scholarly journals Molecular Detection of Phytophthora ramorum, the Causal Agent of Sudden Oak Death in California, and Two Additional Species Commonly Recovered from Diseased Plant Material

2004 ◽  
Vol 94 (6) ◽  
pp. 621-631 ◽  
Author(s):  
Frank N. Martin ◽  
Paul W. Tooley ◽  
Cheryl Blomquist
Keyword(s):  
Plant Species ◽  
The Other ◽  
Sudden Oak Death ◽  
Specific Primer ◽  
Specific Primers ◽  
Marker System ◽  
Phytophthora Spp ◽  
Multiplex Amplification ◽  
Species Specific ◽  
Specific Amplicon

Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. The pathogen causing this is Phy-tophthora ramorum, although species such as P. nemorosa and P. pseudo-syringae often are recovered from symptomatic plants as well. A molecular marker system was developed based on mitochondrial sequences of the cox I and II genes for detection of Phytophthora spp. in general, and P. ramorum, P. nemorosa, and P. pseudosyringae in particular. The first-round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for polymerase chain reaction (PCR) amplification and the other specific for amplification of sequences from Phytophthora spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. Using DNA from purified cultures, the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species. The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species. With the exception of one plant species, the resulting amplicons were smaller than the Phytophthora genus-specific amplicon. The products of the first-round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P. ramorum, P. nemorosa, or P. pseudo-syringae. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P. ramorum, this included 24 isolates from California, Germany, and the Netherlands. Using purified pathogen DNA, the limit of detection for P. ramorum using this marker system was ≈2.0 fg of total DNA. However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure, the sensitivity of detection was reduced by 100- to 1,000-fold, depending on the plant species. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions and, for P. ramorum, amplification with a previously published rDNA internal transcribed spacer species-specific primer pair. Results were compared and validated with three different brands of thermal cyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions. The specificity of the Phytophthora genus-specific primers suggests that they will have utility for pathogen detection in other Phytophthora pathosystems.

scholarly journals A Multiplex PCR for the Detection of Phytophthora nicotianae and P. cactorum, and a Survey of Their Occurrence in Strawberry Production Areas of Japan

Plant Disease ◽  
2011 ◽  
Vol 95 (10) ◽  
pp. 1270-1278 ◽  
Author(s):  
Mingzhu Li ◽  
Takahiro Asano ◽  
Haruhisa Suga ◽  
Koji Kageyama
Keyword(s):  
Multiplex Pcr ◽  
Protein Gene ◽  
Phytophthora Nicotianae ◽  
Soilborne Pathogens ◽  
Chain Reaction ◽  
Specific Primers ◽  
Phytophthora Spp ◽  
Polymerase Chain ◽  
Species Specific ◽  
Production Areas

We aimed to simultaneously detect two pathogens causing strawberry diseases, Phytophthora nicotianae and P. cactorum, by multiplex polymerase chain reaction (PCR), and to survey their occurrence in the main strawberry production areas of Japan. Due to the need to combine different primer pairs for multiplex PCR and the low specificity of published specific primers for P. nicotianae and P. cactorum, new species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions of ribosomal DNA and the ras-related protein gene Ypt1, respectively. Specificity of the designed primers was demonstrated using 68 isolates, including Phytophthora spp., Pythium spp., and other soilborne pathogens. Multiplex PCR discriminated between P. nicotianae and P. cactorum in DNA mixtures of mycelia of the two species. Moreover, both species were detected in artificially and naturally infested soils, indicating that these markers can be used in diagnosis of strawberry diseases. For investigation of the geographic distribution of the two pathogens in Japan, soil samples were collected in 89 strawberry fields from eight prefectures (Gifu, Saga, Nara, Tochigi, Chiba, Shizuoka, Yamanashi, and Hokkaido) of Japan. The method that was developed was successfully applied to survey P. nicotianae and P. cactorum, and distribution of the two pathogens in strawberry plantings in Japan was determined.

scholarly journals Verification of Thai ethnobotanical medicine “Kamlang Suea Khrong” driven by multiplex PCR and powerful TLC techniques

