scholarly journals Molecular Characterization, Intra-Species Diversity and Abundance of Freshwater Plesiomonas shigelloides Isolates

2020 ◽  
Vol 8 (7) ◽  
pp. 1081
Author(s):  
Temitope Ekundayo ◽  
Anthony Okoh
Keyword(s):  
Species Diversity ◽  
Pattern Analysis ◽  
Similarity Index ◽  
Computer Assisted ◽  
Neighbor Joining ◽  
Eastern Cape ◽  
Plesiomonas Shigelloides ◽  
Specific Pcr ◽  
Species Specific ◽  
Pcr Techniques

Molecular signatures of Plesiomonas shigelloides strain specific to pathogenic and nonpathogenic variants are not well established till present. There is a need for intra-species barcoding of P. shigelloides to aid infection control. This study aims at characterizing and assessing intra-species diversity and abundance of P. shigelloides isolated from three freshwaters in the Eastern Cape Province. The study used a Plesiomonas-specific PCR to characterize the isolates. Intra-species (dis)similarities were assessed using ERIC-PCR and (GTG)5-PCR techniques. The DNA fingerprints produced were electrophoresed, digitized, and documented via computer-assisted pattern analysis. The fingerprints were analyzed using neighbor-joining clustering (NJC) based on Euclidean similarity index. Results revealed 80%, 83.64%, and 80% of the water samples from Tyhume, Kat, and Kubusie rivers, respectively, positive for P. shigelloides isolation. The prevalence of P. shigelloides from sites ranged from 13.5% to 88.9%. NJC delineated 48 isolates to 8 clades (ERIC-fingerprints) and 34 isolates into 7 clades ((GTG)5-fingerprints). The relative abundance of unique strains ranged from 6.3% to 22.9% via the two methods. Both fingerprinting approaches have strain-differentiating potential for P. shigelloides, however ERIC-PCR possessed higher resolution (D = 37.46) advantage over (GTG)5-PCR (D = 29.64). In conclusion, the study achieved intra-species diversity and abundance of P. shigelloides from aquatic milieu and provide further opportunity for intra-species-specific barcoding.

scholarly journals Species-Specific PCR as a Tool for the Identification of Burkholderia gladioli

2000 ◽  
Vol 38 (1) ◽  
pp. 282-285
Author(s):  
Paul W. Whitby ◽  
Lauren C. Pope ◽  
Karen B. Carter ◽  
John J. LiPuma ◽  
Terrence L. Stull
Keyword(s):  
Cystic Fibrosis ◽  
Sensitivity And Specificity ◽  
Burkholderia Cepacia ◽  
Bacterial Species ◽  
23S Rrna ◽  
Computer Assisted ◽  
Accurate Identification ◽  
Specific Pcr ◽  
Burkholderia Gladioli ◽  
Species Specific

ABSTRACT Burkholderia gladioli colonizes the respiratory tracts of patients with cystic fibrosis and chronic granulomatous disease. However, due to the high degree of phenotypic similarity between this species and closely related species in the Burkholderia cepacia complex, accurate identification is difficult. Incorrect identification of these species may have serious repercussions for the management of patients with cystic fibrosis. To develop an accurate procedure for the identification of B. gladioli , a molecular method to discriminate between this species and other species commonly isolated from the sputa of patients with cystic fibrosis was investigated. The 23S ribosomal DNA was cloned from several clinical isolates of B. gladioli , and the nucleotide sequence was determined. Computer-assisted sequence comparisons indicated four regions of the 23S rRNA specific for this species; these regions were used to design three primer pairs for species-specific PCR. Two of the primer pairs showed 100% sensitivity and specificity for B. gladioli when tested against a panel of 47 isolates comprising 19 B. gladioli isolates and 28 isolates of 16 other bacterial species. One of the primer pairs was further assessed for species specificity by using a panel of 102 isolates obtained from the Burkholderia cepacia Research Laboratory and Repository. The species-specific PCR was positive for 70 of 74 isolates of B. gladioli and was negative for all other bacterial species examined. Overall, this primer pair displayed a sensitivity and specificity of 96% (89 of 93) and 100%, respectively. These data demonstrate the potential of species-specific PCR for the identification of B. gladioli .

scholarly journals Development of Species-Specific PCR Techniques for the Detection of Tortoise Herpesvirus

