scholarly journals Quantitative Real-Time PCR Assay for the Detection of Pectobacterium parmentieri, a Causal Agent of Potato Soft Rot

Plants ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1880
Author(s):  
Anna A. Lukianova ◽  
Peter V. Evseev ◽  
Alexander A. Stakheev ◽  
Irina B. Kotova ◽  
Sergey K. Zavriev ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Detection Threshold ◽  
Soft Rot ◽  
Taqman Probe ◽  
Separate Species ◽  
Environmental Preferences ◽  
Pure Bacterial Cultures ◽  
Associated Species

Pectobacterium parmentieri is a plant-pathogenic bacterium, recently attributed as a separate species, which infects potatoes, causing soft rot in tubers. The distribution of P. parmentieri seems to be global, although the bacterium tends to be accommodated to moderate climates. Fast and accurate detection systems for this pathogen are needed to study its biology and to identify latent infection in potatoes and other plant hosts. The current paper reports on the development of a specific and sensitive detection protocol based on a real-time PCR with a TaqMan probe for P. parmentieri, and its evaluation. In sensitivity assays, the detection threshold of this protocol was 102 cfu/mL on pure bacterial cultures and 102–103 cfu/mL on plant material. The specificity of the protocol was evaluated against P. parmentieri and more than 100 strains of potato-associated species of Pectobacterium and Dickeya. No cross-reaction with the non-target bacterial species, or loss of sensitivity, was observed. This specific and sensitive diagnostic tool may reveal a wider distribution and host range for P. parmentieri and will expand knowledge of the life cycle and environmental preferences of this pathogen.

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.

New PCR-based assay for the identification of Pectobacterium carotovorum causing potato soft rot

Plant Disease ◽  
2021 ◽  
Author(s):  
M. Belén Suárez ◽  
Marta Diego ◽  
F. J. Feria ◽  
M J Martín-Robles ◽  
Sergio Moreno ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Intergenic Spacer ◽  
Soft Rot ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Accurate Identification ◽  
Intergenic Spacer Region

Soft rot on potato tuber is a destructive disease caused by pathogenic bacterial species of the genera Pectobacterium and Dickeya. Accurate identification of the causal agent is necessary to ensure adequate disease management, since different species may have distinct levels of aggressiveness and host range. One of the most important potato pathogens is P. carotovorum, a highly heterogeneous species capable of infecting multiple hosts. The complexity of this species, until recently divided into several subspecies, has made it difficult to develop precise diagnostic tests. This study proposes a PCR assay based on the new pair of primers Pcar1F/R to facilitate the identification of potato isolates of P. carotovorum according to the most recent taxonomic description of this species. The new primers were designed on a variable segment of the 16S rRNA gene and the intergenic spacer region (ITS) of available DNA sequences from classical and recently established species in the genus Pectobacterium. The results of the PCR analysis of genomic DNA from 32 Pectobacterium and Dickeya strains confirmed that the Pcar1F/R primers have sufficient nucleotide differences to discriminate between P. carotovorum and other Pectobacterium species associated with damage to potato crops, with the exception of P. versatile, which improves the specificity of the currently available primers. The proposed assay was originally developed as a conventional PCR but was later adapted to the real-time PCR format for application in combination with the existing real-time PCR test for the potato-specific pathogen P. parmentieri. This should be useful for the routine diagnosis of potato soft rot.

scholarly journals Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

2021 ◽  
Vol 11 ◽  
Author(s):  
Reza Fotouhi-Ardakani ◽  
Seyedeh Maryam Ghafari ◽  
Paul Donald Ready ◽  
Parviz Parvizi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Taqman Probe ◽  
Amino Acid Permease ◽  
Specific Primers ◽  
Accurate Identification ◽  
Parasitic Load ◽  
Species Specific

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

scholarly journals Pathogenicity and a TaqMan Real-Time PCR for Specific Detection of Pantoea allii, a Bacterial Pathogen of Onions

Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3031-3040 ◽  
Author(s):  
Shabnam Rahimi-Khameneh ◽  
Sanni Hsieh ◽  
Renlin Xu ◽  
Tyler J. Avis ◽  
Sean Li ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Phylogenetic Analyses ◽  
Similarity Index ◽  
Taqman Probe ◽  
Economic Losses ◽  
In Planta ◽  
Pcr Assay ◽  
Blind Test ◽  
Reliable Detection

