A robust method for identification and in-planta detection of Verticillium dahliae in the infected olive trees, using real-time PCR and nested PCR

Pathogen-free certified planting material and accurate detection of Verticillium dahliae pathotypes infecting the plant are key components of successful management of Verticillium wilt of olive. Use of a nested-polymerase chain reaction (PCR) procedure developed in earlier studies for in planta detection of the defoliating (D) and nondefoliating (ND) V. dahliae pathotypes resulted in ambiguous detection of the pathogen in some cases, due to heterologous amplification of the D-associated marker in ND-infected olive plants. In the present study, an improved procedure was developed that eliminates ambiguity and reduces time and cost for detection of D and ND V. dahliae in olive. The improved procedure is based on the simultaneous amplification of both an ND- and a new D-specific marker by means of duplex, nested PCR. The procedure was effective in the rapid and unequivocal detection of the D and ND V. dahliae in both artificially inoculated, own-rooted olive plants and naturally infected adult olive trees of different cultivar, age, and growing conditions. Furthermore, the duplex, nested-PCR procedure detected simultaneously the D and ND pathotypes in adult olive trees naturally infected by both pathotypes and in young olive plants that were double-inoculated with D and ND isolates under controlled conditions.

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Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

Open Medicine ◽

10.2478/s11536-007-0029-z ◽

2007 ◽

Vol 2 (3) ◽

pp. 271-279 ◽

Cited By ~ 1

Author(s):

Koray Ergunay ◽

Gulcin Altinok ◽

Bora Gurel ◽

Ahmet Pinar ◽

Arzu Sungur ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

San Francisco ◽

Real Time Pcr ◽

Nested Pcr ◽

Parvovirus B19 ◽

Etiologic Agent ◽

Hydrops Fetalis ◽

Nucleic Acid Detection

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

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Erratum: 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 ◽

10.1094/pdis-05-14-0528.1-re ◽

2015 ◽

Vol 99 (10) ◽

pp. 1452-1452

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Verticillium Dahliae ◽

Real Time Pcr ◽

Chain Reaction ◽

Accurate Detection ◽

Polymerase Chain

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Detection and Quantification of Human Adenoviruses in Surface Waters by Nested PCR, TaqMan Real-Time PCR and Cell Culture Assays

Water Air & Soil Pollution ◽

10.1007/s11270-007-9608-5 ◽

2007 ◽

Vol 191 (1-4) ◽

pp. 83-93 ◽

Cited By ~ 27

Author(s):

M. Muscillo ◽

M. Pourshaban ◽

M. Iaconelli ◽

S. Fontana ◽

A. Di Grazia ◽

...

Keyword(s):

Cell Culture ◽

Real Time ◽

Surface Waters ◽

Real Time Pcr ◽

Nested Pcr ◽

Human Adenoviruses ◽

Detection And Quantification

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Efficient nested-PCR-based method development for detection and genotype identification of Acanthamoeba from a small volume of aquatic environmental sample

Scientific Reports ◽

10.1038/s41598-021-00968-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tsui-Kang Hsu ◽

Jung-Sheng Chen ◽

Hsin-Chi Tsai ◽

Chi-Wei Tao ◽

Yu-Yin Yang ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Nested Pcr ◽

In Silico ◽

Environmental Samples ◽

Method Development ◽

Small Volume ◽

Detection Efficiency ◽

Genotyping Method ◽

Pcr Method

AbstractAcanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

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A Set of Conventional and Multiplex Real-Time PCR Assays for Direct Detection of Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis in Citrus Fruits

Plant Disease ◽

10.1094/pdis-05-18-0798-re ◽

2019 ◽

Vol 103 (2) ◽

pp. 345-356 ◽

Cited By ~ 4

Author(s):

Yosra Ahmed ◽

Jacqueline Hubert ◽

Céline Fourrier-Jeandel ◽

Megan M. Dewdney ◽

Jaime Aguayo ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Culture Media ◽

