Detection and Identification of Fish-PathogenicAphanomyces piscicidaUsing Polymerase Chain Reaction (PCR) with Species-Specific Primers

2004 ◽  
Vol 16 (4) ◽  
pp. 220-230 ◽  
Author(s):  
Panarat Phadee ◽  
Osamu Kurata ◽  
Kishio Hatai ◽  
Ikuo Hirono ◽  
Takashi Aoki
Keyword(s):  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Specific Primers ◽  
Detection And Identification ◽  
Polymerase Chain ◽  
Species Specific

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.

scholarly journals Detection of Erysiphe necator in Air Samples Using the Polymerase Chain Reaction and Species-Specific Primers

2007 ◽  
Vol 97 (10) ◽  
pp. 1290-1297 ◽  
Author(s):  
Jennifer S. Falacy ◽  
Gary G. Grove ◽  
Walter F. Mahaffee ◽  
Heather Galloway ◽  
Dean A. Glawe ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Powdery Mildew ◽  
Bud Burst ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Specific Primers ◽  
Erysiphe Necator ◽  
Air Samples ◽  
Polymerase Chain ◽  
Species Specific

A polymerase chain reaction (PCR) assay employing species-specific primers was developed to differentiate Erysiphe necator from other powdery mildews common in the northwest United States. DNA was extracted from mycelia, conidia, and/or chasmothecia that were collected from grape leaves with a Burkard cyclonic surface sampler. To differentiate E. necator from other erysiphaeceous fungi, primer pairs Uncin144 and Uncin511 were developed to select unique sequences of the internal transcribed spacer regions of E. necator. Using these primers in PCR amplifications, a 367-bp amplicon specific to E. necator was generated, but no amplicons were generated from other erysiphaceous species collected from 48 disparate hosts representing 26 vascular plant families. The PCR limit of detection was one to five conidia of E. necator placed directly into reaction mixtures or 100 to 250 conidia placed on glass rods coated with silicon grease. During field studies, this PCR assay facilitated the detection of E. necator inoculum in air samples within hours of sample rod collection and prior to disease onset. Amplification of E. necator DNA did not occur when the PCR assay was conducted on vineyard air samples collected while grapes were dormant or during periods when vine growth occurred but E. necator remained dormant. The initial PCR detection of E. necator of the season occurred during seasonal ascospore releases caused by precipitation events between bud burst and the prebloom period during the 3 years of the study. Detection ceased for 7 to 11 days following ascospore release and then resumed several days prior to the observance of microscopic symptoms and signs of powdery mildew in the field. Results of this study represent the initial step toward the goal of incorporating an inoculum availability component into current and future grapevine powdery mildew risk assessment models.

scholarly journals Detection and Identification of Four Common Rust Pathogens of Cereals and Grasses Using Real-Time Polymerase Chain Reaction

2007 ◽  
Vol 97 (6) ◽  
pp. 717-727 ◽  
Author(s):  
C. W. Barnes ◽  
L. J. Szabo
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Internal Control ◽  
Chain Reaction ◽  
Detection And Identification ◽  
Pasture Grasses ◽  
Significant Yield ◽  
Polymerase Chain ◽  
Species Specific ◽  
Rust Pathogens

Puccinia spp. are widespread pathogens of cereals and grasses that annually cause significant yield losses worldwide, especially in barley, oat, and wheat. Urediniospore morphology and early symptom development have limited usefulness for distinguishing Puccinia spp. Therefore, we developed real-time polymerase chain reaction assays for rapid detection of the four rust pathogen species, Puccinia graminis (Pers.:Pers.), P. striiformis (Westend.), P. triticina (Eriks.), and P. recondita (Roberge ex Desmaz.). Duplex assays were constructed for the nuclear rDNA gene, using the variable internal transcribed spacer 1 (ITS1) region to distinguish between species, and the conserved 28S region as an internal control. Species-specific ITS1 primer/probe sets were highly specific and could detect <1 pg of DNA. The species-specific primer/probe sets showed positive results over a linear range of DNA five orders of magnitude or greater. Specificity of the assays was tested using multiple collections representing a range of races and formae speciales within a species. Additionally, assay specificity was evaluated by testing a range of other grass rust pathogens, as well as other fungi. The 28S primer/probe combination was successful in detecting all Puccinia spp. tested within the duplex assays, validating the integrity of each assay. Finally, the assays were used to identify unknown rust fungi infecting pasture grasses.

