scholarly journals Characterization of Colletotrichum Isolates from Tamarillo, Passiflora, and Mango in Colombia and Identification of a Unique Species from the Genus

2003 ◽  
Vol 93 (5) ◽  
pp. 579-587 ◽  
Author(s):  
Lucia Afanador-Kafuri ◽  
Dror Minz ◽  
Marcel Maymon ◽  
Stanley Freeman
Keyword(s):  
Morphological Characterization ◽  
Its2 Region ◽  
Specific Primers ◽  
Molecular Analyses ◽  
Rapd Pcr ◽  
Morphological Criteria ◽  
Polymerase Chain ◽  
Species Specific ◽  
Unique Species

This study was conducted to identify the species of Colletotrichum infecting tamarillo, mango, and passiflora in Colombia and to assess whether cross-infection between host species is occurring. Isolates of Colletotrichum spp. from tamarillo (n = 54), passiflora (n = 26), and mango (n = 15) were characterized by various molecular methods and by morphological criteria. Morphological characterization grouped the tamarillo isolates as C. acutatum and the passiflora and mango isolates as C. gloeosporioides. Species-specific primer analysis was reliable and confirmed grouping of the tamarillo isolates (besides Tom-6) as C. acutatum and the mango isolates (besides Man-76) as C. gloeosporioides. However, DNA of the passiflora isolates was not amplified by either C. acutatum- or C. gloeosporioides-specific primers, but reacted with a new primer, Col1, designed according to the internal transcribed spacer (ITS) 1 region of these isolates. Isolates Tom-6 and Man-76 also reacted positively with the Col1 primer. All the isolates reacting with the C. acutatum- and C. gloeosporioides-specific primers failed to react with primer Col1. Isolate Pass-35 from passiflora did not react with any of the taxon-specific primers. Arbitrarily primed polymerase chain reaction (ap-PCR), random amplified polymerase DNA (RAPD)-PCR, and A+T-rich DNA analyses delineated representative isolates into subgroups within the designated species. Molecular analyses indicated that the C. acutatum tamarillo isolates were uniform or clonal, whereas the C. gloeosporioides mango isolates and Colletotrichum passiflora isolates were heterogeneous. Likewise, sequence analysis of the complete ITS (ITS1-5.8S-ITS2) region identified certain isolates to their respective species: tamarillo isolates as C. acutatum; mango isolates as C. gloeosporioides; passiflora, Tom-6, and Man-76 isolates as a Colletotrichum sp. as yet undefined; and the Pass-35 isolate as an additional undefined Colletot-richum sp. Molecular analyses of the population of Colletotrichum isolates from passiflora, Tom-6 from tamarillo, and Man-76 from mango indicate that this population may not be host specific.

scholarly journals Identification and Characterization of Benomyl-Resistant and -Sensitive Populations of Colletotrichum gloeosporioides from Statice (Limonium spp.)

2006 ◽  
Vol 96 (5) ◽  
pp. 542-548 ◽  
Author(s):  
Marcel Maymon ◽  
Aida Zveibil ◽  
Shimon Pivonia ◽  
Dror Minz ◽  
Stanley Freeman
Keyword(s):  
Colletotrichum Gloeosporioides ◽  
Sequence Analyses ◽  
Specific Primers ◽  
Tubulin Genes ◽  
Colletotrichum Spp ◽  
Polymerase Chain ◽  
Species Specific ◽  
Identification And Characterization ◽  
Propagation Material

Sixty-four isolates of Colletotrichum gloeosporioides were isolated from infected Limonium spp. cultivated in 12 different locations in Israel. All isolates were identified as belonging to the C. gloeosporioides complex by species-specific primers. Of these isolates, 46 were resistant to benomyl at 10 μg/ml and 18 were sensitive to this concentration of fungicide. Based on arbitrarily primed polymerase chain reaction of all isolates and internal transcribed spacer-1 sequence analyses of 12 selected isolates, the benomyl-resistant and -sensitive populations belong to two distinct genotypes. Sequence analyses of the β-tubulin genes, TUB1 and TUB2, of five sensitive and five resistant representative isolates of C. gloeosporioides from Limonium spp. revealed that the benomyl-resistant isolates had an alanine substitute instead of a glutamic acid at position 198 in TUB2. All data suggest that the resistant and sensitive genotypes are two independent and separate populations. Because all Limonium plant propagation material is imported from various geographic regions worldwide, and benomyl is not applied to this crop or for the control of Colletotrichum spp. in Israel, it is presumed that plants are bearing quiescent infections from the points of origin prior to arrival.

scholarly journals Development of specific markers for the detection of Tolypella canadensis eDNA in water samples