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257243
Author(s):  
Suthira Yanaso ◽  
Ampai Phrutivorapongkul ◽  
Darunee Hongwiset ◽  
Sirivipa Piyamongkol ◽  
Aekkhaluck Intharuksa
Keyword(s):  
Chemical Analysis ◽  
Multiplex Pcr ◽  
Plant Species ◽  
Specific Primers ◽  
Discrimination Power ◽  
Plant Materials ◽  
Identification Methods ◽  
Crude Drug ◽  
Crude Drugs ◽  
Species Specific

Kamlang Suea Khrong (KSK) crude drug, a traditional Thai medicine used for oral tonic and analgesic purposes, is obtained from three origins: the inner stem bark of Betula alnoides (BA) or the stems of Strychnos axillaris (SA) or Ziziphus attopensis (ZA). According to the previous reports, SA contains strychnine-type alkaloids that probably cause poisoning; however, only organoleptic approaches are insufficient to differentiate SA from the other plant materials. To ensure the botanical origin of KSK crude drug, powerful and reliable tools are desperately needed. Therefore, molecular and chemical identification methods, DNA barcoding and thin-layer chromatography (TLC), were investigated. Reference databases, i.e., the ITS region and phytochemical profile of the authentic plant species, were conducted. In case of molecular analysis, multiplex polymerase chain reaction (PCR) based on species-specific primers was applied. Regarding species-specific primers designation, the suitability of three candidate barcode regions (ITS, ITS1, and ITS2) was evaluated by genetic distance using K2P model. ITS2 presented the highest interspecific variability was verified its discrimination power by tree topology. Accordingly, ITS2 was used to create primers that successfully specified plant species of authentic samples. For chemical analysis, TLC with toluene:ethyl acetate:ammonia (1:9:0.025) and hierarchical clustering were operated to identify the authentic crude drugs. The developed multiplex PCR and TLC methods were then applied to identify five commercial KSK crude drugs (CK1-CK5). Both methods correspondingly indicated that CK1-CK2 and CK3-CK5 were originated from BA and ZA, respectively. Molecular and chemical approaches are convenient and effective identification methods that can be performed for the routine quality-control of the KSK crude drugs for consumer reliance. According to chemical analysis, the results indicated BA, SA, and ZA have distinct chemical profiles, leading to differences in pharmacological activities. Consequently, further scientific investigations are required to ensure the quality and safety of Thai ethnobotanical medicine known as KSK.

scholarly journals Species-specific RT-PCR amplification of human enteroviruses: a tool for rapid species identification of uncharacterized enteroviruses

2006 ◽  
Vol 87 (1) ◽  
pp. 119-128 ◽  
Author(s):  
M. Steven Oberste ◽  
Kaija Maher ◽  
Alford J. Williams ◽  
Naomi Dybdahl-Sissoko ◽  
Betty A. Brown ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Pcr Primers ◽  
Human Enterovirus ◽  
Rt Pcr ◽  
Specific Primer ◽  
Specific Primers ◽  
Typical Application ◽  
Partial Sequencing ◽  
Specific Pcr ◽  
Species Specific

The 65 serotypes of human enteroviruses are classified into four species, Human enterovirus (HEV) A to D, based largely on phylogenetic relationships in multiple genome regions. The 3′-non-translated region of enteroviruses is highly conserved within a species but highly divergent between species. From this information, species-specific RT-PCR primers were developed that can be used to rapidly screen collections of enterovirus isolates to identify species of interest. The four primer pairs were 100 % specific when tested against enterovirus prototype strains and panels of isolates of known serotype (a total of 193 isolates). For evaluation in a typical application, the species-specific primers were used to screen 186 previously uncharacterized non-polio enterovirus isolates. The HEV-B primers amplified 68·3 % of isolates, while the HEV-A and HEV-C primers accounted for 9·7 and 11·3 % of isolates, respectively; no isolates were amplified with the HEV-D primers. Twelve isolates (6·5 %) were amplified by more than one primer set and eight isolates (4·3 %) were not amplified by any of the four primer pairs. Serotypes were identified by partial sequencing of the VP1 capsid gene, and in every case sequencing confirmed that the species-specific PCR result was correct; the isolates that were amplified by more than one species-specific primer pair were mixtures of two (11 isolates) or three (one isolate) species of viruses. The eight isolates that were not amplified by the species-specific primers comprised four new serotypes (EV76, EV89, EV90 and EV91) that appear to be unique members of HEV-A based on VP1, 3D and 3′-non-translated region sequences.

