Sampling Protocols and Risk of Error Significance in Molecular Detection Tests for Fruit Trees Certification

Kummert, J.; Malice, Marie; Marbot, Sophie; Lepoivre, Philippe; Steyer, Stéphane; Oger, Robert

doi:10.17660/actahortic.2004.657.88

Cited by 3 publications

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“…31 The various nucleic acid-based detection protocols are fast and sensitive; however, they involve rather substantial sample pretreatment and are susceptible to errors. 32 Reliable detection of ASPV is further complicated since, owing to the frequently used grafting procedure, the wide distribution of ASPV is associated with high genetic variability. 33,34 Therefore, efforts were made to generate monoclonal antibodies as a basis of reliable serological enzyme immunoassays for fast routine determinations.…”

Section: Introductionmentioning

confidence: 99%

Aptamer-based biochips for label-free detection of plant virus coat proteins by SPR imaging

Lautner

Balogh

Bardóczy

et al. 2010

Analyst

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Specific detection of virus strains by affinity-based bioassays is often limited by the availability of ligands able to differentiate among close homologues of virus coat proteins. As viruses are prone to mutation, the ligand generation should, in addition, be fast enough to allow rapid identification of new varieties. These two criteria are difficult to be fulfilled by antibodies; however, they open up opportunities for aptamer-based detection. Here we report on the feasibility of selectively detecting the apple stem pitting virus (ASPV) coat proteins (PSA-H, MT32) using original DNA aptamers. Surface plasmon resonance (SPR) imaging was used together with aptamer-modified sensor chips to optimize the aptamer immobilization for highest sensitivity and to characterize the aptamer-virus coat protein binding. Different parameters affecting this binding, such as the aptamer flanking, surface coverage, and type of spacer molecules, were identified and their influence was determined. A direct label-free method is proposed for assessing the ASPV based on the detection of the respective virus coat proteins in plant extracts.

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Section: Introductionmentioning

confidence: 99%

Aptamer-based biochips for label-free detection of plant virus coat proteins by SPR imaging

Lautner

Balogh

Bardóczy

et al. 2010

Analyst

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“…Presently, the routine ASPV detection relies on biological indexing by grafting or mechanical inoculation of infected tissues onto indicator hosts, but several efforts have been made to invent faster, more robust, and flexible detection protocols. The developed nucleic acid‐based detection protocols, such as RT‐PCR (19–21) and molecular beacons (22), are sensitive but cannot fulfill the requirements of high‐throughput virus tests because they involve rather substantial sample pretreatment and are error prone (3). Recently, monoclonal antibodies have been also produced for serological identification of ASPV isolates in apple and pear protein extracts, and an enzyme‐linked immunosorbent assay (ELISA) protocol has been introduced using this monoclonal antibody (23).…”

mentioning

confidence: 99%

“…Direct detection comprises the traditional viral isolation culture, as well as application of more recent methodologies, allowing the measurement of virus-specific antigens and genetic components. Although PCR-related methods, especially real-time PCR, have become widely accepted molecular tools for virus detection, typing and subtyping, their application is limited by a complicated procedure, susceptibility to contamination, and a high false-positive rate (3). Considering these drawbacks, detection of virus-specific antigens with various immunoassay protocols seems to be preferred for screening large number of samples (4).…”

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confidence: 99%

Selection and versatile application of virus‐specific aptamers

et al. 2010

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Although the significance of molecular diagnostics in routine plant virus detection is rapidly growing, the preferred methods are still antibody-based enzyme immunoassays. In the past decade, aptamers have been demonstrated to be viable alternatives of antibodies in many applications. We set out to select apple stem pitting virus (ASPV)-specific aptamers and to apply them as antibody substitutes in various immunoassay methods. The applied systematic evolution of ligands by exponential enrichment (SELEX) procedure resulted in highly discriminative aptamers selectively binding to the target virus coat protein even in complex protein matrixes. We developed protocols for exploitation of aptamers in diverse plant virus diagnosis methods, such as dot and Western blot analyses and enzyme-linked oligonucleotide assay (ELONA). Our selected aptamers proved to be superior to the available antibody in all aspects. In contrast to the antibody, the aptamers decorate both native and denaturated proteins, and ELONA produces higher signal intensity than traditional enzyme-linked immunosorbent assay (ELISA) with virus-infected plant extract. Summarily, our results present the selection and practical utilization of first plant virus-specific aptamers. Most important, the first application of ELONA for virus detection is demonstrated, which proposes a novel, more flexible, and cost-effective means of virus diagnostics.

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Simultaneous infection of sweet cherry with eight virus species including a new foveavirus

Yaegashi

Oyamada

Goto³

et al. 2019

J Gen Plant Pathol

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Sampling Protocols and Risk of Error Significance in Molecular Detection Tests for Fruit Trees Certification

Cited by 3 publications

References 0 publications

Aptamer-based biochips for label-free detection of plant virus coat proteins by SPR imaging

Aptamer-based biochips for label-free detection of plant virus coat proteins by SPR imaging

Selection and versatile application of virus‐specific aptamers

Simultaneous infection of sweet cherry with eight virus species including a new foveavirus

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