scholarly journals A multi-tool approach to assess microalgal diversity in lichens: isolation, Sanger sequencing, HTS and ultrastructural correlations

2018 ◽  
Vol 50 (1) ◽  
pp. 123-138 ◽  
Author(s):  
Arántzazu MOLINS ◽  
Patricia MOYA ◽  
Francisco J. GARCÍA-BREIJO ◽  
José REIG-ARMIÑANA ◽  
Eva BARRENO
Keyword(s):  
Sanger Sequencing ◽  
454 Pyrosequencing ◽  
Genetic Identification ◽  
Specific Pcr ◽  
Transmission Electron ◽  
Ultrastructural Characterization ◽  
Polymerase Chain ◽  
Specific Pcr Primer ◽  
The Relationship ◽  
Pcr Primer

AbstractLichen thalli represent the most conspicuous examples of fungal-algal interactions. Studies that describe phycobiont diversity within entire thalli are based mainly on Sanger sequencing. In some lichen species, this technique could underestimate the intrathalline coexistence of multiple microalgae. In this study different multi-tool approaches were applied to two lichen taxa, Circinaria hispida and Flavoparmelia soredians, to detect algal coexistence. Here, we combined Sanger sequencing, a specific polymerase chain reaction (PCR) primer, 454-pyrosequencing, phycobiont isolation and ultrastructural characterization. Furthermore, we compared pyrenoid ultrastructural features of lichenized phycobionts with microalgae isolated in culture. An improved methodology was used to isolate and propagate phycobionts which, in combination with fast genetic identification, resulted in a considerable reduction in time and cost to complete the process. This isolation method, coupled with a specific PCR primer, allowed for the detection of coexisting algae in C. hispida (four Trebouxia lineages). 454-pyrosequencing detected only a fraction of such diversity, while Sanger sequencing identified only the primary phycobiont. Ultrastructural features of the isolated algae were observed by transmission electron microscopy; the maintenance of the pyrenoid characteristics suggested the existence of different Trebouxia lineages. In F. soredians a single Trebouxia lineage was identified using all these approaches.In cases of lichens with algal coexistence, a combination of different molecular and ultrastructural approaches may be required to reveal the underlying algal diversity within a single thallus. The approach proposed in this study provides information about the relationship between molecular and ultrastructural data, and represents an improvement in the delimitation of taxonomic features which is needed to recognize intrathalline Trebouxia diversity.

scholarly journals A New ‘Candidatus Liberibacter’ Species Associated with Diseases of Solanaceous Crops

Plant Disease ◽  
2009 ◽  
Vol 93 (3) ◽  
pp. 208-214 ◽  
Author(s):  
Lia W. Liefting ◽  
Paul W. Sutherland ◽  
Lisa I. Ward ◽  
Kerry L. Paice ◽  
Bevan S. Weir ◽  
...  
Keyword(s):  
16S Rrna ◽  
Pcr Primers ◽  
23S Rrna ◽  
Rrna Gene ◽  
High Identity ◽  
Specific Pcr ◽  
Transmission Electron ◽  
Candidatus Liberibacter ◽  
Polymerase Chain ◽  
Specific Pcr Primer

A new disease of glasshouse-grown tomato and pepper in New Zealand has resulted in plant decline and yield loss. Affected plants are characterized by spiky, chlorotic apical growth, curling or cupping of the leaves, and overall stunting. Transmission electron microscopy revealed the presence of phloem-limited bacterium-like organisms in symptomatic plants. The strategy used to identify the bacterium involved using specific prokaryote polymerase chain reaction (PCR) primers in combination with universal 16S rRNA primers. Sequence analysis of the 16S rRNA gene, the 16S/23S rRNA spacer region, and the rplKAJL-rpoBC operon revealed that the bacterium shared high identity with ‘Candidatus Liberibacter’ species. Phylogenetic analysis showed that the bacterium is distinct from the three citrus liberibacter species previously described and has been named ‘Candidatus Liberibacter solanacearum’. This is the first report of a liberibacter naturally infecting a host outside the Rutaceae family. A specific PCR primer pair was developed for its detection.

scholarly journals An improved allele-specific PCR primer design method for SNP marker analysis and its application

Plant Methods ◽  
2012 ◽  
Vol 8 (1) ◽  
pp. 34 ◽  
Author(s):  
Jing Liu ◽  
Shunmou Huang ◽  
Meiyu Sun ◽  
Shengyi Liu ◽  
Yumei Liu ◽  
...  
Keyword(s):  
Design Method ◽  
Primer Design ◽  
Snp Marker ◽  
Specific Pcr ◽  
Marker Analysis ◽  
Allele Specific ◽  
Specific Pcr Primer ◽  
Allele Specific Pcr ◽  
Pcr Primer

Molecular identification of zoonotic hookworm species in dog faeces from Tanzania

2018 ◽  
Vol 93 (3) ◽  
pp. 313-318 ◽  
Author(s):  
A. Merino-Tejedor ◽  
P. Nejsum ◽  
E.M. Mkupasi ◽  
M.V. Johansen ◽  
Annette Olsen
Keyword(s):  
Sanger Sequencing ◽  
Molecular Techniques ◽  
Health Concern ◽  
Specific Pcr ◽  
Molecular Approaches ◽  
Faecal Samples ◽  
Pcr Rflp ◽  
Veterinary Importance ◽  
Polymerase Chain ◽  
Species Specific

AbstractThe presence and distribution of various species of canine hookworms in Africa are poorly known. The main objective of this study, therefore, was to identify the hookworm species present in canine faecal samples from Morogoro, Tanzania, using molecular techniques. Faecal samples from 160 local dogs were collected and hookworm positive samples processed to recover larvae for further molecular characterization. DNA was extracted from pools of larvae from individual samples (n = 66), which were analysed subsequently using two different molecular approaches, polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) and species-specific PCR coupled with Sanger sequencing. The PCR-RFLP technique detected only the presence of the ubiquitousAncylostoma caninumin the 66 samples. However, by species-specific PCR coupled with Sanger sequencing we identified ten samples withA. braziliense, two withUncinaria stenocephalaand five withA. ceylanicum. Thus, all four known species of canine hookworms were identified in Morogoro, Tanzania. To our knowledge this is the first report of the detection of the presence ofU. stenocephalaandA. ceylanicumin Africa using molecular techniques. In addition to their veterinary importance, canine hookworms have zoonotic potential and are of public health concern.

