Dataset Information

Dual-Priming Isothermal Amplification (DAMP) for Highly Sensitive and Specific Molecular Detection with Ultralow Nonspecific Signals.

ABSTRACT: Nucleic acid amplification tests have been widely used in clinical diagnostics, food safety monitoring, and molecular biology. Loop-mediated isothermal amplification (LAMP) is a prevailing nucleic acid isothermal amplification method. It has become a powerful alternative to conventional polymerase chain reaction (PCR) for pathogen detection because of its simplicity, rapidity, and high sensitivity. However, the current LAMP methods, especially LAMP with two loop primers, suffer from undesired nonspecific amplification with strong background signals due to the increasing target sites. This nonspecific amplification substantially reduced the reliability of LAMP and limited its applications in clinical diagnostics. Here, we report a "dual-priming" ("self-priming" and "pairing-priming") isothermal amplification (DAMP) assay for rapid nucleic acid detection with ultralow nonspecific signals. This method takes advantage of the "dual-priming" strand extension strategy by adding two pairing-competition primers and designing unique inner primers, enabling highly sensitive and specific molecular detection. As an application demonstration, the DAMP assay was used to detect HIV-1 DNA/RNA and Escherichia coli DNA, showing equal or better sensitivity with shorter detection time compared to conventional LAMP and PCR methods. More importantly, the DAMP assay showed ultralow background signals without false positive signals even after a 2 h incubation. Such a simple, reliable, sensitive, and specific DAMP assay can be well suited for rapid nucleic acid detection as point-of-care testing, particularly in resource-limited settings.

SUBMITTER: Ding X

PROVIDER: S-EPMC6980338 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Dual-Priming Isothermal Amplification (DAMP) for Highly Sensitive and Specific Molecular Detection with Ultralow Nonspecific Signals.

Ding Xiong X Xu Zhiheng Z Yin Kun K Sfeir Maroun M Liu Changchun C

Analytical chemistry 20190926 20

Nucleic acid amplification tests have been widely used in clinical diagnostics, food safety monitoring, and molecular biology. Loop-mediated isothermal amplification (LAMP) is a prevailing nucleic acid isothermal amplification method. It has become a powerful alternative to conventional polymerase chain reaction (PCR) for pathogen detection because of its simplicity, rapidity, and high sensitivity. However, the current LAMP methods, especially LAMP with two loop primers, suffer from undesired no ...[more]

PMID: 31525941

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Project description:The rapidly evolving nature of antibody drug development has resulted in technologies that generate vast numbers (hundreds to thousands) of lead antibody candidates during early discovery. These candidates must be rapidly pared down to identify the most drug-like candidates for in-depth analysis of their safety and efficacy, which can only be performed on a limited number of antibodies due to time and resource requirements. One key biophysical property of successful antibody therapeutics is high specificity, defined as low levels of nonspecific binding or polyspecificity. Although there has been some progress in developing assays for detecting antibody polyspecificity, most of these assays are limited by poor sensitivity or assay formats that require proprietary antibody surface display methods, and some of these assays use complex and poorly defined polyspecificity reagents. Here we report the PolySpecificity Particle (PSP) assay, a sensitive flow cytometry assay for evaluating antibody nonspecific interactions that overcomes previous limitations and can be used for evaluating diverse types of IgGs, multispecific antibodies and Fc-fusion proteins. Our approach uses micron-sized magnetic beads coated with Protein A to capture antibodies at extremely dilute concentrations (<0.02 mg/mL). Flow cytometry analysis of polyspecificity reagent binding to these conjugates results in sensitive detection of differences in nonspecific interactions for clinical-stage antibodies. Our PSP assay strongly discriminates between antibodies with different levels of polyspecificity using previously reported polyspecificity reagents that are either well-defined proteins or highly complex protein mixtures. Moreover, we also find that a unique reagent, namely ovalbumin, results in the best assay sensitivity and specificity. Importantly, our assay is much more sensitive than standard assays such as ELISAs. We expect that our simple, sensitive, and high-throughput PSP assay will accelerate the development of safe and effective antibody therapeutics.

Dataset Information

Dual-Priming Isothermal Amplification (DAMP) for Highly Sensitive and Specific Molecular Detection with Ultralow Nonspecific Signals.

Publications

Dual-Priming Isothermal Amplification (DAMP) for Highly Sensitive and Specific Molecular Detection with Ultralow Nonspecific Signals.

Similar Datasets

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