Dataset Information

Tuberculosis Biomarker Extraction and Isothermal Amplification in an Integrated Diagnostic Device.

ABSTRACT: In this study, we integrated magnetic bead-based sample preparation and isothermal loop mediated amplification (LAMP) of TB in a single tube. Surrogate sputum samples produced by the Program for Appropriate Technology in Health containing inactivated TB bacteria were used to test the diagnostic. In order to test the sample preparation method, samples were lysed, and DNA was manually extracted and eluted into water in the tube. In a thermal cycler, LAMP amplified TB DNA from 103 TB cells/mL of sputum at 53.5 ± 3.3 minutes, 104 cells/mL at 46.3 ± 2.2 minutes, and 105 cells/mL at 41.6 ± 1.9 minutes. Negative control samples did not amplify. Next, sample preparation was combined with in-tubing isothermal LAMP amplification by replacing the water elution chamber with a LAMP reaction chamber. In this intermediate configuration, LAMP amplified 103 cells/mL at 74 ± 10 minutes, 104 cells/mL at 60 ± 9 minutes, and 105 TB cells/mL of sputum at 54 ± 9 minutes. Two of three negative controls did not amplify; one amplified at 100 minutes. In the semi-automated system, DNA was eluted directly into an isothermal reaction solution containing the faster OptiGene DNA polymerase. The low surrogate sputum concentration, 103 TB cells/mL, amplified at 52.8 ± 3.3 minutes, 104 cells/mL at 45.4 ± 11.3 minutes, and 105 cells/mL at 31.8 ± 2.9 minutes. TB negative samples amplified at 66.4 ± 7.4 minutes. This study demonstrated the feasibility of a single tube design for integrating sample preparation and isothermal amplification, which with further development could be useful for point-of-care applications, particularly in a low-resource setting.

SUBMITTER: Creecy A

PROVIDER: S-EPMC4488445 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Tuberculosis Biomarker Extraction and Isothermal Amplification in an Integrated Diagnostic Device.

Creecy Amy A Russ Patricia K PK Solinas Francesca F Wright David W DW Haselton Frederick R FR

PloS one 20150701 7

In this study, we integrated magnetic bead-based sample preparation and isothermal loop mediated amplification (LAMP) of TB in a single tube. Surrogate sputum samples produced by the Program for Appropriate Technology in Health containing inactivated TB bacteria were used to test the diagnostic. In order to test the sample preparation method, samples were lysed, and DNA was manually extracted and eluted into water in the tube. In a thermal cycler, LAMP amplified TB DNA from 103 TB cells/mL of sp ...[more]

PMID: 26132307

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Project description:Campylobacter jejuni is recognized as the most common species in the genus Campylobacter that causes foodborne diseases. The main reservoirs harboring C. jejuni are poultry products, which are associated with most illnesses, creating a demand for effective detection methods to achieve point-of-need diagnostics. We developed an easy-to-use, hybrid paper/polymer-based microfluidic device that integrates paper-based DNA extraction, isothermal nucleic acid amplification, and lateral flow detection. Overall, the recombinase polymerase amplification (RPA) reaction was completed in 20 min and demonstrated 100% specificity to C. jejuni, including 2 reference strains and 6 wild strains isolated from the agroecosystem, 9 other Campylobacter subspecies strains, and 11 non-Campylobacter strains. The limit of detection (LOD) was 46 CFU/mL with DNA extracted on the cellulose paper. The sensitivity was reduced to 460 CFU/mL on the integrated hybrid paper/polymer-based microfluidic device. This device could detect C. jejuni spiked at concentrations ranging from 101 to 102 CFU/g in chicken meat after an enrichment of 5 to 10 h. For C. jejuni levels of >102 CFU/g, it managed to confirm positive results immediately, without bacterial enrichment. RPA reagents and primers remained stable on the paper platform at 22°C for 12 h. After lyophilization and storage on paper, the RPA reaction showed consistent sensitivity for 3 days, and the LOD was reduced to 103 CFU/mL when storage was extended to 25 days. The use of this hybrid paper/polymer-based microfluidic device enabled detection of Campylobacter in foods with high specificity and sensitivity, demonstrating its potential as a reliable point-of-need diagnostic platform for on-site conditions due to its low cost, portability, and simplicity. IMPORTANCE The global health and economic burden of Campylobacter prompts the development of novel detection techniques that can be implemented in resource-limited and on-site settings. This study described point-of-need identification of C. jejuni using a hybrid paper/polymer-based microfluidic device that is easy to operate. This device had high specificity and sensitivity toward C. jejuni and significantly reduced the total analysis time compared to conventional culture-based methods. Nucleic acid extraction was simplified from intensive pipetting to a paper dipstick, making it more convenient for use in the field as a promising tool for future routine surveillance and outbreak investigation.

Dataset Information

Tuberculosis Biomarker Extraction and Isothermal Amplification in an Integrated Diagnostic Device.

Publications

Tuberculosis Biomarker Extraction and Isothermal Amplification in an Integrated Diagnostic Device.

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