2021 ◽  
Vol 4 ◽  
Author(s):  
Christina Wiebe ◽  
Petra Nowak ◽  
Hendrik Schubert
Keyword(s):  
Rare Species ◽  
Environmental Samples ◽  
Large Scale ◽  
Morphological Characters ◽  
Its2 Region ◽  
Specific Marker ◽  
Specific Primers ◽  
Species Specific ◽  
Taxonomic Groups ◽  
Scale Integration

Assessing the biodiversity of an ecosystem plays a major role in ecosystem management. However, proper determination on species-level is often tricky when morphological features are scarce and especially rare species require huge sampling efforts to be detected in the aquatic realm. As an alternative to conventional methods, environmental samples can be examined via the eDNA method, allowing for large-scale integration as well as taxa resolution independent from expression of morphological characters. However, to apply this technique genetic markers that are specific to a species or at least a genus are required. Such markers until now have been successfully developed only for a few well studied taxonomic groups like, e.g., fishes and amphibians, but are still missing for others, especially plants and algae (e.g. Bista et al. 2017). This project focusses on the development of species-specific markers for the macrophytic green algae Tolypella canadensis (Characeae, Charophyta), a rare alga preferring deep water and known so far mainly from remote places. Tolypella canadensis is a circumpolar species and prefers oligotrophic lakes, where it grows in depths up to 13 m (Langangen 2002; Romanov and Kopyrina 2016). In addition, proper determination of Tolypella-species is a field of a few specialists, further complicating monitoring or even detection of this rare species. The design of the species-specific primers was based on reference nucleotide sequences of the chloroplast genes rbcL, psbC and atpB and of the ribosomal internal transcribed spacer regions ITS1 and ITS2, obtained from GenBank (Perez et al. 2017). To determine the specificity of the newly designed primers, DNA isolates obtained from T. canadensis specimens collected from the Torneträsk (Sweden, 2018) and other charophyte species were prepared in different proportions. The sensitivity of the primers was experimentally assayed by using serial dilutions of T. canadensis DNA. Additionally, a mock test comprised of a sample with the DNA of several charophyte species was conducted and finally, the markers were tested on environmental samples from the Torneträsk. Tolypella canadensis-specific primers of the ITS2 region yielded positive PCR amplifications of one single band when T. canadensis was present in a sample. Cross-amplification was not found during the mock test; other charophyte species did not yield positive amplification. The eDNA samples from the Torneträsk validated the performance of the ITS2 marker. The T. canadensis-specific marker designed in this project was proven to be sensitive and accurate. It could be recommended as a useful tool to detect the presence of T. canadensis DNA, even at low concentration and in complex samples containing other charophyte species.

Microsporidial infections due toEncephalitozoon intestinalisin non-HIV-infected patients with chronic diarrhoea

2011 ◽  
Vol 140 (10) ◽  
pp. 1773-1779 ◽  
Author(s):  
J. YAKOOB ◽  
Z. ABBAS ◽  
M. ASIM BEG ◽  
W. JAFRI ◽  
S. NAZ ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Stool Examination ◽  
Chronic Diarrhoea ◽  
Healthy Controls ◽  
Chain Reaction ◽  
Specific Primers ◽  
Specific Pcr ◽  
Stool Samples ◽  
Polymerase Chain ◽  
Species Specific

SUMMARYWe determined the prevalence of microsporidiaEnterocytozoon(Ent.)bieneusiandEncephalitozoon(E.)intestinalisinfection in patients with chronic diarrhoea and hepatocellular carcinoma (HCC). A total of 330 stool samples were examined from 171 (52%) patients with chronic diarrhoea, 18 (5%) with HCC while 141 (43%) were controls. Stool microscopy, polymerase chain reaction (PCR) with species-specific primers forEnt. bieneusiandE. intestinalisand sequencing were carried out. Microsporidia were found by trichrome staining in 11/330 (3%) andE. intestinalisby PCR in 13/330 (4%) whileEnt. bieneusiwas not detected. PCR forE. intestinaliswas positive in 8/171 (5%) stool samples from patients with chronic diarrhoea, 2/141 (1·4%) samples from healthy controls and in 3/18 (17%) samples from patients with HCC. In the chronic diarrhoea group,E. intestinaliswas positive in 4/171 (2·3%) (P=0·69) stool samples compared to 2/18 (11%) (P=0·06) in the HCC group and 2/141 (1·4%) from healthy controls.E. intestinalisinfection was significantly associated with chronic diarrhoea and HCC in these patients who were negative for HIV. Stool examination with trichrome or species-specific PCR for microsporidia may help establish the cause of chronic diarrhoea.

scholarly journals Molecular Analyses of Colletotrichum Species from Almond and Other Fruits

