scholarly journals Real-Time Fluorescent Polymerase Chain Reaction Detection of Phytophthora ramorum and Phytophthora pseudosyringae Using Mitochondrial Gene Regions

2006 ◽  
Vol 96 (4) ◽  
pp. 336-345 ◽  
Author(s):  
Paul W. Tooley ◽  
Frank N. Martin ◽  
Marie M. Carras ◽  
Reid D. Frederick
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Multiplex Assay ◽  
Phytophthora Ramorum ◽  
Chain Reaction ◽  
Plant Dna ◽  
Field Samples ◽  
Polymerase Chain ◽  
Fluorescent Polymerase Chain Reaction

A real-time fluorescent polymerase chain reaction (PCR) detection method for the sudden oak death pathogen Phytophthora ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg of genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a three-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae, and plant DNA in a single tube. P. ramorum was detected down to a 10-5 dilution of extracted tissue of artificially infected rhododendron ‘Cunningham's White’, and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.

scholarly journals Quantitative Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 588
Author(s):  
Wipaporn Wongfieng ◽  
Rungtiwa Nutalai ◽  
Amonrat Jumnainsong ◽  
Chanvit Leelayuwat
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Expression Patterns ◽  
Multiplex Assay ◽  
Clinical Samples ◽  
Lower Sensitivity ◽  
Chain Reaction ◽  
Kir Genes ◽  
Polymerase Chain

(1) Background: KIR2DL4/KIR3DL3 are the framework genes present in all KIR haplotypes, with unique expression patterns being present only in women and CD56bright NK cells. KIR genes have a high degree of DNA sequence identity. Consequently, they are one of the most challenging genes for molecular detection—especially regarding expressions; (2) Methods: We developed an effective method to determine KIR3DL3/KIR2DL4 expressions based on a multiplex quantitative real-time Reverse transcription polymerase chain reaction (qRT-PCR )with fluorescent probes using NK92; (3) Results: Standardizations of the singleplex KIR2DL4 and KIR3DL3 were performed to evaluate the sensitivity and specificity for further development of the multiplex assay. The limit of detection was at 500 copies each. There was cross-amplification with the presence of related KIR genes at a level of 5 × 107 copies. This is not biologically significant because this high level of KIR expression has not been found in clinical samples. The multiplex assay was reproducible equivalent to its singleplex (KIR2DL4; R2 = 0.995, KIR3DL3; R2 = 0.996, but lower sensitivity of 103 copies). Furthermore, the validation of the developed method on samples of blood donors showed high sensitivity (100%) and specificity (99.9%); (4) Conclusions: The developed method is reliable and highly specific suitable for evaluation of the KIR2DL4/3DL3 mRNA expressions in further applications.

scholarly journals Development of a One-Step Real-Time Polymerase Chain Reaction Assay for Diagnosis of Phytophthora ramorum

2006 ◽  
Vol 96 (9) ◽  
pp. 975-981 ◽  
Author(s):  
Kelvin J. D. Hughes ◽  
Jennifer A. Tomlinson ◽  
Ruth L. Griffin ◽  
Neil Boonham ◽  
Alan J. Inman ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Host Plant ◽  
Real Time ◽  
Plant Material ◽  
Limit Of Detection ◽  
Phytophthora Ramorum ◽  
Chain Reaction ◽  
Simple Method ◽  
Isolation Technique ◽  
Polymerase Chain

Phytophthora ramorum is a recently described pathogen causing bleeding cankers, dieback, and leaf blight on trees and shrubs in parts of Europe and North America, where the disease is commonly known as sudden oak death. This article describes the development of a single-round real-time polymerase chain reaction (PCR) assay based on TaqMan chemistry, designed within the internal transcribed spacer 1 region of the nuclear ribosomal (nr)RNA gene for detection of P. ramorum in plant material. Unlike previously described methods for the molecular detection of P. ramorum, this assay involves no post amplification steps or multiple rounds of PCR. The assay was found to have a limit of detection of 10 pg of P. ramorum DNA, and could detect P. ramorum in plant material containing 1% infected material by weight within 36 cycles of PCR. The assay also was used to test DNA from 28 other Phytophthora spp. to establish its specificity for P. ramorum. A quick and simple method was used to extract DNA directly from host plant material, and detection of P. ramorum was carried out in multiplex with an assay for a gene from the host plant in order to demonstrate whether amplifiable DNA had been extracted. Amplifiable DNA was extracted from 84.4% of samples, as demonstrated by amplification of host plant DNA. The real-time protocol was used to test 320 plant samples (from 19 different plant species) from which DNA extraction had been successful, and was shown to give results comparable with a traditional isolation technique for diagnosis of P. ramorum in plant material from common U.K. hosts.

scholarly journals Development of Quantitative Real-Time Polymerase Chain Reaction for Detection of and Discrimination between Erysipelothrix Rhusiopathiae and Other Erysipelothrix Species

2009 ◽  
Vol 21 (5) ◽  
pp. 701-706 ◽  
Author(s):  
Ho To ◽  
Tomohiro Koyama ◽  
Shinya Nagai ◽  
Kotaro Tuchiya ◽  
Tetsuo Nunoya
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Enrichment Culture ◽  
Bacterial Species ◽  
Erysipelothrix Rhusiopathiae ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Practical Applications ◽  
Polymerase Chain

Quantitative real-time polymerase chain reaction (qPCR) assays were developed and validated in combination with enrichment culture for the detection and discrimination of Erysipelothrix rhusiopathiae and other Erysipelothrix species from tissue samples. The targets for SYBR green qPCR assays were the 16S ribosomal RNA gene for Erysipelothrix species and a gene involved in capsular formation for E. rhusiopathiae. The specificity of the assays was assessed with Erysipelothrix species and other related bacterial species. The limit of detection was found to be 5 colony-forming units per reaction. Amplification of DNA extracted from spleen and joint samples spiked with increasing quantities of Erysipelothrix cells was shown to be equally sensitive to DNA extracted from a pure bacterial culture. The assays were evaluated with 88 tissue samples from 3 experimentally infected pigs and 50 mice and with 36 tissue samples from 3 naturally infected pigs and 11 noninfected pigs. Results were compared with those of direct qPCR and conventional culture. The qPCR after enrichment increased the diagnostic sensitivity over that of culture and qPCR, thereby significantly reducing the total time taken for the detection of E. rhusiopathiae and other Erysipelothrix species. Therefore, this technique could be used for practical applications.

scholarly journals Evaluation of Polymerase Chain Reaction (PCR)-Based Methods for Rapid, Accurate Detection and Monitoring of Verticillium dahliae in Woody Hosts by Real-Time PCR

Plant Disease ◽  
2015 ◽  
Vol 99 (6) ◽  
pp. 866-873 ◽  
Author(s):  
Baker Aljawasim ◽  
Paul Vincelli
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Verticillium Dahliae ◽  
Sugar Maple ◽  
Broad Host Range ◽  
Chain Reaction ◽  
Accurate Detection ◽  
Field Samples ◽  
Vascular Discoloration ◽  
Polymerase Chain

Verticillium wilt, caused by Verticillium dahliae, is one of the most economically important diseases of woody hosts such as ash (Fraxinus spp.), sugar maple (Acer saccharum), and redbud (Cercis canadensis). The causal agent has a broad host range, including not only woody hosts but also important vegetable and field crops, and it is distributed worldwide. Diagnosis of V. dahliae in infected woody hosts is often based on the occurrence of vascular discoloration and time-consuming isolation. However, not all woody hosts exhibit vascular discoloration, and not all vascular discoloration symptoms are due to infection by V. dahliae. In this study, real-time polymerase chain reaction (PCR)-based assays were evaluated and employed for rapid and accurate detection of V. dahliae in different woody hosts. High-quality DNA was extracted in large quantities from presumptively infected woody hosts by collecting drill-press shavings from sample tissue, bead beating, and extracting using a cetyltrimethylammonium bromide method. Six published primer sets were evaluated against genomic DNA of V. dahliae as well as selected negative controls, and two sets (VertBt-F/VertBt-R and VDS1/VDS2) showed promise for further evaluation using DNA extracts from field samples. The VertBt primers amplified a species-specific 115-bp fragment of the expected size, while the VDS primers amplified the expected specific 540-bp fragment. However, the VertBt primer set exhibited higher sensitivity in detection of V. dahliae even in asymptomatic trees. The PCR-based methods developed here could be used as rapid tools for pathogen detection and monitoring, thus informing plant pathogen management decisions.

Detection of Naegleria fowleri, Acanthamoeba spp, and Balamuthia mandrillaris in Formalin-Fixed, Paraffin-Embedded Tissues by Real-Time Multiplex Polymerase Chain Reaction

2019 ◽  
Vol 152 (6) ◽  
pp. 799-807 ◽  
Author(s):  
Andrew P Norgan ◽  
Lynne M Sloan ◽  
Bobbi S Pritt
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Polymerase Chain ◽  
Formalin Fixed

Abstract Objectives Pathogenic free-living amebae (FLAs) cause skin, ocular, and central nervous system (CNS) infections with significant morbidity and mortality. Diagnosis of FLA infections by pathologic examination of tissue sections can be aided using molecular assays. This study investigated the performance characteristics of a multiplex real-time polymerase chain reaction (PCR) assay (FLA-PCR) for detection and differentiation of FLAs in clinical specimens. Methods FLA-PCR was performed on 39 human specimens comprising one cutaneous, 14 corneal, and 24 CNS formalin-fixed, paraffin-embedded (FFPE) tissues with a histopathologic diagnosis of FLA infection and four CNS FFPE tissues with inflammation but no evidence of FLAs. In addition, clinical specificity and assay limit of detection were determined. Results FLA detection sensitivities ranged from 79% to 84% in FFPE tissues. No cross-reactivity was observed. Conclusions While sensitivity is limited, FLA-PCR assay may serve as a useful adjunct for detection or confirmation of FLA infections in FFPE tissues.

scholarly journals Real-time fluorogenic reverse transcription polymerase chain reaction assay for the specific detection of Bagaza virus

2012 ◽  
Vol 24 (5) ◽  
pp. 959-963 ◽  
Author(s):  
Dolores Buitrago ◽  
Ana Rocha ◽  
Cristina Tena-Tomás ◽  
Marta Vigo ◽  
Montserrat Agüero ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Diagnostic Methods ◽  
Nucleotide Sequencing ◽  
Alectoris Rufa ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Field Samples ◽  
Polymerase Chain

In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ring-necked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a commercial 5’-Taq nuclease-3’ minor groove binder DNA probe and primers targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely related flaviviruses ( Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated using field samples of red-legged partridges dead during the outbreak ( n = 11), as well as samples collected from partridges during surveillance programs ( n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RT-PCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid technique for BAGV genome detection, useful in both diagnosis and surveillance studies.

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