Descriptive assessment of uncertainties of qualitative real-time PCR for detection of plant pathogens and quality performance monitoring

EPPO Bulletin ◽  
2014 ◽  
Vol 44 (3) ◽  
pp. 502-509 ◽  
Author(s):  
N. Mehle ◽  
T. Dreo ◽  
C. Jeffries ◽  
M. Ravnikar
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Performance Monitoring ◽  
Plant Pathogens ◽  
Quality Performance ◽  
Descriptive Assessment

Evaluation of droplet digital PCR for the detection of black canker disease in tomato

Plant Disease ◽  
2021 ◽  
Author(s):  
Li Wang ◽  
Tian Qian ◽  
Pei Zhou ◽  
Wenjun Zhao ◽  
Xianchao Sun
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Plant Protection ◽  
Detection Threshold ◽  
Digital Pcr ◽  
Taqman Probe ◽  
Tomato Seed ◽  
Canker Disease ◽  
Probe Set

Clavibacter michiganensis subsp. michiganensis (Cmm), the cause of bacterial canker disease, is one of the most destructive pathogens in greenhouse and field tomato. The pathogen is now present in all main production areas of tomato and is quite widely distributed in the EPPO（European and Mediterranean Plant Protection Organization）region. The inspection and quarantine of the plant pathogens relies heavily on accurate detection tools. Primers and probes reported in previous studies do not distinguish the Cmm pathogen from other closely related subspecies of C. michiganensis, especially the non-pathogenic subspecies that were identified from tomato seeds recently. Here, we have developed a droplet digital polymerase chain reaction (ddPCR) method for the identification of this specific bacterium with primers/TaqMan probe set designed based on the pat-1 gene of Cmm. This new primers/probe set has been evaluated by qPCRthe real time PCR（qPCR） and ddPCR. The detection results suggest that the ddPCR method established in this study was highly specific for the target strains. The result showed the positive amplification for all 5 Cmm strains，and no amplification was observed for the other 43 tested bacteria, including the closely related C. michiganensis strains. The detection threshold of ddPCR was 10.8 CFU/mL for both pure Cmm cell suspensions and infected tomato seed, which was 100 times-fold more sensitive than that of the real-time PCR (qPCR ) performed using the same primers and probe. The data obtained suggest that our established ddPCR could detect Cmm even with low bacteria load, which could facilitate both Cmm inspection for pathogen quarantine and the routine pathogen detection for disease control of black canker in tomato.

scholarly journals Digital PCR Technology for Detection of Palm-Infecting Phytoplasmas Belonging to Group 16SrIV that Occur in Florida

Plant Disease ◽  
2018 ◽  
Vol 102 (5) ◽  
pp. 1008-1014 ◽  
Author(s):  
Brian W. Bahder ◽  
Ericka E. Helmick ◽  
De-Fen Mou ◽  
Nigel A. Harrison ◽  
Robert Davis
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Plant Pathogens ◽  
Rapid Determination ◽  
Disease Spread ◽  
Taqman Assay ◽  
Insect Vector ◽  
Palm Trees ◽  
Pcr Assays

Phytoplasmas are an economically important group of plant pathogens that negatively impact a wide variety of plants in agricultural and natural ecosystems. In Florida, palm trees are essential elements in the nursery and landscaping industries that suffer from diseases caused by phytoplasmas that are related to each other but are classified in two different subgroups, 16SrIV-A and 16SrIV-D. In this study, a TaqMan assay was developed for digital polymerase chain reaction (dPCR) to detect both palm-infecting phytoplasmas found in Florida. When compared with real-time PCR assays and nested PCR assays, dPCR was capable of detecting the phytoplasmas at much lower concentrations than was possible by using real-time PCR and nested PCR. Additionally, the assay was capable of detecting 16SrIV-B phytoplasma as well as isolates representing the 16SrI and 16SrIII phytoplasma groups. Due to sequence identity of primer annealing regions across diverse phytoplasmas, the assay is likely to be successful for detection of a wide variety of phytoplasmas. The increased sensitivity of this dPCR assay over real-time PCR will allow for earlier detection of phytoplasma infection in palm trees, as well as for screening of salivary glands of candidate insect vector species. These advantages should aid timely management decisions to reduce disease spread and rapid determination of phytoplasma transmission by vectors.