scholarly journals Environmental DNA and Specific Primers for Detecting the Invasive Species Ectopleura crocea (Hydrozoa: Anthoathecata) in Seawater Samples

2020 ◽  
Vol 12 (6) ◽  
pp. 2360 ◽  
Author(s):  
Philjae Kim ◽  
Tae Joong Yoon ◽  
Sook Shin
Keyword(s):  
Invasive Species ◽  
Survey Methods ◽  
Environmental Dna ◽  
Visual Assessment ◽  
Molecular Techniques ◽  
Mitochondrial Cytochrome ◽  
Specific Primer ◽  
Specific Primers ◽  
Seawater Samples ◽  
Species Specific

In marine environments, environmental DNA (eDNA) can be effectively detected and possibly quantified when combined with molecular techniques, as demonstrated by several recent studies. In this study, we developed a species-specific primer set and a probe to detect the distribution and biomass of an invasive hydrozoan in South Korea, Ectopleura crocea. These molecular markers were designed to amplify a 187 bp region based on mitochondrial cytochrome c oxidase subunit I (COI) of E. crocea and were tested on seawater samples from 35 Korean harbors in 2017. Of the 35 sites we investigated, only nine harbors returned positive detections when using traditional survey methods, while surveys based on the use of eDNA techniques detected E. crocea DNA in all seawater samples. These results suggest that eDNA surveys based on molecular techniques are more effective at identifying species distribution and estimating biomass than traditional surveys based on visual assessment of morphology.

scholarly journals Two-Step PCR-Based Assay for Identification of Bacterial Etiology of Otitis Media with Effusion in Infected Lebanese Children

1998 ◽  
Vol 36 (5) ◽  
pp. 1185-1188 ◽  
Author(s):  
Ghassan M. Matar ◽  
Nada Sidani ◽  
Michel Fayad ◽  
Usamah Hadi
Keyword(s):  
Otitis Media ◽  
Otitis Media With Effusion ◽  
Bacterial Species ◽  
Cost Effective ◽  
Universal Primers ◽  
Rrna Gene ◽  
Specific Primer ◽  
Specific Primers ◽  
Bacterial Dna ◽  
Species Specific

We developed and evaluated a two-step PCR-based assay with universal primers and genus- or species-specific primers for the detection of the most prevalent bacterial etiologies of otitis media with effusion (OME) in children from Lebanese hospitals. These etiologies included Haemophilus, Streptococcus, and Moraxella (Branhamella)catarrhalis, which were detected in middle-ear effusion (MEE) samples taken from children with OME. A total of 47 MEE samples were aspirated from 36 patients during insertion of a tympanostomy tube performed particularly for OME. The duration of effusion in all patients was ≥2 months. DNA was extracted from MEE samples, and PCR was initially done with DNA extracts by using the universal primers RW01 and DG74, which flank an ∼370-bp fragment found in the 16S rRNA gene of all bacterial species. For the identification of specific bacteria, we used in three separate reaction mixtures the following genus- or species-specific primers: (i) aHaemophilus-specific probe (probe RDR125) as a primer along with DG74, (ii) a Streptococcus-specific primer (primer STR1; designed by us) along with DG74, and (iii) an M. catarrhalis-specific primer pair (primer pair MCA1-MCA2). Thirty-five MEE samples (74.5%) gave the expected 370-bp band, indicating the presence of bacterial DNA in the tested samples. Of the 35 PCR-positive samples tested, 33 (94.3%) were positive forHaemophilus, 3 (8.6%) were positive forStreptococcus, and 10 (28.6%) were positive for M. catarrhalis. Ten samples (28.6%) exhibited a mixed infection and were positive for both Haemophilus and M. catarrhalis. Culture was simultaneously performed for all 47 MEE samples. Ten of the 47 MEE samples (21.3%) exhibited bacterial growth. These 10 were PCR positive for bacterial DNA. The remaining 25 PCR-positive samples were negative by culture, thus showing about 53% discordance between PCR results and those of culture. The PCR assay proved to be more sensitive than culture, more rapid, less cumbersome, and more cost-effective than the available PCR-Southern hybridization-based assays.

