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

Creecy A, Russ PK, Solinas F, Wright DW, Haselton FR - PLoS ONE (2015)

Bottom Line: Negative control samples did not amplify.Two of three negative controls did not amplify; one amplified at 100 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Integrated DNA extraction and amplification device.The extraction tubing was raised and lowered between attracting magnets to move the binding beads through the solutions into the isothermal reaction chamber. After the DNA eluted from the beads, the LAMP solution chamber was positioned for amplification in a copper heat block. The block held the reaction chamber at 65°C while the detector measured fluorescence over time. Diagram not to scale.
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pone.0130260.g002: Integrated DNA extraction and amplification device.The extraction tubing was raised and lowered between attracting magnets to move the binding beads through the solutions into the isothermal reaction chamber. After the DNA eluted from the beads, the LAMP solution chamber was positioned for amplification in a copper heat block. The block held the reaction chamber at 65°C while the detector measured fluorescence over time. Diagram not to scale.

Mentions: A. Lysed PATH samples were loaded into the extraction tubing as the first chamber. An external magnet was used to pull the binding beads through the solutions, and the DNA was eluted in the final chamber. B. Low-resource DNA extraction was combined with in-tube amplification. After the lysed sputum sample was introduced into the tubing, an oil valve was added to the opposite end to prevent evaporation of the LAMP reaction solution that followed. C. The tubing for automated DNA extraction and amplification is the same as the tubing in B with the addition of a leader section that guided the tubing through the instrument during the assay, as shown in Fig 2 and SI1.


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

Creecy A, Russ PK, Solinas F, Wright DW, Haselton FR - PLoS ONE (2015)

Integrated DNA extraction and amplification device.The extraction tubing was raised and lowered between attracting magnets to move the binding beads through the solutions into the isothermal reaction chamber. After the DNA eluted from the beads, the LAMP solution chamber was positioned for amplification in a copper heat block. The block held the reaction chamber at 65°C while the detector measured fluorescence over time. Diagram not to scale.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488445&req=5

pone.0130260.g002: Integrated DNA extraction and amplification device.The extraction tubing was raised and lowered between attracting magnets to move the binding beads through the solutions into the isothermal reaction chamber. After the DNA eluted from the beads, the LAMP solution chamber was positioned for amplification in a copper heat block. The block held the reaction chamber at 65°C while the detector measured fluorescence over time. Diagram not to scale.
Mentions: A. Lysed PATH samples were loaded into the extraction tubing as the first chamber. An external magnet was used to pull the binding beads through the solutions, and the DNA was eluted in the final chamber. B. Low-resource DNA extraction was combined with in-tube amplification. After the lysed sputum sample was introduced into the tubing, an oil valve was added to the opposite end to prevent evaporation of the LAMP reaction solution that followed. C. The tubing for automated DNA extraction and amplification is the same as the tubing in B with the addition of a leader section that guided the tubing through the instrument during the assay, as shown in Fig 2 and SI1.

Bottom Line: Negative control samples did not amplify.Two of three negative controls did not amplify; one amplified at 100 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus