Use of a Biomimetic Peptide in the Design of a Competitive Binding Assay for Biotin and Biotin Analogues

Gregory, Kalvin J.; Bachas, Leonidas G.

doi:10.1006/abio.2000.4907

Cited by 7 publications

(2 citation statements)

References 22 publications

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“…In our assay, DNA amplicons that could possibly interfere with array hybridization specificity and sensitivity were removed by the incorporation of streptavidindesthiobiotin-mediated PLP purification after the ligation reaction. The rationale of using desthiobiotin instead of biotin is the approximately 1,000-fold lower affinity for streptavidin (20,25), which permits subsequent release of the PLPs (26,53). The fluorescent signals of the reversibly captured PLP samples were not significantly different from the control treatments, indicating that this procedure did not compromise the final assay sensitivity.…”

Section: Discussionmentioning

confidence: 99%

Robust Detection and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay

Doorn

Slawiak

Szemes

et al. 2009

Appl Environ Microbiol

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Simultaneous detection and identification of multiple pathogenic microorganisms in complex environmental samples are required in numerous diagnostic fields. Here, we describe the development of a novel, backgroundfree ligation detection (LD) system using a single compound detector probe per target. The detector probes used, referred to as padlock probes (PLPs), are long oligonucleotides containing asymmetric target complementary regions at both their 5 and 3 ends which confer extremely specific target detection. Probes also incorporate a desthiobiotin moiety and an internal endonuclease IV cleavage site. DNA samples are PCR amplified, and the resulting products serve as potential targets for PLP ligation. Upon perfect target hybridization, the PLPs are circularized via enzymatic ligation, captured, and cleaved, allowing only the originally ligated PLPs to be visualized on a universal microarray. Unlike previous procedures, the probes themselves are not amplified, thereby allowing a simple PLP cleavage to yield a background-free assay. We designed and tested nine PLPs targeting several oomycetes and fungi. All of the probes specifically detected their corresponding targets and provided perfect discrimination against closely related nontarget organisms, yielding an assay sensitivity of 1 pg genomic DNA and a dynamic detection range of 10 4 . A practical demonstration with samples collected from horticultural water circulation systems was performed to test the robustness of the newly developed multiplex assay. This novel LD system enables highly specific detection and identification of multiple pathogens over a wide range of target concentrations and should be easily adaptable to a variety of applications in environmental microbiology.

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Section: Discussionmentioning

confidence: 99%

Robust Detection and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay

Doorn

Slawiak

Szemes

et al. 2009

Appl Environ Microbiol

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“…Between the primer sites we introduced a thymine-linked desthiobiotin molecule for specific capturing and release with streptavidin-coated magnetic beads. The rationale of using desthiobiotin instead of biotin is the approximately 1000 times lower affinity for streptavidin [ 22 , 39 ], which permits the reversible release of the PRI-lock probes (see below).…”

Section: Methodsmentioning

confidence: 99%

Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™

Doorn

Szemes

Bonants³

et al. 2007

BMC Genomics

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Background: Diagnostics and disease-management strategies require technologies to enable the simultaneous detection and quantification of a wide range of pathogenic microorganisms. Most multiplex, quantitative detection methods available suffer from compromises between the level of multiplexing, throughput and accuracy of quantification. Here, we demonstrate the efficacy of a novel, high-throughput, ligation-based assay for simultaneous quantitative detection of multiple plant pathogens. The ligation probes, designated Plant Research International-lock probes (PRI-lock probes), are long oligonucleotides with target complementary regions at their 5' and 3' ends. Upon perfect target hybridization, the PRI-lock probes are circularized via enzymatic ligation, subsequently serving as template for individual, standardized amplification via unique probe-specific primers. Adaptation to OpenArrays™, which can accommodate up to 3072 33 nl PCR amplifications, allowed high-throughput real-time quantification. The assay combines the multiplex capabilities and specificity of ligation reactions with high-throughput real-time PCR in the OpenArray™, resulting in a flexible, quantitative multiplex diagnostic system.

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Rapid Identification and Detection of Pathogenic Fungi by Padlock Probes

Tsui

Wang

Schoen³

et al. 2012

Laboratory Protocols in Fungal Biology

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Use of a Biomimetic Peptide in the Design of a Competitive Binding Assay for Biotin and Biotin Analogues

Cited by 7 publications

References 22 publications

Robust Detection and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay

Robust Detection and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay

Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™

Rapid Identification and Detection of Pathogenic Fungi by Padlock Probes

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