Molecular assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera

Abstract/KeywordsTo determine if living microorganisms of phytosanitary concern are present in wood after eradication treatment and to evaluate the efficacy of such treatments, the method of choice is to grow microbes in petri dishes for subsequent identification. However, some plant pathogens are difficult or impossible to grow in axenic cultures. A molecular methodology capable of detecting living fungi and fungus-like organisms in situ can provide a solution. RNA represents the transcription of genes and can therefore only be produced by living organisms, providing a proxy for viability. We designed and used RNA-based molecular diagnostic assays targeting genes essential to vital processes and assessed their presence in wood colonized by fungi and oomycetes through reverse transcription and real-time polymerase chain reaction (PCR). A stability analysis was conducted by comparing the ratio of mRNA to gDNA over time following heat treatment of wood infected with the Oomycete Phytophthora ramorum and the fungus Grosmannia clavigera. The real-time PCR results indicated that the DNA remained stable over a period of 10 days post treatment in heat-treated wood samples, whereas mRNA could not be detected after 24 hours for P. ramorum or 96 hours for G. clavigera. Therefore, this method provides a reliable way to evaluate the viability of these pathogens and test the effectiveness of existing and emerging wood treatments. This can have important phytosanitary impacts on assessing both timber and non-timber forest products of commercial value in international wood trade.

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A Comparative Analysis of Detection Techniques Used in US Regulatory Programs to Determine Presence of Phytophthora ramorum in Camellia japonica ‘Nucio's Gem’ in an Infested Nursery in Southern California

Plant Health Progress ◽

10.1094/php-2006-1016-01-rs ◽

2006 ◽

Vol 7 (1) ◽

pp. 9 ◽

Cited By ~ 7

Author(s):

Russ Bulluck ◽

Pat Shiel ◽

Phil Berger ◽

David Kaplan ◽

Greg Parra ◽

...

Keyword(s):

Growth Medium ◽

Phytophthora Ramorum ◽

Molecular Diagnostic ◽

Medium Ph ◽

Camellia Japonica ◽

Disease Symptoms ◽

Diagnostic Assays ◽

Detection Techniques ◽

The Usa ◽

Diagnostic Sensitivity And Specificity

Phytophthora ramorum (Pram) is a pathogen of regulatory concern in the USA, and accurate diagnostics is a key component in the response to potential pathogen outbreaks. Although the molecular diagnostic protocols used in regulatory programs have been evaluated using regulatory samples, to date, no direct comparison of these methods has been analyzed within a nursery setting. A block of 300 camellia plants within a California nursery known to be infested with Pram was simultaneously assayed for visual symptoms, growth medium pH, and moss presence as well as culture isolation and molecular analysis prior to plant destruction. Disease symptoms such as foliar lesions and leaf drop were recorded for each plant prior to foliar and growth medium sampling. All diagnostic assays were highly correlated with one another and disease symptoms, with nested PCR having the best correlation with symptoms, followed by Real-Time PCR then culture. No correlation with disease or diagnostic assays was observed with moss presence or medium pH. Analysis of results allowed diagnostic sensitivity and specificity of the assays to be determined and the performance of each method for diagnosis of Phytophthora spp. or Phytophthora ramorum in camellia tissues and associated potting medium could be compared. Accepted for publication 12 July 2006. Published 16 October 2006.

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

Plant Disease ◽

10.1094/pdis-08-21-1639-re ◽

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.

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Comparison of multiplexed-tandem real-time PCR panel with reference real-time PCR molecular diagnostic assays for detection of Giardia intestinalis and Tritrichomonas foetus in cats

Veterinary Parasitology ◽

10.1016/j.vetpar.2018.12.009 ◽

2019 ◽

Vol 266 ◽

pp. 12-17 ◽

Cited By ~ 4

Author(s):

Maira N. Meggiolaro ◽

Florian Roeber ◽

Victoria Kobylski ◽

Damien P. Higgins ◽

Jan Šlapeta

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Giardia Intestinalis ◽

Molecular Diagnostic ◽

Tritrichomonas Foetus ◽

Diagnostic Assays

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Serology- and Blood-PCR-Based Screening for Schistosomiasis in Pregnant Women in Madagascar—A Cross-Sectional Study and Test Comparison Approach

Pathogens ◽

10.3390/pathogens10060722 ◽

2021 ◽

Vol 10 (6) ◽

pp. 722

Author(s):

Tanja Hoffmann ◽

Imke Carsjens ◽

Raphaël Rakotozandrindrainy ◽

Mirko Girmann ◽

Njary Randriamampionona ◽

...