scholarly journals Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™

BMC Genomics ◽  
2007 ◽  
Vol 8 (1) ◽  
pp. 276 ◽  
Author(s):  
Ronald van Doorn ◽  
Marianna Szemes ◽  
Peter Bonants ◽  
George A Kowalchuk ◽  
Joana F Salles ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Multiplex Detection

A new real-time PCR assay for detecting fungi in genus Ceratocystis

Plant Disease ◽  
2021 ◽  
Author(s):  
Karthikeyan Dharmaraj ◽  
Alice Merrall ◽  
Julie A. Pattemore ◽  
Joanne Mackie ◽  
Brett J.R Alexander ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Stem Tissue ◽  
Natural Ecosystems ◽  
Pcr Assay ◽  
Practical Applications ◽  
Wide Range

The genus Ceratocystis contains several significant plant pathogens, causing wilt and canker disease on a wide range of plants species. Currently, there are over 40 known species of Ceratocystis, some of which are becoming increasingly important in agricultural or natural ecosystems. The diagnostics for most Ceratocystis species currently relies on time consuming and labour-intensive culturing approaches. To provide more time efficient and sensitive molecular diagnostic tools for Ceratocystis, a generic Taq-Man real-time PCR assay was developed using the ITS gene. This novel two-probe Taq-man assay amplified DNA from all tested Ceratocystis species. Some non-specific amplification of a few species from closely related genera was observed under certain conditions; however, these false positive detections could be ruled out using the additional PCR primers developed for further sequence based identification of the detected species. The assay was highly sensitive as it detected 0.2 pg/µl of Ceratocystis DNA in water as well as in host DNA matrix. Further validation with artificially inoculated fig stem tissue demonstrated that the assay was also able to effectively detect the pathogen in infected asymptomatic stem tissue. This newly developed real-time PCR assay has practical applications in biosecurity, conservation, and agriculture, enabling to detect Ceratocystis species directly from plant material, to facilitate more sensitive screening of imported plant germplasm, and allow rapid tracking of pathogens in case of disease outbreaks.

scholarly journals Detection of plant pathogens using real-time PCR: how reliable are lateCtvalues?

2016 ◽  
Vol 66 (3) ◽  
pp. 359-367 ◽  
Author(s):  
M. Grosdidier ◽  
J. Aguayo ◽  
B. Marçais ◽  
R. Ioos
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens

scholarly journals Real-time PCR applied to study on plant pathogens: potential applications in diagnosis – a review

2016 ◽  
Vol 51 (No. 4) ◽  
pp. 177-190 ◽  
Author(s):  
S.M. Mirmajlessi ◽  
E. Loit ◽  
M. Mänd ◽  
S.M. Mansouripour
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Potential Applications

scholarly journals The Allele-Specific Probe and Primer Amplification Assay, a New Real-Time PCR Method for Fine Quantification of Single-Nucleotide Polymorphisms in Pooled DNA

2011 ◽  
Vol 78 (4) ◽  
pp. 1063-1068 ◽  
Author(s):  
A. Billard ◽  
V. Laval ◽  
S. Fillinger ◽  
P. Leroux ◽  
H. Lachaise ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Amplification Refractory Mutation System ◽  
Specific Probe ◽  
Monitoring Methods ◽  
Single Nucleotide ◽  
Allele Specific ◽  
Amplification Assay ◽  
Pcr Method

ABSTRACTThe evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.

scholarly journals Establishment of a quadruplex real-time PCR assay to distinguish the fungal pathogens Diaporthe longicolla, D. caulivora, D. eres, and D. novem on soybean