2000 ◽  
Vol 90 (6) ◽  
pp. 608-614 ◽  
Author(s):  
Stanley Freeman ◽  
Dror Minz ◽  
Edouard Jurkevitch ◽  
Marcel Maymon ◽  
Ezra Shabi
Keyword(s):  
United States ◽  
Sequence Analysis ◽  
Its Region ◽  
The United States ◽  
Morphological Identification ◽  
Molecular Analyses ◽  
Colletotrichum Species ◽  
Morphological Criteria ◽  
Colletotrichum Spp ◽  
Polymerase Chain

Isolates of Colletotrichum spp. from almond, avocado, and strawberry from Israel and isolates of the pink subpopulation from almond from the United States were characterized by various molecular methods and compared with morphological identification. Taxon-specific primer analysis grouped the avocado isolates within the species C. gloeosporioides and the U.S. almond and Israeli strawberry isolates within the species C. acutatum. However, the Israeli almond isolates, previously identified morphologically as C. gloeosporioides, reacted with C. acutatum-specific primers. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses determined that each population from almond and strawberry was distinct and clonal. Sequence analysis of the complete internal transcribed spacer (ITS) region (ITS 1–5.8S–ITS 2) revealed a similarity of between 97.03 and 98.72% among almond isolates from Israel, C. acutatum almond isolates from the United States, and C. acutatum strawberry isolates from Israel. Similarity of the above populations to that of C. gloeosporioides of avocado was between 92.42 and 92.86%. DNA sequence analysis of the entire ITS region supported the phylogeny inferred from the ITS 1 tree of 14 different Colletotrichum species. Although morphological criteria indicated that the Israeli isolates from almond are unique, this population was grouped within the C. acutatum species according to molecular analyses.

a Novel Method for the Detection of Minimal Residual Disease in B-Cell Malignancies: Ion Semiconductor Sequencing for the Evaluation of Igh Gene Rearrangements

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2983-2983
Author(s):  
Silvia Gimondi ◽  
Alessandra Cavanè ◽  
Antonio Vendramin ◽  
Giulia Biancon ◽  
Paolo Corradini ◽  
...  
Keyword(s):  
B Cell ◽  
Multiplex Pcr ◽  
Residual Disease ◽  
Chain Reaction ◽  
B Cell Malignancies ◽  
Specific Primers ◽  
Namalwa Cell ◽  
Polymerase Chain ◽  
Minimal Residual

Abstract Background: Minimal residual disease (MRD) detection is of high clinical relevance in patients with B-cell malignancies and is generally a surrogate parameter to evaluate treatment response and long-term prognosis. IgH gene rearrangements can be used as molecular marker in approximately 80% of lymphoma and myeloma patients since they represent lineage-specific markers and the complementarity determining region 3 (CDR3) is unique to each clone. To date, allele specific oligonucleotide polymerase chain reaction (ASO-PCR) and real-time quantitative polymerase chain reaction (RQ-PCR) are considered the most sensitive and widely applicable methods for MRD detection. A major disadvantage of ASO-PCR and RQ-PCR assays, is the use of specific primers and probes for every individual patient. Clone-specific primers and probes are not only expensive but also time-consuming to design and to test, which limits their wide applicability in the clinical setting. The recent major improvements in next generation sequencing (NGS) technologies, provide the opportunity to identify and quantify clonotypes with consensus primers combining the benefits of high sensitivity and universal applicability. The present work was designed to overcome ASO-PCR and RQ-PCR limitations by developing a feasible method for rearranged IgH genes amplification, NGS and analysis using Ion Torrent Personal Genome Machine (IT-PGM). Methods: To define a multiplex PCR protocol, DNA from 7 CLL patients, previously shown to bare a family specific clonal VDJ rearrangement, was amplified with a pool of the seven different family-specific IgH-V primers, and a consensus JH primer (Voena et al., Leukemia 1997). After Sanger sequencing, results were compared to the ones obtained with singleplex PCR protocol. Once validated, the multiplex PCR protocol was used to amplify DNA from patients and serially diluted (up to 10-8 ) DNA from Namalwa cell line (bearing a known IgH rearrangement) and subsequently sequenced on the IT-PGM using the 316 Ion-chip. NGS data were analyzed by using the IMGT-High V-Quest web server tool and the statistical software R. RQ-PCR was used to quantify the specific VDJ rearrangement in the serially diluted Namalwa DNA solutions and in DNA from patients as previously described (Farina et al, Haematologica 2009). RQ-PCR data were analyzed through a relative quantification procedure. Results: The multiplex PCR reactions we have tested, demonstrated the same specificity as the standard singleplex PCR protocol and therefore was used to construct the DNA library required for IT-PGM-based sequencing. The IT-PGM sequencing output is represented by at least 400000 reads per sample with a minimum average coverage of the VDJ repertoire of 500x. The IMGT-High V-quest tool allows a user-friendly web based analysis and a deep molecular characterization of the IgH recombinatorial repertoire. Namalwa clonal CRD3 sequences were detected up to a dilution of 10-5 without the need for specific CDR3 primers. Comparability of NGS and ASO RQ-PCR results was assessed. The use of CDR3 specific primers, along with the specific IgH-V family fluorescent probe, enabled the identification of clonal VDJ rearrangements with a sensitivity up to 10-5 (2/3 replicates) and 10-6 (just 1/3 replicates) in Namalwa Cell Line. Similar results were obtained when we characterized the IgH recombination repertoire of two CLL patients over time. Conclusions: IgH sequencing with the IT-PGM platform showed at least the same level of sensitivity as ASO RQ-PCR, without the need for patient-specific reagents. It also allows specific and detailed molecular characterization of the clonal rearrangements and could be easily incorporated into clinical laboratories for routine testing of MRD in B-cell malignancies. Disclosures No relevant conflicts of interest to declare.