2001 ◽  
Vol 13 (6) ◽  
pp. 513-516 ◽  
Author(s):  
Masaru Murakami ◽  
Chikako Matsuba ◽  
Yumi Une ◽  
Yasuo Nomura ◽  
Hideo Fujitani
Keyword(s):  
Specific Pcr ◽  
Species Specific ◽  
Pcr Techniques

scholarly journals Detection of Phomopsis sclerotioides in Commercial Cucurbit Field Soil by Nested Time-Release PCR

Plant Disease ◽  
2012 ◽  
Vol 96 (4) ◽  
pp. 515-521 ◽  
Author(s):  
T. Ito ◽  
S. Fuji ◽  
E. Sato ◽  
Y. Iwadate ◽  
T. Toda ◽  
...  
Keyword(s):  
Minimum Concentration ◽  
Disease Symptoms ◽  
Specific Pcr ◽  
Phomopsis Sclerotioides ◽  
Fluorescent Pcr ◽  
Pcr Products ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Species Specific ◽  
Pcr Techniques

A polymerase chain reaction (PCR)-based molecular method to detect Phomopsis sclerotioides in soil was developed using a species-specific primer pair. To improve sensitivity of the detection, three PCR techniques were used; namely, nested PCR using the primer pair internal transcribed spacer (ITS)1 and ITS4, time-release PCR using two different DNA polymerases (recombinant Taq and AmpliTaq Gold), and fluorescent PCR to obtain fluorescent-labeled PCR products that can be analyzed by capillary electrophoresis. The latter two techniques were combined and termed nested time-release fluorescent (NTRF)-PCR. The minimum concentration of DNA required to obtain species-specific PCR products successfully was 50 fg/μg. Using the NTRF-PCR method, the fungus could be detected in sandy soil that was artificially infested at a density of 10 CFU/g. The pathogen was detected in most soil samples collected from commercial cucumber fields in which visual disease symptoms had appeared, and even in samples collected from fields where visual disease symptoms had not appeared. To prevent the invasion and establishment of root-inhabiting pathogens such as P. sclerotioides, it is critical to detect the fungus in soil as soon as possible after its introduction into a cucumber-growing region.

scholarly journals Survey on Perkinsus Species in Manila Clam Ruditapes philippinarum in Korean Waters Using Species-Specific PCR

2017 ◽  
Vol 52 (4) ◽  
pp. 202-205 ◽  
Author(s):  
Hyun-Sil Kang ◽  
Hyun-Sung Yang ◽  
Kimberly S. Reece ◽  
Young-Ghan Cho ◽  
Hye-Mi Lee ◽  
...  
Keyword(s):  
Ruditapes Philippinarum ◽  
Manila Clam ◽  
Korean Waters ◽  
Specific Pcr ◽  
Species Specific

scholarly journals Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

2003 ◽  
Vol 69 (11) ◽  
pp. 6380-6385 ◽  
Author(s):  
R. Temmerman ◽  
L. Masco ◽  
T. Vanhoutte ◽  
G. Huys ◽  
J. Swings
Keyword(s):  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Temporal Changes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Gradient Gel Electrophoresis ◽  
Species Specific ◽  
Taxonomic Characterization

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

scholarly journals Eutypella parasitica and Other Frequently Isolated Fungi in Wood of Dead Branches of Young Sycamore Maple (Acer pseudoplatanus) in Slovenia

Forests ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 467 ◽  
Author(s):  
Ana Brglez ◽  
Barbara Piškur ◽  
Nikica Ogris
Keyword(s):  
Species Diversity ◽  
Fungal Community ◽  
Sampling Site ◽  
Outer Part ◽  
Similarity Index ◽  
Asymptomatic Infection ◽  
Fungal Species ◽  
Acer Pseudoplatanus ◽  
Pure Cultures ◽  
Sycamore Maple