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257225
Author(s):  
Behnoush Hosseini ◽  
Ralf T. Voegele ◽  
Tobias I. Link
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Economic Damage ◽  
Pcr Assay ◽  
Plant Materials ◽  
Fungal Plant Pathogens ◽  
Pure Cultures ◽  
Soybean Leaves

Diaporthe species are fungal plant pathogens of many important crops. Seed decay is one of the most important diseases on soybean. It is caused by various species of the genus Diaporthe and responsible for significant economic damage. In central Europe the four species D. longicolla, D. caulivora, D. eres, and D. novem are considered the principal species of Diaporthe on soybean. Fast and accurate detection of these pathogens is of utmost importance. In this study four species-specific TaqMan primer-probe sets that can be combined into a quadruplex assay were designed based on TEF sequences. The specificity and efficiency of the primer-probe sets were tested using PCR products and genomic DNA from pure cultures of the four Diaporthe species and other soybean fungal pathogens. Our results indicate that the primer-probe sets DPCL, DPCC, DPCE, and DPCN allow discrimination of D. longicolla, D. caulivora, D. eres, and D. novem, respectively, and can be used to detect and quantify these four Diaporthe species in parallel using quadruplex real-time PCR. In addition, the quadruplex real-time PCR assay was evaluated on different plant materials including healthy and infected soybean seeds or seed lots, soybean stems, and soybean leaves. This assay is a rapid and effective method to detect and quantify Diaporthe species from samples relevant for disease control.

Validation of Real-time PCR Assays for Bioforensic Detection of Model Plant Pathogens

2013 ◽  
Vol 59 (2) ◽  
pp. 463-469 ◽  
Author(s):  
Mindy James ◽  
Trenna Blagden ◽  
Ian Moncrief ◽  
James P. Burans ◽  
Katherine Schneider ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Model Plant ◽  
Pcr Assays

Diagnostics of banana Blood disease

Plant Disease ◽  
2021 ◽  
Author(s):  
Vivian A Rincon-Florez ◽  
Jane D Ray ◽  
Lilia Costa Carvalhais ◽  
Cecilia A O'Dwyer ◽  
Siti Subandiyah ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Limit Of Detection ◽  
Diagnostic Methods ◽  
Detection Accuracy ◽  
Pcr Assay ◽  
Blood Disease ◽  
Specificity And Sensitivity ◽  
Validation Parameters

Blood disease in bananas caused by Ralstonia syzygii subsp. celebesensis (Rsce) is a bacterial wilt disease that causes major yield losses of banana in Indonesia and peninsular Malaysia. The disease has significantly increased its geographic distribution in the last decade. Diagnostic methods are an important component of disease management in vegetatively propagated crops such as banana to constrain incursions of plant pathogens. Therefore, the objectives of this study were: i) to design and rigorously validate a novel banana Blood disease (BBD) real-time PCR assay with a high level of specificity and sensitivity of detection. ii) to validate published PCR based diagnostic methods targeting either the intergenic region in the megaplasmid (“121 assay” with primer set 121) or the phage tail protein coding sequence in the bacterial chromosome (“Kubota assay” and “BDB2400 assay” with primer set BDB2400). Assay validation included 339 samples (174 Blood disease bacterium, 51 bacteria associated with banana plants, 51 members of the Ralstonia solanacearum species complex and 63 samples from symptomatic and healthy plant material). Validation parameters were analytical specificity (inclusivity and exclusivity), selectivity, limit of detection, accuracy, and ruggedness. The “121 assay” and our newly developed “BBD real-time PCR assay” detected all Rsce strains with no cross specificity during validation. Two different PCR assays using the primer set BDB2400 lacked specificity and selectivity. This study reveals that our novel “BBD real-time PCR assay” and the conventional PCR “121 assay” are reliable methods for Blood disease diagnostics as they comply with all tested validation parameters.

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