Development and Application of a New PCR Method for Detection of Blumeria graminis f. sp. tritici

2018 ◽  
Vol 28 (3) ◽  
pp. 137-146 ◽  
Author(s):  
Adam Kuzdraliński ◽  
Hubert Szczerba ◽  
Anna Kot ◽  
Agnieszka Ostrowska ◽  
Michał Nowak ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Puccinia Triticina ◽  
Blumeria Graminis ◽  
Pyrenophora Tritici Repentis ◽  
Field Samples ◽  
Expected Length ◽  
Pcr Assays ◽  
Primer Sets ◽  
Wheat Leaves ◽  
Species Specific

We developed new PCR assays that target beta-tubulin (<i>TUB2</i>) and 14 alpha-demethylase (<i>CYP51</i>) genes and used them for the species-specific detection of <i>Blumeria graminis</i> f. sp. <i>tritici</i> (<i>Bgt</i>). Based on fungi DNA sequences available in the NCBI (National Center for Biotechnology Information) GenBank database we developed simplex and duplex PCR assays. The specificities of the primer sets were evaluated using environmental samples of wheat leaves collected during the 2015/2016 growing season across Poland. Primer sets<i></i> LidBg17/18 and LidBg21/22 strongly amplified fragments of the expected length for all 67 tested samples. Primer specificity was confirmed using field samples of <i>Zymoseptoria tri­tici</i>, <i>Puccinia triticina</i> (syn.<i> P. recondita</i> f. sp.<i> tritici</i>), <i>P. striiformis</i> f. sp.<i> tritici</i>, and <i>Pyrenophora tritici-repentis</i>.

scholarly journals DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

2015 ◽  
Vol 8 ◽  
pp. MBI.S29736 ◽  
Author(s):  
Kenjiro Nagamine ◽  
Guo-Chiuan Hung ◽  
Bingjie Li ◽  
Shyh-Ching Lo
Keyword(s):  
Safety Concern ◽  
Rapid Development ◽  
High Sensitivity ◽  
High Specificity ◽  
Clostridium Tetani ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Pcr Assays ◽  
Primer Sets ◽  
Species Specific

Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents.

Novel PCR Assays for the Detection of Biological Agents Responsible for Wheat Rust Diseases: Puccinia triticina and Puccinia striiformis f. sp. tritici

2017 ◽  
Vol 27 (5) ◽  
pp. 299-305 ◽  
Author(s):  
Adam Kuzdraliński ◽  
Anna Kot ◽  
Hubert Szczerba ◽  
Agnieszka Ostrowska ◽  
Michał Nowak ◽  
...  
Keyword(s):  
Puccinia Striiformis ◽  
Infected Plant ◽  
Simultaneous Detection ◽  
Puccinia Triticina ◽  
Biological Agents ◽  
Plant Materials ◽  
Rust Diseases ◽  
Pcr Assays ◽  
Primer Sets ◽  
Wheat Rust

The species <i>Puccinia triticina</i> (Pt) and <i>Puccinia striiformis</i> f. sp. <i>tritici </i>(Pst) are devastating cereal pathogens that cause leaf and stripe rust diseases. We developed PCR assays for the species-specific detection of Pt and Pst, 2 biological agents that cause wheat rust disease. For each pathogen, we validated 3 primer sets that target the second largest subunits of the RNA polymerase II (<i>rpb2</i>) and β-tubulin 1 (<i>tub1</i>) genes. The specificities of the primers were verified using naturally infected plant materials with visual symptoms of disease. All primer sets amplified a single DNA fragment of the expected length. The primer sets LidPr15/16, LidPr1/2, and LidPs13/14 were able to detect small amounts of pure fungal DNA with sensitivities of 0.1, 1, and 10 pg/μL, respectively. A sufficient detection limit (1 pg/μL to 5 ng/μL) was observed for all assays when the sensitivity test was performed with host plant DNA. The study also evaluated the simultaneous detection of both rust pathogens, and the multiplex PCR assay generated amplicons of 240 and 144 bp in length for Pts (LidPs9/10) and Pt (LidPr1/2), respectively.

