Development of a Rapid, Sensitive, and Field-Deployable Razor Ex BioDetection System and Quantitative PCR Assay for Detection of Phymatotrichopsis omnivora Using Multiple Gene Targets

Arif, Mohammad; Fletcher, Jacqueline; Marek, Stephen M.; Melcher, Ulrich; Ochoa-Corona, F. M.

doi:10.1128/aem.03239-12

Cited by 31 publications

(27 citation statements)

References 23 publications

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“…Assay specificity, accuracy, and reproducibility are critical for applications in quarantine situations, microbial forensics, biosecurity, and diagnostics (10). The specificity of designed primers was confirmed in silico after positive BLASTn matching with five accessions and positive-endpoint RT-PCR with 30 HPV-infected plant samples collected from six states of the Great Plains (Table 1).…”

Section: Discussionmentioning

confidence: 94%

“…In some diagnostics applications it can be important for users having minimal expertise or training to be able to perform the assay in the field. The battery operated Razor Ex is suitable for detecting several plant pathogens in the field, and results can be obtained in about 25 to 40 min (10,16). To minimize sample cross-contamination, commercially available Razor Ex pouches filled with lyophilized PCR components eliminate the need for cold storage.…”

Section: Discussionmentioning

confidence: 99%

“…PCR-based techniques, which are generally more sensitive than immunological methods and have high specificity and powerful discriminatory capabilities, are most widely used (10). Quantitative PCR (qPCR)-based methods eliminate the need for post-PCR product analysis (electrophoresis) and, among these formats, TaqMan and SYBR green qPCR are the most widely used (11).…”

mentioning

confidence: 99%

“…Use of the Razor Ex BioDetection System (Razor Ex; Idaho Technology, Inc., Salt Lake City, UT), a portable, battery operated, compact real-time qPCR system, allows sensitive in-field pathogen detection by a minimally trained operator without the need for a laboratory facility. Other portable thermocyclers include the Smart Cycler (Cepheid, Sunnyvale, CA), the LightCycler (Roche Applied Science, Indianapolis, IN), and the Bio-Seeq instrument (Smiths Detection, Edgewood, MD), but the Razor Ex has the advantages of very compact size (11 lb) and rapid cycling (only 30 to 40 min) (10,16). Originally, the Razor Ex was designed for military use, to detect high-consequence organisms on-site.…”

mentioning

confidence: 99%

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Primer Modification Improves Rapid and Sensitive In Vitro and Field-Deployable Assays for Detection of High Plains Virus Variants

Arif

Aguilar-Moreno

Wayadande

et al. 2014

Appl Environ Microbiol

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A high consequence pathogen, High plains virus (HPV) causes considerable damage to wheat if the crop is infected during early stages of development. Methods for the early, accurate, and sensitive detection of HPV in plant tissues are needed for the management of disease outbreaks and reservoir hosts. In this study, the effectiveness of five methods-real-time SYBR green and TaqMan reverse transcription-quantitative PCR (RT-qPCR), endpoint RT-PCR, RT-helicase dependent amplification (RT-HDA) and the Razor Ex BioDetection System (Razor Ex)-for the broad-range detection of HPV variants was evaluated. Specific PCR primer sets and probes were designed to target the HPV nucleoprotein gene. Primer set HPV6F and HPV4R, which amplifies a product of 96 bp, was validated in silico against published sequences and in vitro against an inclusivity panel of infected plant samples and an exclusivity panel of near-neighbor viruses. The primers were modified by adding a customized 22 nucleotide long tail at the 5= terminus, raising the primers' melting temperature (T m ; ca. 10°C) to make them compatible with RT-HDA (required optimal T m ‫؍‬ 68°C), in which the use of primers lacking such tails gave no amplification. All of the methods allowed the detection of as little as 1 fg of either plasmid DNA carrying the target gene sequence or of infected plant samples. The described in vitro and in-field assays are accurate, rapid, sensitive, and useful for pathogen detection and disease diagnosis, microbial quantification, and certification and breeding programs, as well as for biosecurity and microbial forensics applications.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Primer Modification Improves Rapid and Sensitive In Vitro and Field-Deployable Assays for Detection of High Plains Virus Variants

Arif

Aguilar-Moreno

Wayadande

et al. 2014

Appl Environ Microbiol

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“…In contrast to published real-time PCR protocols designed for other plant-pathogenic bacteria, we used a double-quencher probe with an internal quencher named ZEN (Integrated DNA Technologies, Inc., USA). This kind of probe has been reported to improve amplification efficiency, reduce background noise, and increase the fluorescence level of the probe, enhancing PCR sensitivity for plant-pathogenic fungi (32). In addition, by using this type of probe for other bacteria, such as "Candidatus Liberibacter asiaticus" and "Candidatus Liberibacter solanacearum," or some plant viruses, such as Plum pox virus, it is possible to reduce the number of cycles in the amplification curves (unpublished data).…”

Section: Discussionmentioning

confidence: 99%

Conventional and Real-Time PCRs for Detection of Erwinia piriflorinigrans Allow Its Distinction from the Fire Blight Pathogen, Erwinia amylovora

