initiation protein revealed high IncP-1 plasmid diversity and an increase in the abundance of IncP-1β and a decrease in the abundance of IncP-1ε over time. The data of the chemical analysis showed increasing concentrations of various pesticides over the course of the agricultural season. As an increase in the relative abundances of bacteria carrying IncP-1β plasmids also occurred, this might point to a role of these plasmids in the degradation of many different pesticides.

scholarly journals The use of cytochrome b and ryanodine polymorphism to identify DNA of animal and human origin

2019 ◽  
Author(s):  
Małgorzata Natonek-Wiśniewska ◽  
Anna Radko
Keyword(s):  
Cytochrome B ◽  
Red Deer ◽  
Dna Analysis ◽  
Roe Deer ◽  
Restriction Analysis ◽  
Dna Profile ◽  
Applied Method ◽  
Gene Coding ◽  
Pcr Product ◽  
Pcr Products

The aim of this study was to determine a match between DNA recovered from evidence material, such as knocked down red deer, and from comparative material in form of two brown traces on the bonnet of a car driven by a person suspected of knocking down the animal. The spots coming from the car provided no DNA profile, which questioned that they originated from a red deer and ruled out performance of a comparative DNA analysis. For this reason, the material obtained from the blood smear was analyzed for species identification. The method applied can discriminate between cattle, red deer and roe deer based on restriction analysis (Tsp509I) of PCR product (195bp), obtained by amplifying a fragment of the cytochrome b coding gene. Because the obtained restriction profile confirmed the match with red deer DNA for one trace, and in the second case ruled out that the biological traces originated from the species mentioned above, the PCR products were subjected to sequencing. In both cases, 195bp PCR products that were 98% homologous with red deer DNA sequence-NC_007704.2-trace1 and with the gene coding for the human ryanodine receptor-NC_008799.2-trace2. The quantity and quality of DNA obtained from the traces collected from the car bonnet did not allow confirmation of the involvement of a specific animal in the event, but the applied method made it possible to determine the species from which the obtained traces originated. Furthermore, the applied method, which was used earlier to determine cervine DNA, was successfully used to detect human DNA.

Enzymatic amplification of mini-exon-derived RNA gene spacers of Leishmania donovani: primers and probes for DNA diagnosis

Parasitology ◽  
1993 ◽  
Vol 107 (5) ◽  
pp. 509-517 ◽  
Author(s):  
Q. Hassan ◽  
A. Ghosh ◽  
S. S. Ghosh ◽  
M. Gupta ◽  
D. Basu ◽  
...  
Keyword(s):  
Leishmania Donovani ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Dna Diagnosis ◽  
Intergenic Spacers ◽  
Kala Azar ◽  
Pcr Product ◽  
Pcr Products ◽  
Dilution Experiments ◽  
Skin Biopsies

SUMMARYThe multicopy mini-exon-derived RNA (med RNA) locus of Leishmania donovani was enzymatically amplified by the polymerase chain reaction (PCR). The major 180 bp PCR product contained conserved med RNA gene sequences flanking the variable intergenic spacer from the med RNA gene tandem repeat. The oligonucleotide primers cross-reacted with other Leishmania species. In serial dilution experiments, positivity in the PCR assay was observed down to the genomic DNA equivalent of less than a single Leishmania cell. When the major PCR products from Indian L. donovani isolates were cloned and used as probes in dot hybridization analyses, they discriminated between L. donovani and L. amazonensis, L. major and L. infantum under high stringency conditions. DNA from spleen biopsies and blood samples of confirmed kala azar patients was positive, as were two skin biopsies from patients with post-kala azar dermal leishmaniasis (PKDL). These observations demonstrate that PCR amplification of med RNA intergenic spacers is sufficiently sensitive for clinical diagnosis of kala azar and PKDL, and furthermore, that cloned intergenic spacer probes may be useful for identification and classification of L. donovani.

scholarly journals Detection and Diversity of Expressed Denitrification Genes in Estuarine Sediments after Reverse Transcription-PCR Amplification from mRNA

2002 ◽  
Vol 68 (10) ◽  
pp. 5017-5025 ◽  
Author(s):  
Balbina Nogales ◽  
Kenneth N. Timmis ◽  
David B. Nedwell ◽  
A. Mark Osborn
Keyword(s):  
Reverse Transcription ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Estuarine Sediments ◽  
Rt Pcr ◽  
Sediment Samples ◽  
Reverse Transcription Pcr ◽  
Nitrogen Inputs ◽  
Denitrification Genes

ABSTRACT The expression of five denitrification genes coding for two nitrate reductases (narG and napA), two nitrite reductases (nirS and nirK), and nitrous oxide reductase (nosZ) was analyzed by reverse transcription (RT)-PCR of mRNA extracted from two sediment samples obtained in the River Colne estuary (United Kingdom), which receives high nitrogen inputs and for which high denitrification rates have been observed. The presence of all five genes in both sediment samples was confirmed by PCR amplification from extracted DNA prior to analysis of gene expression. Only nirS and nosZ mRNAs were detected; nirS was detected directly as an RT-PCR amplification product, and nosZ was detected following Southern blot hybridization. This indicated that active expression of at least the nirS and nosZ genes was occurring in the sediments at the time of sampling. Amplified nirS RT-PCR products were cloned and analyzed by sequencing, and they were compared with amplified nirS gene sequences from isolates obtained from the same sediments. A high diversity of nirS sequences was observed. Most of the cloned nirS sequences retrieved were specific to one site or the other, which underlines differences in the compositions of the bacterial communities involved in denitrifrification in the two sediments analyzed.

scholarly journals A Simple PCR Method for Rapid Genotype Analysis ofMycobacterium ulcerans

2000 ◽  
Vol 38 (4) ◽  
pp. 1482-1487 ◽  
Author(s):  
Timothy Stinear ◽  
John K. Davies ◽  
Grant A. Jenkin ◽  
Françoise Portaels ◽  
Bruce C. Ross ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Geographic Region ◽  
Mycobacterium Ulcerans ◽  
Geographic Origins ◽  
Pcr Products ◽  
Total Dna ◽  
Pcr Method ◽  
Clonal Population Structure ◽  
Tissue Specimens ◽  
First Time

Two high-copy-number insertion sequences, IS2404 and IS2606, were recently identified in Mycobacterium ulcerans and were shown by Southern hybridization to possess restriction fragment length polymorphism between strains from different geographic origins. We have designed a simple genotyping method that captures these differences by PCR amplification of the region between adjacent copies of IS2404 and IS2606. We have called this system 2426 PCR. The method is rapid, reproducible, sensitive, and specific for M. ulcerans, and it has confirmed previous studies suggesting a clonal population structure ofM. ulcerans within a geographic region. M. ulcerans isolates from Australia, Papua New Guinea, Malaysia, Surinam, Mexico, Japan, China, and several countries in Africa were easily differentiated based on an array of 4 to 14 PCR products ranging in size from 200 to 900 bp. Numerical analysis of the banding patterns suggested a close evolutionary link between M. ulceransisolates from Africa and southeast Asia. The application of 2426 PCR to total DNA, extracted directly from M. ulcerans-infected tissue specimens without culture, demonstrated the sensitivity and specificity of this method and confirmed for the first time that both animal and human isolates from areas of endemicity in southeast Australia have the same genotype.

Chromosome Rearrangement with No Apparent Gene Mutation in Familial Adenomatous Polyposis and Hepatocellular Neoplasia

2002 ◽  
Vol 5 (1) ◽  
pp. 69-75 ◽  
Author(s):  
Jean-Pierre de Chadarévian ◽  
Stephen Dunn ◽  
J. Jeffrey Malatack ◽  
Arupa Ganguly ◽  
Uwe Blecker ◽  
...  
Keyword(s):  
Familial Adenomatous Polyposis ◽  
Chromosome Rearrangement ◽  
Position Effect ◽  
Pcr Amplification ◽  
Adenomatous Polyposis ◽  
Gene Coding ◽  
Pcr Products ◽  
Hepatic Neoplasia ◽  
Polymerase Chain ◽  
Mexican Family

We have identified a constitutional inversion in chromosome 5 associated with familial adenomatous polyposis in three generations of a Mexican family. Two of three siblings developed hepatic neoplasia in infancy. The gene truncation assay failed to demonstrate a truncated protein in the segment harboring the adenomatous polyposis coli (APC) genes. Polymerase chain reaction (PCR) amplification of APC gene coding exons and sequencing of PCR products did not reveal any significant mutation. The data suggest that in this family, the phenotype may be the result of a “position effect.”