Keyword(s):

Pregnant Women ◽

Real Time ◽

Real Time Pcr ◽

Disease Burden ◽

Cross Sectional Study ◽

Molecular Diagnostic ◽

Sectional Study ◽

Cross Sectional ◽

Diagnostic Assays ◽

Edta Plasma

This work was conducted as a cross sectional study to define the disease burden of schistosomiasis in pregnant Madagascan women and to evaluate serological and molecular diagnostic assays. A total of 1154 residual EDTA blood samples from pregnant Madagascan women were assessed. The nucleic acid extractions were subjected to in-house real-time PCRs specifically targeting S. mansoni complex, S. haematobium complex, and African Schistosoma spp. on genus level, while the EDTA plasma samples were analyzed using Schistosoma-specific IgG and IgM commercial ELISA and immunofluorescence assays. The analyses indicated an overall prevalence of schistosomiasis in Madagascan pregnant women of 40.4%, with only minor regional differences and differences between serology- and blood PCR-based surveillance. The S. mansoni specific real-time PCR showed superior sensitivity of 74% (specificity 80%) compared with the genus-specific real-time PCR (sensitivity 13%, specificity 100%) in blood. The laborious immunofluorescence (sensitivity IgM 49%, IgG 87%, specificity IgM 85%, IgG 96%) scored only slightly better than the automatable ELISA (sensitivity IgM 38%, IgG 88%, specificity IgM 78%, IgG 91%). Infections with S. mansoni were detected only. The high prevalence of schistosomiasis recorded here among pregnant women in Madagascar calls for actions in order to reduce the disease burden.

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Can Testing Predict SARS-CoV-2 Infectivity? The Potential for Certain Methods to be a Surrogate for Replication-Competent Virus

Journal of Clinical Microbiology ◽

10.1128/jcm.00469-21 ◽

2021 ◽

Author(s):

Matthew J. Binnicker

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Viral Rna ◽

Molecular Diagnostic ◽

Public Health Agencies ◽

Diagnostic Assays ◽

Molecular Tests ◽

Primary Means ◽

Clinical Illness ◽

Antigen Tests

Since the beginning of the COVID-19 pandemic, molecular methods (e.g., real-time PCR) have been the primary means of diagnosing the disease. It is now well-established that molecular tests can continue to detect SARS-CoV-2 genomic RNA for weeks or months following the resolution of clinical illness. This has prompted public health agencies to recommend a symptom and/or time-based strategy for discontinuation of isolation precautions, which for hospitalized patients, results in significant use of personal protective equipment. Due to the inability of current molecular diagnostic assays to differentiate between the presence of remnant viral RNA (i.e., non-infectious) and replication-competent (i.e., infectious) virus, there has been interest in determining whether laboratory tests can be used to predict an individual’s likelihood of transmitting the virus to others. This review will highlight what is currently known about the potential for existing assays, such as real-time PCR and antigen tests, to predict active viral infection. In addition, data on the performance of new methods, such as molecular tests targeting viral RNA intermediates (e.g., subgenomic RNA), will be discussed.

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Illegal rattan extraction trends in the Ankasa Conservation Area in Ghana

Journal of Energy and Natural Resource Management ◽

10.26796/jenrm.v2i3.49 ◽

2018 ◽

Vol 2 (3) ◽

pp. 84-92

Author(s):

R. Obour, D. Amankwaa, A. Asare

Keyword(s):