Eutypella parasitica R.W. Davidson and R.C. Lorenz is the causative agent of Eutypella canker of maple, a destructive disease of maples in Europe and North America. The fungus E. parasitica infects the trunk through a branch stub or bark wound. Because the fungal community may have an impact on infection and colonization by E. parasitica, the composition of fungi colonizing wood of dead branches of sycamore maple (Acer pseudoplatanus L.) was investigated in five sampling sites in Slovenia. Forty samples from each sampling site were collected between the November 2017 and March 2018 period. Isolations were made from the wood in the outer part of dead branches and from discoloured wood in the trunk that originated from a dead branch. Pure cultures were divided into morphotypes, and one representative culture per morphotype was selected for further molecular identification. From a total of 2700 cultured subsamples, 1744 fungal cultures were obtained, which were grouped into 212 morphotypes. The investigated samples were colonized by a broad spectrum of fungi. The most frequently isolated species were Eutypa maura (Fr.) Sacc., Eutypa sp. Tul. and C. Tul., Fusarium avenaceum (Fr.) Sacc., Neocucurbitaria acerina Wanas., Camporesi, E.B.G. Jones and K.D. Hyde and E. parasitica. In this study, we distinguished species diversity and the fungal community. There were no significant differences in the diversity of fungal species between the five sampling sites, and branch thickness did not prove to be a statistically significant factor in fungal species diversity. Nevertheless, relatively low Jaccard similarity index values suggested possible differences in the fungal communities from different sampling sites. This was confirmed by an analysis of similarities, which showed that the isolated fungal community distinctly differed between the five sampling sites and between the different isolation sources. Eutypella parasitica was isolated from all five investigated sampling sites, although Eutypella cankers were observed in only three sampling sites, indicating the possibility of asymptomatic infection.

scholarly journals First Report of Orange Rust of Sugarcane Caused by Puccinia kuehnii in Colombia

Plant Disease ◽  
2012 ◽  
Vol 96 (1) ◽  
pp. 143-143 ◽  
Author(s):  
M. Cadavid ◽  
J. C. Ángel ◽  
J. I. Victoria
Keyword(s):  
Internal Transcribed Spacer ◽  
Pcr Primers ◽  
The United States ◽  
Optical Microscope ◽  
First Report ◽  
5.8S Rdna ◽  
Specific Pcr ◽  
Plant Pathol ◽  
Species Specific ◽  
New Cultivars

Symptoms of sugarcane orange rust were first observed in July 2010 on sugarcane (interspecific hybrid of Saccharum L. species) cv. CC 01-1884 planted in the La Cabaña Sugar Mill, Puerto Tejada, Colombia. Morphological features of uredinial lesions and urediniospores inspected with an optical microscope and scanning electron microscopy were distinct from common rust of sugarcane caused by Puccinia melanocephala Syd. & P. Syd., revealing spores identical morphologically to those described for the fungus P. kuehnii (Kruger) E. Butler, causal agent of sugarcane orange rust (1,3). Uredinial lesions were orange and distinctly lighter in color than pustules of P. melanocephala. Urediniospores were orange to light cinnamon brown, mostly ovoid to pyriform, variable in size (27.3 to 39.2 × 16.7 to 21.2 μm), with pronounced apical wall and moderately echinulate with spines evenly distributed. Paraphyses, telia, and teliospores were not observed. Species-specific PCR primers designed from the internal transcribed spacer (ITS)1, ITS2, and 5.8S rDNA regions of P. melanocephala and P. kuehnii were used to differentiate the two species (2). The primers Pm1-F and Pm1-R amplified a 480-bp product from P. melanocepahala DNA in leaf samples with symptoms of common rust. By contrast, the primers Pk1-F and Pk1-R generated a 527-bp product from presumed P. kuehnii DNA in leaf samples with signs of orange rust, confirming the identity as P. kuehnii. The Centro de Investigación de la Caña de Azúcar de Colombia (Cenicaña) started a survey of different cultivars in nurseries and experimental and commercial fields in the Cauca River Valley and collected leaf samples for additional analyses. Experimental cvs. CC 01-1884, CC 01-1866, and CC 01-1305 were found to be highly susceptible to orange rust and were eliminated from regional trials, whereas commercial cvs. CC 85-92 and CC 84-75, the most widely grown cultivars, were resistant. With the discovery of orange rust of sugarcane in Colombia, Cenicaña has incorporated orange rust resistance in the selection and development of new cultivars. To our knowledge, this is the first report of P. kuehnii on sugarcane in Colombia. Orange rust has also been reported from the United States, Cuba, Mexico, Guatemala, Nicaragua, El Salvador, Costa Rica, Panama, Ecuador, and Brazil. References: (1) J. C. Comstock et al. Plant Dis. 92:175, 2008. (2) N. C. Glynn et al. Plant Pathol. 59:703, 2010. (3) E. V. Virtudazo et al. Mycoscience 42:167, 2001.

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