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 A Species-Specific Polymerase Chain Reaction Assay for Rapid Detection of Phytophthora nicotianae in Irrigation Water

2003 ◽  
Vol 93 (7) ◽  
pp. 822-831 ◽  
Author(s):  
Ping Kong ◽  
Chuanxue Hong ◽  
Steven N. Jeffers ◽  
Patricia A. Richardson
Keyword(s):  
Polymerase Chain Reaction ◽  
Irrigation Water ◽  
Phytophthora Nicotianae ◽  
Detection Sensitivity ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Specific Polymerase Chain Reaction ◽  
Wide Range ◽  
Polymerase Chain ◽  
Species Specific

Phytophthora nicotianae is a common and destructive pathogen of numerous ornamental, agronomic, and horticultural crops such as tobacco, tomato, and citrus. We have developed a species-specific polymerase chain reaction (PCR) assay for rapid and accurate detection of this pathogen in irrigation water, a primary source of inoculum and an efficient means of propagule dissemination. This PCR assay consists of a pair of species-specific primers (PN), customization of a commercial soil DNA extraction kit for purification of DNA from propagules in irrigation water, and efficient PCR protocols for primer tests and sample detection. The PN primers proved adequately specific for P. nicotianae in evaluations with 131 isolates of P. nicotianae, 102 isolates from 15 other species of Phytophthora, and 64 isolates from a variety of other oomycetes, true fungi, and bacteria. These isolates originated from a wide range of host plants, three substrates (plant tissue, soil, and irrigation water), and numerous geographic locations. The detection sensitivity is between 80 and 800 fg DNA/μl. The assay detected the pathogen in naturally infested water samples from Virginia and South Carolina nurseries more rapidly and accurately than standard isolation methods. Use of this PCR assay can assist growers in making timely disease management decisions with confidence.

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 Species specific polymerase chain reaction (PCR) assay for identification of pig (Sus domesticus) meat

2012 ◽  
Vol 11 (89) ◽  
pp. 15590-15595 ◽  
Author(s):  
Kumar Arun ◽  
Ranjan Kumar Rajiv ◽  
Deo Sharma Brahma ◽  
Kumar Mendiratta Sanjod ◽  
Sharma Deepak ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Specific Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Species Specific ◽  
Sus Domesticus

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 Efficacy of a Modified Polymerase Chain Reaction Assay for Detection of Ehrlichia Canis infection

2000 ◽  
Vol 12 (5) ◽  
pp. 456-459 ◽  
Author(s):  
John S. Mathew ◽  
Sidney A. Ewing ◽  
Jerry R. Malayer ◽  
Joseph C. Fox ◽  
Katherine M. Kocan
Keyword(s):  
Polymerase Chain Reaction ◽  
Ehrlichia Canis ◽  
New Method ◽  
Conventional Pcr ◽  
Serologic Test ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Specific Primers ◽  
Specificity And Sensitivity ◽  
Polymerase Chain

Detection of Ehrlichia canis in acutely infected and convalescent dogs is important for effective treatment and control. However, accurate detection has been difficult to achieve, in part because dogs that have been treated therapeutically often remain seropositive for extended periods. A new method, polymerase chain reaction (PCR) assay using biotinylated E. canis-specific primers (PCR-BP), was developed for detection of E. canis. Four dogs experimentally infected with E. canis by intravenous inoculation of whole blood from carrier dogs and 2 naturally infected convalescent carriers were used to compare the specificity and sensitivity of the new method with that of microscopy/blood smear evaluation, serologic test, and conventional PCR assay using E. canis-specific primers. In experimentally infected animals, infection was detected as early as 7 days postexposure using PCR-BP. Although the 2 naturally infected dogs were positive by serologic test and PCR-BP, both were negative by conventional PCR. Results suggest that the new method is a sensitive assay for detection of E. canis infection. In addition, results were obtained more rapidly than with other PCR-based assays.

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