Detection of cow milk paneer in mixed/buffalo milk paneer through conventional species specific Polymerase Chain Reaction

2016 ◽  
Author(s):  
Tanmay Hazra ◽  
Vivek Sharma ◽  
Rekha Sharma ◽  
S. De ◽  
Sumit Arora ◽  
...  
Keyword(s):  
Buffalo Milk ◽  
Cow Milk ◽  
Market Demand ◽  
Chain Reaction ◽  
Specific Primers ◽  
Mt Dna ◽  
Milk Adulteration ◽  
D Loop ◽  
Polymerase Chain ◽  
Species Specific

Due to higher market demand of buffalo milk paneer, lower price cow milk is often adulterated with higher cost buffalo milk for preparation of paneer. Till date no rapid technique is available in market to ensure that paneer is made from buffalo milk. Currently a PCR based method has been developed to authenticate the buffalo milk paneer. DNA was isolated from paneer by DNeasy Mericon food kit. A set of bovine specific primers (P1) targeting D-loop (displacement loop) of mt- DNA was selected and standardized to amplify cow DNA resulted 126bp amplicon. Using this PCR based approach even upto 1% level of cow milk adulteration in buffalo milk paneer could be detected.

scholarly journals Phenotypic and Genotypic Characterization of Uromyces appendiculatus from Phaseolus vulgaris in the Americas

Plant Disease ◽  
2004 ◽  
Vol 88 (8) ◽  
pp. 830-836 ◽  
Author(s):  
C. M. Araya ◽  
A. T. Alleyne ◽  
J. R. Steadman ◽  
K. M. Eskridge ◽  
D. P. Coyne
Keyword(s):  
Phaseolus Vulgaris ◽  
Central America ◽  
Random Amplified Polymorphic Dna ◽  
Parallel Evolution ◽  
Uromyces Appendiculatus ◽  
Bean Rust ◽  
Rapd Pcr ◽  
Polymerase Chain ◽  
Clear Differentiation

Populations of 90 Uromyces appendiculatus isolates were collected from throughout the Americas and evaluated for virulence on 19 standard bean rust differentials, and also on 12 landraces of Phaseolus vulgaris from South and Central America. The landrace differentials represented geographical centers of bean domestication. Three groups were observed. Two groups were isolates from centers of bean domestication and a third heterogeneous group comprised isolates from countries in South and Central America. Molecular analysis using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was also conducted on these isolates. Cluster analysis of the molecular profiles showed three groups that corresponded to those obtained by virulence tests. These results show a clear differentiation of the pathogen population along similar lines as its host and suggest parallel evolution in the bean rust pathosystem.

scholarly journals Detection of Pythium ultimum Using Polymerase Chain Reaction with Species-Specific Primers

Plant Disease ◽  
1997 ◽  
Vol 81 (10) ◽  
pp. 1155-1160 ◽  
Author(s):  
K. Kageyama ◽  
A. Ohyama ◽  
M. Hyakumachi
Keyword(s):  
Polymerase Chain Reaction ◽  
Internal Transcribed Spacer ◽  
Pcr Amplification ◽  
Its Region ◽  
Pythium Ultimum ◽  
Pythium Species ◽  
Chain Reaction ◽  
Specific Primers ◽  
Polymerase Chain ◽  
Species Specific

This study was conducted to sequence the rDNA internal transcribed spacer (ITS) region of Pythium ultimum and Pythium group HS, design species-specific primers for polymerase chain reaction (PCR), and detect P. ultimum from diseased seedlings using PCR. The sequence of the ITS region of P. ultimum was identical with that of Pythium group HS. The results support the reports that the HS group is an asexual strain of P. ultimum. Using PCR, the primer pair K1+K3, designed on portions of the sequence of the ITS region, amplified isolates of P. ultimum and the HS group but not isolates of 20 other Pythium species. DNA extracts from damped-off seedlings were not amplified, but a 10-fold dilution of the extracts with Tris-EDTA (TE) buffer diluted the inhibitors and allowed PCR amplification. The primer pair used detected P. ultimum from a single diseased seedling.

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