Direct Detection ◽

Elongation Factor ◽

Single Copy ◽

The United States ◽

Performance Criteria ◽

In Planta ◽

Conventional Pcr

Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis are causal agents of citrus scab and spot diseases. The three pathogens are listed as quarantine pests in many countries and are subject to phytosanitary measures to prevent their entry. Diagnosis of these diseases based on visual symptoms is problematic, as they could be confused with other citrus diseases. Isolation of E. fawcettii, E. australis, and P. angolensis from infected tissues is challenging because they grow slowly on culture media. This study developed rapid and specific detection tools for the in planta detection of these pathogens, using either conventional PCR or one-tube multiplex real-time PCR. Primers and hybridization probes were designed to target the single-copy protein-coding gene MS204 for E. fawcettii and E. australis and the translation elongation factor (Tef-1α) gene for P. angolensis. The specificity of the assays was evaluated by testing against DNA extracted from a large number of isolates (102) collected from different citrus-growing areas in the world and from other hosts. The newly described species E. citricola was not included in the specificity test due to its unavailability from the CBS collection. The detection limits of conventional PCR for the three pathogens were 100, 100, and 10 pg μl−1 gDNA per reaction for E. fawcettii, E. australis, and P. angolensis, respectively. The quadruplex qPCR was fully validated assessing the following performance criteria: sensitivity, specificity, repeatability, reproducibility, and robustness. The quadruplex real-time PCR proved to be highly sensitive, detecting as low as 243, 241, and 242 plasmidic copies (pc) μl−1 of E. fawcettii, E. australis, and P. angolensis, respectively. Sensitivity and specificity of this quadruplex assay were further confirmed using 176 naturally infected citrus samples collected from Ethiopia, Cameroon, the United States, and Australia. The quadruplex assay developed in this study is robust, cost-effective, and capable of high-throughput detection of the three targets directly from citrus samples. This new detection tool will substantially reduce the turnaround time for reliable species identification and allow rapid response and appropriate action.

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

Plant Disease ◽

10.1094/pdis-03-19-0563-re ◽

2019 ◽

Vol 103 (12) ◽

pp. 3031-3040 ◽

Cited By ~ 1

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.

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In Planta Distribution of ‘Candidatus Liberibacter asiaticus’ as Revealed by Polymerase Chain Reaction (PCR) and Real-Time PCR

Phytopathology ◽

10.1094/phyto-98-5-0592 ◽

2008 ◽

Vol 98 (5) ◽

pp. 592-599 ◽

Cited By ~ 151

Author(s):

Satyanarayana Tatineni ◽

Uma Shankar Sagaram ◽

Siddarame Gowda ◽

Cecile J. Robertson ◽

William O. Dawson ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Citrus Tree ◽

Candidatus Liberibacter Asiaticus ◽

In Planta ◽

Chain Reaction ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Polymerase Chain

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic α-proteobacterium that is yet to be cultured. In this study, a combination of traditional polymerase chain reaction (PCR) and real-time PCR targeting the putative DNA polymerase and 16S rDNA sequence of ‘Candidatus Liberibacter asiaticus,’ respectively, were used to examine the distribution and movement of the HLB pathogen in the infected citrus tree. We found that ‘Ca. Liberibacter asiaticus’ was distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. Quantification analysis of the HLB bacterium indicated that it was distributed unevenly in planta and ranged from 14 to 137,031 cells/μg of total DNA in different tissues. A relatively high concentration of ‘Ca. Liberibacter asiaticus’ was observed in fruit peduncles. Our data from greenhouse-infected plants also indicated that ‘Ca. Liberibacter asiaticus’ was transmitted systemically from infection site to different parts of the plant. Understanding the distribution and movement of the HLB bacterium inside an individual citrus tree is critical for discerning its virulence mechanism and to develop management strategies for HLB.

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