scholarly journals Prebiotic Combinations Effects on the Colonization of Staphylococcal Skin Strains

2020 ◽  
Vol 9 (1) ◽  
pp. 37
Author(s):  
Silvia Di Lodovico ◽  
Franco Gasparri ◽  
Emanuela Di Campli ◽  
Paola Di Fermo ◽  
Simonetta D’Ercole ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Commensal Bacteria ◽  
The Other ◽  
Bacteriostatic Effect ◽  
Skin Microbiota ◽  
Relevant Effect ◽  
Planktonic Phase ◽  
Species Specific ◽  
Biomass Quantification ◽  
General Reduction

Background: An unbalanced skin microbiota due to an increase in pathogenic vs. commensal bacteria can be efficiently tackled by using prebiotics. The aim of this work was to identify novel prebiotic combinations by exerting species-specific action between S. aureus and S. epidermidis strains. Methods: First, the antimicrobial/antibiofilm effect of Xylitol-XYL and Galacto-OligoSaccharides–GOS combined with each other at different concentrations (1, 2.5, 5%) against S. aureus and S. epidermidis clinical strains was evaluated in time. Second, the most species-specific concentration was used to combine XYL with Fructo-OligoSaccharides–FOS, IsoMalto-Oligosaccharides–IMO, ArabinoGaLactan–LAG, inulin, dextran. Experiments were performed by OD600 detection, biomass quantification and LIVE/DEAD staining. Results: 1% XYL + 1% GOS showed the best species-specific action with an immediate antibacterial/antibiofilm action against S. aureus strains (up to 34.54% ± 5.35/64.68% ± 4.77) without a relevant effect on S. epidermidis. Among the other prebiotic formulations, 1% XYL plus 1% FOS (up to 49.17% ± 21.46/37.59% ± 6.34) or 1% IMO (up to 41.28% ± 4.88/36.70% ± 10.03) or 1% LAG (up to 38.21% ± 5.31/83.06% ± 5.11) showed antimicrobial/antibiofilm effects similar to 1% XYL+1% GOS. For all tested formulations, a prevalent bacteriostatic effect in the planktonic phase and a general reduction of S. aureus biofilm formation without loss of viability were recorded. Conclusion: The combinations of 1% XYL with 1% GOS or 1% FOS or 1% IMO or 1% LAG may help to control the balance of skin microbiota, representing good candidates for topic formulations.

scholarly journals Species-Specific Segregation of Gender-Associated Mitochondrial DNA Types in an Area Where Two Mussel Species (Mytilus edulis and M. trossulus) Hybridize

Genetics ◽  
1996 ◽  
Vol 143 (3) ◽  
pp. 1359-1367 ◽  
Author(s):  
Carlos Saavedra ◽  
Donald T Stewart ◽  
Rebecca R Stanwood ◽  
Eleftherios Zouros
Keyword(s):  
Mytilus Edulis ◽  
The Other ◽  
Allozyme Analysis ◽  
Mussel Species ◽  
Species Origin ◽  
Species Pairs ◽  
Nuclear Background ◽  
Species Specific ◽  
Dna Types ◽  
Fertile Hybrids

Abstract In each of the mussel species Mytilus edulis and M. trossulus there exist two types of mtDNA, the F type transmitted through females and the M type transmitted through males. Because the two species produce fertile hybrids in nature, F and M types of one may introgress into the other. We present the results from a survey of a population in which extensive hybridization occurs between these two species. Among specimens classified as “pure” M. edulis or “pure” M. trossulus on the basis of allozyme analysis, we observed no animal that carried the F or the M mitotype of the other species. In most animals of mixed nuclear background, an individual's mtDNA came from the species that contributed the majority of the individual's nuclear genes. Most importantly, the two mtDNA types in post-F1 male hybrids were of the same species origin. We interpret this to mean that there are intrinsic barriers to the exchange of mtDNA between these two species. Because such barriers were not noted in other hybridizing species pairs (many being even less interfertile than M. edulis and M. trossulus), their presence in Mytilus could be another feature of the unusual mtDNA system in this genus.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

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