scholarly journals Sensitive and Direct Detection of Heterodera filipjevi in Soil and Wheat Roots by Species-Specific SCAR-PCR Assays

Plant Disease ◽  
2013 ◽  
Vol 97 (10) ◽  
pp. 1288-1294 ◽  
Author(s):  
Huan Peng ◽  
Xiaoli Qi ◽  
Deliang Peng ◽  
Haibo Long ◽  
Xufeng He ◽  
...  
Keyword(s):  
Direct Detection ◽  
Parasitic Nematodes ◽  
Cyst Nematodes ◽  
Specific Sequence ◽  
Wheat Roots ◽  
Sequence Characterized Amplified Region ◽  
Field Samples ◽  
Pcr Assays ◽  
Heterodera Filipjevi ◽  
Species Specific

Cereal cyst nematodes are the most important plant-parasitic nematodes on cereal crops in wheat producing areas of the world. Heterodera filipjevi was first reported in China in 2010. In this study, species-specific sequence characterized amplified region–polymerase chain reaction (SCAR-PCR) assays for detection and identification of H. filipjevi from infected wheat roots and soil were developed. The species-specific primers were designed according to the randomly amplified polymorphic DNA (RAPD) markers amplified with random primer OPK16. A 646-bp specific fragment of sequence was generated, which characterized amplified regions in H. filipjevi. The detection limitation of the PCR assay was as low as 0.125 μl second-stage juvenile (J2) lysate, 3.9 × 10–3 μl adult female lysate, and 10–3 μl cyst lysate. The method was able to detect the various stages (J2, J3, J4, and female) of H. filipjevi, and a single of nematode in 0.5 g of soil. H. filipjevi was detected by the method in two of six field samples, and one of those samples contained a mixed population of H. filipjevi and H. avenae. This study is the first to provide a definitive diagnostic assay for H. filipjevi in wheat roots and soil.

Identification of Meloidogyne incognita, M. javanica and M. arenaria using sequence characterised amplified region (SCAR) based PCR assays

Nematology ◽  
2000 ◽  
Vol 2 (8) ◽  
pp. 847-853 ◽  
Author(s):  
Dorine T.H.M. Donkers-Venne ◽  
Mireille Fargette ◽  
Carolien Zijlstra
Keyword(s):  
Dna Sequences ◽  
Nematode Species ◽  
Life Cycle Stage ◽  
Meloidogyne Arenaria ◽  
Root Knot Nematode ◽  
Rapd Pcr ◽  
Identification Technique ◽  
Length Variant ◽  
Pcr Assays ◽  
Species Specific

AbstractThree randomly amplified polymorphic DNA (RAPD) markers, OPA-12420, OPB-061200 and OPA-01700, species specific to the root-knot nematode species Meloidogyne arenaria, M. incognita and M. javanica respectively, were identified. After sequencing these RAPD-PCR products, longer primers of 18 to 23 nucleotides were designed to complement the terminal DNA sequences of the DNA fragments. This resulted in three pairs of species specific primers that were used to amplify the sequence characterised amplified regions (SCARs). The developed sets of SCAR primers were successfully used in straightforward, fast and reliable PCR assays to identify M. incognita, M. javanica and M. arenaria. The length variant SCAR markers can be amplified from DNA from egg masses, second stage juveniles and females. This species identification technique is therefore independent of the nematode's life cycle stage. Moreover the SCAR-PCR assay was successfully applied using DNA extracts from infested plant material. The method has potential to be optimised for routine practical diagnostic tests facilitating the control of these economically important pest organisms. Identification de Meloigyne incognita, M. javanica et M. arenaria au moyen de l'amplification de régions de séquences caractéristiques (SCAR) par une technique PCR - Trois marqueurs d'ADN polymorphique amplifiée au hasard (RAPD) OPA-12420, OPB-O61200 et OPA-OI700, respectivement spécifiques des espèces de nématodes Meloidogyne arenaria, M. incognita et M. javanica, ont été identifiés. Après le séquençage de ces produits RAPD-PCR, les amorces les plus longues de 18 à 23 nucléotides ont été choisies pour compléter les séquences terminales d'ADN des fragments d'ADN. Cela a conduit à trois paires d'amorces spécifiques de l'espèce, utilisées pour amplifier les régions des séquences caractéristiques (SCAR). Les lots d'amorces SCAR mis au point ont été utilisés avec succès lors d'essais directs, rapides et surs pour identifier M. incognita, M. javanica et M. arenia. Les marqueurs peuvent être amplifiés à partir de l'ADN des masses d'oeufs, des juvéniles de deuxième stade ou des femelles. Cette technique d'identification spécifique est donc indépendante des différents états de développement du nématode. De plus la technique SCAR-PCR a été appliquée avec succès à l'ADN extrait du matériel végétal infesté. Cette méthode présente des potentialités d'amélioration permettant d'envisager des tests pratiques d'identification de routine, facilitant ainsi le contrôle de ces parasites économiquement importants.