Protected Areas ◽

Biological Diversity ◽

Forest Products ◽

Simple Random Sampling ◽

Sampling Techniques ◽

Snowball Sampling ◽

Conservation Area ◽

Forest Reserve ◽

The North ◽

Non Timber Forest Products

Protected Areas (PAs) are created for the protection and maintenance of biological diversity, but many of Ghana’s PAs are subjectto severe pressures and threats, the main pressures being the illegal extraction of natural resources. Rattans are indisputablyone of the most important Non-Timber Forest Products (NTFPs) in Ghana’s Protected Areas that is without doubt one of thereasons for which it has drawn the attention of researchers. In this study the illegal rattan extraction patterns in the AnkasaConservation Area (ACA) in Ghana was inspected. Simple random sampling and Snowball sampling techniques were used. Datacollection employed the use of semi-structured questionnaires, interviews and field enumeration of rattans as well as an analysisof Effective Patrol Man-days (EPMDS) from 2004 to 2012. The results showed a significant positive correlation (r = 0.75, p<0.05, r2 = 0.557) between patrol effort and rattan extraction encounters. In addition, there was a general reduction in illegalrattan extraction encounters from 2004 to 2012 at a rate of 4.3 per year. The highest illegal rattan extraction incidences wererecorded in 2006 (76 encounters), 2005 (35 encounters), 2008 (22 encounters), 2004 (18 encounters) and the least incidencewere recorded in both 2010 (3 encounters) and 2011 (3 encounters).The research also revealed that Eremospatha macrocarpawas the most extracted rattan species followed by Laccosperma secundiflorum. The major rattan extraction and trade routesoriginate in the northern parts and in the area east of the reserve and also south of Draw River Forest Reserve. Generally, rattanpoaching in Ankasa Conservation Area has declined, but there are still human incursions in the northern part of the reserve. Thestudy recommended an intensification of patrols in the north of the reserve. Also, enrichment planting and Agroforestry practicesof inter-cropping rattans with seasonal crops should be pursued vigorously for the local communities.

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Market Survey of Non-Timber Forest Products in the Sunyani Municipality

Journal of Energy and Natural Resource Management ◽

10.26796/jenrm.v3i2.58 ◽

2018 ◽

Vol 3 (2) ◽

pp. 44-53

Author(s):

S.D. Akoto

Keyword(s):

Building Materials ◽

Significant Proportion ◽

Forest Products ◽

Packaging Materials ◽

Medicinal Products ◽

Monthly Basis ◽

Rural Livelihood ◽

Non Timber Forest Products ◽

Communal Lands ◽

Forest Lands

This study sought to: (1) identify the types and sources of Non-Timber Forest Products (NTFPs) traded; (2) find the frequencyof the NTFPs trade and (3) identify the challenges in NTFPs trading in the Sunyani Municipality. The survey was carried outfrom February, 2014 to April, 2014 mainly at the Sunyani Central and Nana Bosoma Markets in the Sunyani Municipality. Thetarget population comprised NTFPs collectors (gatherers), sellers and consumers. Respondents were purposively sampled. Atotal of 100 respondents were engaged in this study. The NTFPs were grouped into six categories namely; food, medicine,building materials, packaging materials, artefacts and domestic utensils. Key informants’ interviews were also conducted atthe Sunyani Forest Services Division to triangulate the data already gathered. Statistical Package for Social Sciences was usedto analyze the data obtained. The study demonstrated that domestic utensils (37%), food (33%), medicinal products (12%),packaging materials (9%), artefacts (6%) and building materials (3%) were the types of NTFPs traded in the two market centers.The results also showed that majority of the respondents (77%) harvest their NTFPs from forest lands as against 23% whoharvest from communal lands. A significant proportion of the respondents (52%) traded in above 40 kg of NTFPs and only 4%were seen trading in 10 kg of NTFPs. The study further highlighted that food (28%) and domestic utensils (26%) were regularlybrought to the market centers on weekly basis whilst significant proportions of medicinal products (9%), building materials(3%) and artefacts (4%) were brought to the market venues on monthly basis. Cumbersome permit procedure (40%), increasedmarket demand (15%) and financial constraints (20%) were identified as some of the challenges encountered in NTFPs tradingin the Sunyani Municipality. To ensure strict monitoring and sustainability of the resource, there is the need for sensitizationprogramme on the importance of NTFPs in rural livelihood and why their conservation is vital in meeting the needs of thepresent generation whilst not undermining their potential in supplying the needs of future generations.

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Comparison of nine different commercially available molecular assays for detection of SARS-CoV-2 RNA

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-021-04159-9 ◽

2021 ◽

Author(s):

Ute Eberle ◽

◽

Clara Wimmer ◽

Ingrid Huber ◽

Antonie Neubauer-Juric ◽

...

Keyword(s):

Real Time ◽

Rt Pcr ◽

Diagnostic Assays ◽

Molecular Assays ◽

Pcr Assays ◽

Laboratory Experiences

AbstractTo face the COVID-19 pandemic, the need for fast and reliable diagnostic assays for the detection of SARS-CoV-2 is immense. We describe our laboratory experiences evaluating nine commercially available real-time RT-PCR assays. We found that assays differed considerably in performance and validation before routine use is mandatory.

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