scholarly journals Identification and Quantification of Pathogenic Pythium spp. from Soils in Eastern Washington Using Real-Time Polymerase Chain Reaction

2006 ◽  
Vol 96 (6) ◽  
pp. 637-647 ◽  
Author(s):  
K. L. Schroeder ◽  
P. A. Okubara ◽  
J. T. Tambong ◽  
C. A. Lévesque ◽  
T. C. Paulitz
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Group Iv ◽  
Chain Reaction ◽  
Specific Primers ◽  
Variable Regions ◽  
Group I ◽  
Polymerase Chain ◽  
Species Specific ◽  
Eastern Washington

Traditional methods of quantifying Pythium spp. rely on the use of selective media and dilution plating. However, high variability is inherent in this type of enumeration and counts may not be representative of the pathogenic population of Pythium spp. Variable regions of the internal transcribed spacer of the rDNA were used to design species-specific primers for detection and quantification of nine Pythium spp. from soils in eastern Washington. Primer pairs were designed for Pythium abappressorium, P. attrantheridium, P. heterothallicum, P. irregulare group I, P. irregulare group IV, P. paroecandrum, P. rostratifingens, P. sylvaticum, and P. ultimum and used with real-time polymerase chain reaction. Standard curves were generated for each of the species using SYBR Green I fluorescent dye for detection of amplification. Seventy-seven isolates of Pythium were screened to confirm specificity of each primer set. DNA was extracted from soil and standard curves were generated for P. irregulare group I, P. irregulare group IV, and P. ultimum to correlate populations of each species in the soil with quantities of DNA amplified from the same soil. Examination of raw field soils revealed results similar to those observed in previous studies. This new technique for the quantification of Pythium spp. is rapid and accurate, and will be a useful tool in the future study of these pathogenic Pythium spp.

scholarly journals Detection of Rhodococcus Equi by Polymerase Chain Reaction Using Species-Specific Nonproprietary Primers

2002 ◽  
Vol 14 (4) ◽  
pp. 347-353 ◽  
Author(s):  
José Miguel Arriaga ◽  
Noah D. Cohen ◽  
James N. Derr ◽  
M. Keith Chaffin ◽  
Ronald J. Martens
Keyword(s):  
Polymerase Chain Reaction ◽  
Rhodococcus Equi ◽  
Chromosomal Dna ◽  
Chain Reaction ◽  
Specific Primers ◽  
Pcr Product ◽  
Random Amplification ◽  
Rhodococcus Species ◽  
Polymerase Chain ◽  
Species Specific

Species-specific primers for the polymerase chain reaction (PCR) for the detection of Rhodococcus equi were developed. These primers were based on unique DNA fragments produced from R. equi reference strains and field isolates. Following random amplification of polymorphic DNA from R. equi and R. rhodochrous with a set of 40 arbitrary 10–base pair (bp) primers, a pair of species-specific primers was designed to detect a unique 700-bp fragment of R. equi chromosomal DNA. This PCR product was limited to R. equi and was not detectable in other Rhodococcus species or in a panel of additional gram-positive and gram-negative bacteria.

Rapid Detection and Identification of Lactobacillus spp. in Dairy Products by Using the Polymerase Chain Reaction

1996 ◽  
Vol 59 (10) ◽  
pp. 1031-1036 ◽  
Author(s):  
MARYANNE DRAKE ◽  
CHRISTOPHER L. SMALL ◽  
KEMET D. SPENCE ◽  
BARRY G. SWANSON
Keyword(s):  
Polymerase Chain Reaction ◽  
Dairy Products ◽  
Lactobacillus Helveticus ◽  
Lactobacillus Delbrueckii ◽  
Lactobacillus Species ◽  
Chain Reaction ◽  
Bacterial Dna ◽  
Detection And Identification ◽  
Polymerase Chain ◽  
Species Specific

Species-specific primers for use in the polymerase chain reaction (PCR) were designed to differentially amplify DNA from the common dairy lactobacillus species Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus helveticus, and Lactobacillus acidophilus. A method for rapid extraction of bacterial DNA from dairy products was developed. The sensitivity of bacterial DNA extraction from food and subsequent amplification by PCR was 100 cells total. Lactobacillus DNA was extracted and identified from commercial yoghurts, acidophilus milk, and cheeses. The methodology allows the presumptive identification of dairy lactobacilli in less than 6 hours.

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