scholarly journals Injuriousness of Blumeria graminis and Pyrenophora tritici-repentis in wheat crops and measures of its operation monitoring

2020 ◽  
Vol 176 ◽  
pp. 04008
Author(s):  
Alyona Kekalo ◽  
Vladimir Nemchenko ◽  
Anatoly Filippov
Keyword(s):  
Host Plants ◽  
Blumeria Graminis ◽  
Biological Efficiency ◽  
Operational Control ◽  
Effective Protection ◽  
Pyrenophora Tritici Repentis ◽  
Infectious Origin ◽  
Wheat Leaves ◽  
Favorable Conditions ◽  
Active Substances

Blumeria graminis and Pyrenophora tritici-repentis of wheat are deleterious infections that often require operation monitoring. The probability of mass lesion of crops by phytopathogens is determined primarily by the presence of pathogens' infectious origin, favorable conditions for their development and spread and host plants susceptibility. Protection strategy and tactics should be based on each particular field and conditions of the growing season. Effective protection of wheat from powdery mildew (75 -87%) was provided by fungicides based on 2-3 active substances, especially the drug Falcon (spiroxamine + tebuconazole + triadimenol). The biological fungicide Phytosporin-M (Bacillus subtilis) provided an average biological efficiency of 58% in years with moderate wheat lesion. Operational control of wheat leaves yellow speckle is more advisable to carry out with preparations on the basis of such active substances as “azoxystrobin + epoxyconazole”, “tebuconazole + propiconazole”.

scholarly journals Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Neeraja Punde ◽  
Jennifer Kooken ◽  
Dagmar Leary ◽  
Patricia M. Legler ◽  
Evelina Angov
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Codon Usage ◽  
Dna Sequences ◽  
Structural Integrity ◽  
Host Cells ◽  
Loss Of Function ◽  
Species Specific ◽  
And Function ◽  
The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

Species-diagnostic and species-specific DNA sequences evenly distributed throughout pine and spruce chromosomes

Genome ◽  
2010 ◽  
Vol 53 (10) ◽  
pp. 769-777 ◽  
Author(s):  
Melanie Mehes-Smith ◽  
Paul Michael ◽  
Kabwe Nkongolo
Keyword(s):  
Dna Sequences ◽  
Random Amplified Polymorphic Dna ◽  
Inter Simple Sequence Repeat ◽  
Issr Markers ◽  
Pinus Monticola ◽  
The Family ◽  
Species Specific ◽  
Rapd And Issr ◽  
Specific Sequences

Genome organization in the family Pinaceae is complex and largely unknown. The main purpose of the present study was to develop and physically map species-diagnostic and species-specific molecular markers in pine and spruce. Five RAPD (random amplified polymorphic DNA) and one ISSR (inter-simple sequence repeat) species-diagnostic or species-specific markers for Picea mariana , Picea rubens , Pinus strobus , or Pinus monticola were identified, cloned, and sequenced. In situ hybridization of these sequences to spruce and pine chromosomes showed the sequences to be present in high copy number and evenly distributed throughout the genome. The analysis of centromeric and telomeric regions revealed the absence of significant clustering of species-diagnostic and species-specific sequences in all the chromosomes of the four species studied. Both RAPD and ISSR markers showed similar patterns.

