scholarly journals Detection and Quantification of Phytophthora ramorum from California Forests Using a Real-Time Polymerase Chain Reaction Assay

2004 ◽  
Vol 94 (10) ◽  
pp. 1075-1083 ◽  
Author(s):  
Katherine J. Hayden ◽  
David Rizzo ◽  
Justin Tse ◽  
Matteo Garbelotto
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Pathogen Detection ◽  
Phytophthora Species ◽  
Phytophthora Ramorum ◽  
Plant Diseases ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
New Host ◽  
Polymerase Chain

The timely and accurate detection of pathogens is a critical aid in the study of the epidemiology and biology of plant diseases. In the case of regulated organisms, the availability of a sensitive and reliable assay is essential when trying to achieve early detection of the pathogen. We developed and tested a real-time, nested polymerase chain reaction (PCR) assay for the detection of Phytophthora ramorum, causal agent of sudden oak death. This technique then was implemented as part of a widespread environmental screen throughout California. The method here described is sensitive, detecting less than 12 fg of pathogen DNA, and is specific for P. ramorum when tested across 21 Phytophthora spp. Hundreds of symptomatic samples from 33 sites in 14 California counties were assayed, resulting in the discovery of 10 new host species and 23 infested areas, including 4 new counties. With the exception of a single host, PCR-based discovery of new hosts and infested areas always was confirmed by traditional pathogen isolations and inoculation studies. Nonetheless, molecular diagnostics were key in early pathogen detection, and steered the direction of further research on this newly discovered and generalist Phytophthora species.

scholarly journals Real-Time Polymerase Chain Reaction for One-Hour On-Site Diagnosis of Pierce's Disease of Grape in Early Season Asymptomatic Vines

2002 ◽  
Vol 92 (7) ◽  
pp. 721-728 ◽  
Author(s):  
N. W. Schaad ◽  
D. Opgenorth ◽  
P. Gaush
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Xylella Fastidiosa ◽  
Molecular Techniques ◽  
Plant Diseases ◽  
Pierce's Disease ◽  
Chain Reaction ◽  
Pierce’S Disease ◽  
Polymerase Chain

Molecular-based techniques, such as polymerase chain reaction (PCR), can reduce the time needed for diagnosis of plant diseases when compared with classical isolation and pathogenicity tests. However, molecular techniques still require 2 to 3 days to complete. To the best of our knowledge, we describe for the first time a real-time PCR technique using a portable Smart Cycler for one-hour on-site diagnosis of an asymptomatic plant disease. Pierce's disease (PD) of grape, caused by the fastidious bacterium Xylella fastidiosa, causes serious losses in grapes in California and the southeastern United States. The disease has been difficult to diagnose because typical leaf scorching symptoms do not appear until late (June and after) in the season and the organism is very difficult to isolate early in the season. Sap and samples of macerated chips of secondary xylem from trunks of vines were used in a direct real-time PCR without extraction of DNA. Using two different sets of primers and probe, we diagnosed PD in 7 of 27 vines (26%) from four of six vineyards sampled 10 to 12 days after bud break in Kern, Tulare, and Napa counties of California. The diagnosis was confirmed by isolation of Xylella fastidiosa from two of the original PCR positive samples and later from symptomatic leaf petioles of four out of four vines from one vineyard that were originally PCR positive.

scholarly journals Evaluation of pathogen detection from clinical samples by real-time polymerase chain reaction using a sepsis pathogen DNA detection kit

Critical Care ◽  
2010 ◽  
Vol 14 (4) ◽  
pp. R159 ◽  
Author(s):  
Katsunori Yanagihara ◽  
Yuko Kitagawa ◽  
Masao Tomonaga ◽  
Kunihiro Tsukasaki ◽  
Shigeru Kohno ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Pathogen Detection ◽  
Dna Detection ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Pathogen Dna

scholarly journals Detection of Bovine Leukemia Virus in Blood and Milk by Nested and Real-Time Polymerase Chain Reactions

2003 ◽  
Vol 15 (1) ◽  
pp. 72-76 ◽  
Author(s):  
Christopher J. Kuckleburg ◽  
Christopher C. Chase ◽  
Eric A. Nelson ◽  
Salvatore A. E. Marras ◽  
Matthew A. Dammen ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Early Detection ◽  
Real Time ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Polymerase Chain

Concerns about retroviruses in livestock and products derived from them have necessitated the development of tests to detect the bovine leukemia virus (BLV) in blood and milk from cattle. Dairy cattle ( n = 101) from 5 different geographical areas were used for this study. A nested polymerase chain reaction (PCR) identified 98% of BLV seropositive cattle ( n = 80) from blood and 65% from milk, whereas real-time PCR detected 94% of BLV seropositive cattle from blood and 59% from milk. Bovine leukemia virus was also detected by PCR in approximately 10% of seronegative cattle ( n = 21), most likely because of early detection before seroconversion.

scholarly journals 3′ Tth Endonuclease Cleavage Polymerase Chain Reaction (3TEC-PCR) Technology for Single-Base-Specific Multiplex Pathogen Detection using a Two-Oligonucleotide System

2021 ◽  
Vol 22 (11) ◽  
pp. 6061
Author(s):  
Owen Higgins ◽  
Terry J. Smith
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Target Detection ◽  
Pathogen Detection ◽  
Multiplex Detection ◽  
Chain Reaction ◽  
Single Base ◽  
Base Specificity ◽  
Pcr Technology ◽  
Polymerase Chain

Polymerase chain reaction (PCR) is the standard in nucleic acid amplification technology for infectious disease pathogen detection and has been the primary diagnostic tool employed during the global COVID-19 pandemic. Various PCR technology adaptations, typically using two-oligonucleotide dye-binding methods or three-oligonucleotide hydrolysis probe systems, enable real-time multiplex target detection or single-base specificity for the identification of single-nucleotide polymorphisms (SNPs). A small number of two-oligonucleotide PCR systems facilitating both multiplex detection and SNP identification have been reported; however, these methods often have limitations in terms of target specificity, production of variable or false-positive results, and the requirement for extensive optimisation or post-amplification analysis. This study introduces 3′ Tth endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, Tth endonuclease IV, for real-time multiplex detection and SNP identification. Complete analytical specificity, low limits of detection, single-base specificity, and simultaneous multiple target detection have been demonstrated in this study using 3TEC-PCR to identify bacterial meningitis associated pathogens. This is the first report of a two-oligonucleotide, real-time multiplex PCR technology with single-base specificity using Tth endonuclease IV.

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