Updating specific PCR primer for detection of Cryptocaryon irritans from reared Larimichthys polyactis

2021 ◽  
Vol 223 ◽  
pp. 108081
Author(s):  
Xiao Xie ◽  
Chao Zheng ◽  
Aysha Zahid ◽  
Jindong Kong ◽  
Bushra ◽  
...  
Keyword(s):  
Larimichthys Polyactis ◽  
Cryptocaryon Irritans ◽  
Specific Pcr ◽  
Specific Pcr Primer ◽  
Pcr Primer

Investigation of the relationship between CE cyst characteristics and genetic diversity of Echinococcus granulosus sensu lato in humans from Turkey

Parasitology ◽  
2020 ◽  
Vol 147 (14) ◽  
pp. 1712-1717
Author(s):  
Serra Örsten ◽  
Türkmen Çiftçi ◽  
Aynur Azizova ◽  
Gökhan Yüce ◽  
Aycan Uysal ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Echinococcus Granulosus ◽  
Rural Areas ◽  
Sensu Stricto ◽  
Genetic Identification ◽  
Cyst Volume ◽  
Gene Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
The Relationship ◽  
Echinococcus Granulosus Sensu Lato

AbstractCystic echinococcosis (CE) is one of the most common zoonotic diseases worldwide, particularly in rural areas. This study aimed at the identification of the genotype/species belonging to Echinococcus granulosus sensu lato (s.l.) specimens in retrieved percutaneously from the human host and to investigate their relationship with cyst characteristics. The genetic identification of cyst material was performed by mt-CO1 gene polymerase chain reaction, and confirmed via sequencing. A total of 110 CE cysts were identified as E. granulosus s.l. In detail, 104 belonged to E. granulosus sensu stricto (G1 and G3) and six isolates were in the E. canadensis cluster (G6/7). All clusters were tested for the relationship between demographics, cyst features and genetic diversity. The relationship between genetic variation and certain clinical characteristics such as cyst volume and location were statistically significant for G6/7 cluster. Further studies are required with a larger sample set to investigate the relationship between the genetic variability of E. granulosus s.l. and cyst features.

scholarly journals PCRTiler: automated design of tiled and specific PCR primer pairs

2010 ◽  
Vol 38 (suppl_2) ◽  
pp. W308-W312 ◽  
Author(s):  
Alain L. Gervais ◽  
Maud Marques ◽  
Luc Gaudreau
Keyword(s):  
Automated Design ◽  
Specific Pcr ◽  
Specific Pcr Primer ◽  
Pcr Primer

scholarly journals Comparison of Three Methods for Monitoring Populations of Different Genotypes of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens in the Rhizosphere

2002 ◽  
Vol 92 (2) ◽  
pp. 129-137 ◽  
Author(s):  
Blanca B. Landa ◽  
Henricus A. E. de Werd ◽  
Brian B. McSpadden Gardener ◽  
David M. Weller
Keyword(s):  
Pseudomonas Fluorescens ◽  
Limit Of Detection ◽  
Root Weight ◽  
Population Densities ◽  
Colony Hybridization ◽  
Specific Pcr ◽  
Hybridization Procedure ◽  
Polymerase Chain ◽  
Endpoint Assay ◽  
The Relationship

Pseudomonas fluorescens strains producing the antibiotic 2,4-diacetylphloroglucinol (DAPG) have biocontrol activity against a broad spectrum of root and seedling diseases. In this study, we determined the effect of genotype on the ability to isolate and quantify introduced 2,4-DAPG producers from the rhizosphere of wheat using three different methods: traditional dilution plating on selective media, colony hybridization followed by polymerase chain reaction (PCR), and phlD-specific PCR-based dilution endpoint assay. Regression analysis of the population densities of 10 2,4-DAPG-producing P. fluorescens, representing five genotypes, determined by the three different methods demonstrated that the relationship was linear (P < 0.001) and the techniques were very similar (i.e., slopes equal to 1.0). The phlD-specific PCR-based assay had a slightly lower limit of detection than the other two methods (log 3.3 versus log 4.0 CFU/g of fresh root weight). With the colony hybridization procedure, we observed that the phlD probe, derived from strain P. fluorescens Q8r1-96, hybridized more strongly to colonies of BOX-PCR genotypes D (strains W2-6, L5.1-96, Q8r1-96, and Q8r2-96) and K (strain F113) compared with strains of genotypes A (Pf-5 and CHA0), B (Q2-87), and L (1M1-96 and W4-4). Colony hybridization alone overestimated the actual densities of some strains, thus requiring an additional PCR step to obtain accurate estimates. In contrast, population densities estimated for three of the bacterial treatments (strains CHA0, W2-6, and Q8r2-96) with the PCR-based assay were significantly (P < 0.041) smaller by 7.6 to 9.2% and 6.4 to 9.4% than population densities detected by the dilution plating and colony hybridization techniques, respectively. In this paper, we discuss the relative advantages of the different methods for detecting 2,4-DAPG producers.

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