scholarly journals Development of Molecular Markers for Detection of Acidovorax citrulli Strains Causing Bacterial Fruit Blotch Disease in Melon

2019 ◽  
Vol 20 (11) ◽  
pp. 2715 ◽  
Author(s):  
Md. Rafiqul Islam ◽  
Mohammad Rashed Hossain ◽  
Hoy-Taek Kim ◽  
Denison Michael Immanuel Jesse ◽  
Md. Abuyusuf ◽  
...  
Keyword(s):  
Infected Leaf ◽  
Bacterial Fruit Blotch ◽  
Early Disease Detection ◽  
Acidovorax Citrulli ◽  
Seed Health ◽  
Field Samples ◽  
Whole Genomes ◽  
Pathogenesis Related ◽  
Primer Sets ◽  
Inoculation Assay

Acidovorax citrulli (A. citrulli) strains cause bacterial fruit blotch (BFB) in cucurbit crops and affect melon significantly. Numerous strains of the bacterium have been isolated from melon hosts globally. Strains that are aggressively virulent towards melon and diagnostic markers for detecting such strains are yet to be identified. Using a cross-inoculation assay, we demonstrated that two Korean strains of A. citrulli, NIHHS15-280 and KACC18782, are highly virulent towards melon but avirulent/mildly virulent to the other cucurbit crops. The whole genomes of three A. citrulli strains isolated from melon and three from watermelon were aligned, allowing the design of three primer sets (AcM13, AcM380, and AcM797) that are specific to melon host strains, from three pathogenesis-related genes. These primers successfully detected the target strain NIHHS15-280 in polymerase chain reaction (PCR) assays from a very low concentration of bacterial gDNA. They were also effective in detecting the target strains from artificially infected leaf, fruit, and seed washing suspensions, without requiring the extraction of bacterial DNA. This is the first report of PCR-based markers that offer reliable, sensitive, and rapid detection of strains of A. citrulli causing BFB in melon. These markers may also be useful in early disease detection in the field samples, in seed health tests, and for international quarantine purposes.

scholarly journals A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1358
Author(s):  
Brigitte Sigrist ◽  
Jessica Geers ◽  
Sarah Albini ◽  
Dennis Rubbenstroth ◽  
Nina Wolfrum
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Epidemiological Studies ◽  
Virus Species ◽  
Rt Pcr ◽  
P Gene ◽  
Field Samples ◽  
Causative Agents ◽  
Species Specific ◽  
Proventricular Dilatation Disease

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.

scholarly journals Retention of relict satellite DNA sequences in Anemone (Ranunculaceae)

2013 ◽  
Vol 72 (1) ◽  
pp. 1-133 ◽  
Author(s):  
Višnja Besendorfer ◽  
Jelena Mlinarec
Keyword(s):  
Dna Sequences ◽  
Southern Hybridization ◽  
Repeat Unit ◽  
Similar Species ◽  
Genomic Component ◽  
Library Hypothesis ◽  
Species Specific ◽  
Eukaryotic Organisms ◽  
Fish Signal

Abstract Satellite DNAis a genomic component present in virtually all eukaryotic organisms. The turnover of highly repetitive satellite DNAis an important element in genome organization and evolution in plants. Here we study the presence, physical distribution and abundance of the satellite DNAfamily AhTR1 in Anemone. Twenty-two Anemone accessions were analyzed by PCR to assess the presence of AhTR1, while fluorescence in situ hybridization and Southern hybridization were used to determine the abundance and genomic distribution of AhTR1. The AhTR1 repeat unit was PCR-amplified only in eight phylogenetically related European Anemone taxa of the Anemone section. FISH signal with AhTR1 probe was visible only in A. hortensis and A. pavonina, showing localization of AhTR1 in the regions of interstitial heterochromatin in both species. The absence of a FISH signal in the six other taxa as well as weak signal after Southern hybridization suggest that in these species AhTR1 family appears as relict sequences. Thus, the data presented here support the »library hypothesis« for AhTR1 satellite evolution in Anemone. Similar species-specific satellite DNAprofiles in A. hortensis and A. pavonina support the treatment of A. hortensis and A. pavonina as one species, i.e. A. hortensis s.l.

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