Bacterial diseases of onion are reported to cause significant economic losses. Pantoea allii Brady, one of the pathogens causing the center rot on onions, has not yet been reported in Canada. We report the pathogenicity of P. allii on commercially available Canadian green onions (scallions). All P. allii-inoculated plants, irrespective of the inoculum concentration, exhibited typical leaf chlorotic discoloration on green onion leaves, which can reduce their marketability. Reisolation of P. allii from infected scallion tissues and reidentification by sequencing and phylogenetic analyses of the leuS gene suggest that the pathogen can survive in infected tissues 21 days after inoculation. This is the first report of P. allii as a potential pathogen of green onions. This study also reports the development and validation of a TaqMan real-time PCR assay targeting the leuS gene for reliable detection of P. allii in pure cultures and in planta. A 642-bp leuS gene fragment was targeted because it showed high nucleotide diversity and positively correlated with genome-based average nucleotide identity with respect to percent similarity index and identity of Pantoea species. The assay specificity was validated using 61 bacterial and fungal strains. Under optimal conditions, the selected primers and FAM-labeled TaqMan probe were specific for the detection of nine reference P. allii strains by real-time PCR. The 52 strains of other Pantoea spp. (n = 25), non-Pantoea spp. (n = 20), and fungi/oomycetes (n = 7) tested negative (no detectable fluorescence). Onion tissues spiked with P. allii, naturally infested onion bulbs, greenhouse infected green onion leaf samples, as well as an interlaboratory blind test were used to validate the assay specificity. The sensitivities of a 1-pg DNA concentration and 30 CFU are comparable to previously reported real-time PCR assays of other bacterial pathogens. The TaqMan real-time PCR assay developed in this study will facilitate reliable detection of P. allii and could be a useful tool for screening onion imports or exports for the presence of this pathogen.

scholarly journals Airway dysbiosis: Haemophilus influenzae and Tropheryma in poorly controlled asthma

2015 ◽  
Vol 47 (3) ◽  
pp. 792-800 ◽  
Author(s):  
Jodie L. Simpson ◽  
Joshua Daly ◽  
Katherine J. Baines ◽  
Ian A. Yang ◽  
John W. Upham ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Bacterial Diversity ◽  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Inflammatory Disorder ◽  
Eosinophilic Asthma ◽  
Chronic Inflammatory Disorder ◽  
16S Rrna Pyrosequencing ◽  
Airway Microbiome

Asthma is a chronic inflammatory disorder of the airways where bacteria may act as protagonists of chronic inflammation. Little is known about the relation of airway inflammation to the presence of specific bacterial taxa. We sought to describe the sputum microbiome in adults with poorly controlled asthma.DNA was extracted from induced sputum and microbial communities were profiled using 16S rRNA pyrosequencing. Bacterial species were characterised, and the relationship between microbial populations, asthma inflammatory subtypes and other covariates was explored. Real-time PCR was used to identify Tropheryma whipplei and Haemophilus influenzae in sputum.Adults with neutrophilic asthma had reduced bacterial diversity and species richness. Tropheryma was identified and confirmed with real-time PCR in 12 (40%) participants. Haemophilus occurred most often in a group of younger atopic males with an increased proportion of neutrophils. PCR confirmed the presence of H. influenzae in 35 (76%) participants with poorly controlled asthma.There are phenotype-specific alterations to the airway microbiome in asthma. Reduced bacterial diversity combined with a high prevalence of H. influenzae was observed in neutrophilic asthma, whereas eosinophilic asthma had abundant T. whipplei.

Quantification of Fusarium graminearum in infected wheat by species specific real-time PCR applying a TaqMan Probe

2004 ◽  
Vol 59 (1) ◽  
pp. 141-146 ◽  
Author(s):  
G.H. Reischer ◽  
M. Lemmens ◽  
A. Farnleitner ◽  
A. Adler ◽  
R.L. Mach
Keyword(s):  
Real Time ◽  
Fusarium Graminearum ◽  
Real Time Pcr ◽  
Taqman Probe ◽  
Species Specific
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