Barbé

Bertolini

Roselló

et al. 2014

Appl Environ Microbiol

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bErwinia piriflorinigrans is a new pathogenic species of the bacterial genus Erwinia that has been described recently in Spain. Accurate detection and identification of E. piriflorinigrans are challenging because its symptoms on pear blossoms are similar to those caused by Erwinia amylovora, the causal agent of fire blight. Moreover, these two species share phenotypic and molecular characteristics. Two specific and sensitive conventional and real-time PCR protocols were developed to identify and detect E. piriflorinigrans and to differentiate it from E. amylovora and other species of this genus. These protocols were based on sequences from plasmid pEPIR37, which is present in all strains of E. piriflorinigrans analyzed. After the stability of the plasmid was demonstrated, the specificities of the protocols were confirmed by the amplification of all E. piriflorinigrans strains tested, whereas 304 closely related pathogenic and nonpathogenic Erwinia strains and microbiota from pear trees were not amplified. In sensitivity assays, 10 3 cells/ml extract were detected in spiked plant material by conventional or real-time PCR, and 10 2 cells/ml were detected in DNA extracted from spiked plant material by real-time PCR. The protocols developed here succeeded in detecting E. piriflorinigrans in 102 out of 564 symptomatic and asymptomatic naturally infected pear samples (flowers, cortex stem tissue, leaves, shoots, and fruitlets), in necrotic Pyracantha sp. blossoms, and in necrotic pear and apple tissues infected with both E. amylovora and E. piriflorinigrans. Therefore, these new tools can be used in epidemiological studies that will enhance our understanding of the life cycle of E. piriflorinigrans in different hosts and plant tissues and its interaction with E. amylovora. Erwinia piriflorinigrans is a Gram-negative plant-pathogenic bacterium causing pear blossom necrosis (1). The disease affects pear production and can reduce fruit yield. It was first observed in Valencia, Spain, in 1999 (2, 3). After the first identification, the disease was observed at the same place in later years (1). Due to its recent discovery, the biology, ecology, and life cycle of this new species are still poorly understood.Unlike Erwinia amylovora, the causal agent of fire blight, E. piriflorinigrans affects blossoms and not other parts of plants (3). Nevertheless, E. piriflorinigrans colonies can be easily mistaken for E. amylovora in culture media, e.g., CCT medium (4), King's medium B (5), and sucrose nutrient agar (SNA) or Levan medium, which are usually utilized and recommended for the isolation of E. amylovora (6, 7). These two pathogens show similar metabolic profiles by use of the commercial API 20E, API 20NE, API ZYM, and API 50CH strips (bioMérieux, France), as well as close fatty acid profiles. E. piriflorinigrans can react with several antisera, and even with one of the monoclonal antibodies, used to detect E. amylovora (3). Moreover, E. piriflorinigrans reacts positively in PCR using the 23S rRNA gene sequence-based primers de...

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Monitoring and Surveillance of Forest Insects

Brockerhoff

Corley

Jactel

et al. 2023

Forest Entomology and Pathology

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Monitoring of insect populations is widely used in forest entomology in the context of biodiversity studies, as an aspect of pest management, and for the detection and surveillance of non-native invasive species. In particular, monitoring is undertaken to obtain information on the presence or abundance of particular species, to study their phenology (e.g. the time of oviposition or flight periods), to predict pest population size, spread and damage, or to determine if pest management activities are required. A wide variety of methods are being used for these purposes including physical surveys, the use of insect traps, molecular methods, as well as aerial surveys and remote sensing. This chapter focusses on some of the more important methods to provide an overview of the objectives and applications of monitoring and surveillance of forest insects. The principles of each method and common uses are explained and illustrated with case studies on prominent forest insects including the pine processionary moth (Thaumetopoea pityocampa), the Sirex wood wasp (Sirex noctilio), spongy moth (Lymantria dispar), bark beetles such as Ips typographus, and the brown spruce longhorn beetle (Tetropium fuscum). The chapter also explores statistical considerations and issues such as imperfect relationships between trap catch and the local population size of target species. Niche methods that are not widely used but have strengths in some situations (e.g. detector dogs for detection of Anoplophora glabripennis and other invasive species) or are still in development (e.g. e-noses and acoustic detection) are also discussed.

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Development of a Rapid, Sensitive, and Field-Deployable Razor Ex BioDetection System and Quantitative PCR Assay for Detection of Phymatotrichopsis omnivora Using Multiple Gene Targets

Cited by 31 publications

References 23 publications

Primer Modification Improves Rapid and Sensitive In Vitro and Field-Deployable Assays for Detection of High Plains Virus Variants

Primer Modification Improves Rapid and Sensitive In Vitro and Field-Deployable Assays for Detection of High Plains Virus Variants

Conventional and Real-Time PCRs for Detection of Erwinia piriflorinigrans Allow Its Distinction from the Fire Blight Pathogen, Erwinia amylovora

Monitoring and Surveillance of Forest Insects

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