Pediococcus parvulus gtf Gene Encoding the GTF Glycosyltransferase and Its Application for Specific PCR Detection of β-d-Glucan–Producing Bacteria in Foods and Beverages

2006 ◽  
Vol 69 (1) ◽  
pp. 161-169 ◽  
Author(s):  
MARIA LAURA WERNING ◽  
IDOIA IBARBURU ◽  
MARIA TERESA DUEÑAS ◽  
ANA IRASTORZA ◽  
JESÚS NAVAS ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Oenococcus Oeni ◽  
Alcoholic Beverages ◽  
Gene Encoding ◽  
Specific Pcr ◽  
Genomic Locations

Exopolysaccharide production by lactic acid bacteria is beneficial in the dairy and oat-based food industries and is used to improve the texture of the fermented products. However, β-D-glucan–producing bacteria are considered spoilage microorganisms in alcoholic beverages because their secreted exopolysaccharides alter the viscosity of cider, wine, and beer, rendering them unpalatable. The plasmidic glycosyltransferase (gtf) gene of the Pediococcus parvulus 2.6 strain isolated from ropy cider has been cloned and sequenced, and its GTF product was functionally expressed in Streptococcus pneumoniae. The GTF protein, which has glycosyltransferase activity, belongs to the COG1215 membrane-bound glycosyltransferase family, and agglutination tests revealed that the enzyme enables S. pneumoniae to synthesize β-D-glucan. PCR amplification and Southern blot hybridization showed that the gtf gene is also present at different genomic locations in the β-d-glucan producers Lacto-bacillus diolivorans G77 and Oenococcus oeni I4 strains, also isolated from ropy cider. A PCR assay has been developed for the detection of exopolysaccharide-producing bacteria. Forward and reverse primers, included respectively in the coding sequences of the putative glycosyltransferase domain and the fifth trans-membrane segment of the GTF, were designed. Analysis of 76 ropy and nonropy lactic acid bacteria validated the method for specific detection of β-d-glucan homopolysaccharide producer Pediococcus, Lactobacillus, and Oenococcus strains. The limit of the assay in cider was 3 × 102 CFU/ml. This molecular method can be useful for the detection of ropy bacteria in cider before spoilage occurs, as well as for isolation of new exopolysaccharide-producing strains of industrial interest.

scholarly journals Differences in Virulence and Genomic Features of Strains of ‘Candidatus Phytoplasma mali’, the Apple Proliferation Agent

2007 ◽  
Vol 97 (8) ◽  
pp. 964-970 ◽  
Author(s):  
Erich Seemüller ◽  
Bernd Schneider
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Southern Blot Hybridization ◽  
Apple Proliferation ◽  
Candidatus Phytoplasma Mali ◽  
Polymerase Chain

Root and shoot samples from 24 symptomatic or nonsymptomatic apple trees infected with ‘Candidatus Phytoplasma mali’ were collected at different locations in Germany and France and used to inoculate rootstock M11 top grafted with cv. Golden Delicious. Inoculated trees were monitored over a 12-year period for apple proliferation (AP) symptoms and categorized as not or slightly, moderately, or severely affected. Based on symptomatology, the phytoplasma strains were defined as being avirulent to mildly, moderately, or highly virulent. Determination of phytoplasma titers by quantitative polymerase chain reaction (PCR) with DNA from roots revealed similar phytoplasma concentrations in all virulence groups. Molecular characterization of the strains by differential PCR amplification with five sets of primers resulted in 13 profiles. Six strains that were maintained in periwinkle and tobacco were molecularly characterized in more detail. The genome sizes of these strains as determined by pulsed-field gel electrophoresis using yeast chromosomes as size references ranged between 640 and 680 kb. Cleavage of the chromosome with the rare cutting restriction enzymes ApaI, BamHI, BssHII, MluI, and SmaI resulted in macro fragment patterns distinctly different in all strains. Similar results were obtained by Southern blot hybridization with three probes derived from strain AT. Differential PCR amplification at an annealing temperature of 52°C using eight primer pairs derived from strain AT revealed heterogeneity of target sequences among all strains. Based on these results, there is considerable variability in virulence and genomic traits in ‘Ca. P. mali’. However, correlations between molecular markers and virulence or phytoplasma titer could not be identified.

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