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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

Low-resource DNA extraction technique.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.
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pone.0130260.g001: Low-resource DNA extraction technique.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.

Mentions: Chemical lysis was performed to release the bacterial DNA into the sputum [20–22]. In 2 mL tubes, 500 uL of surrogate sputum were mixed with 500 uL of lysis and binding buffer (4M guanidine thiocyanate, 25 mM sodium citrate, 4.9% Triton X-100, 0.2% sodium dodecyl sulfate) and 0.8 mg of MyOne Silane Dynal beads. This mixture was agitated for 10 minutes on a Fisher Vortex Genie 2 at speed 4. After agitation, 1 mL of lysed sample was pipetted into the tubing for extraction (Fig 1). The procedure was repeated for each concentration of TB in surrogate sputum: 0, 103, 104, and 105 cells/mL.


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

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

Low-resource DNA extraction technique.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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130260.g001: Low-resource DNA extraction technique.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.
Mentions: Chemical lysis was performed to release the bacterial DNA into the sputum [20–22]. In 2 mL tubes, 500 uL of surrogate sputum were mixed with 500 uL of lysis and binding buffer (4M guanidine thiocyanate, 25 mM sodium citrate, 4.9% Triton X-100, 0.2% sodium dodecyl sulfate) and 0.8 mg of MyOne Silane Dynal beads. This mixture was agitated for 10 minutes on a Fisher Vortex Genie 2 at speed 4. After agitation, 1 mL of lysed sample was pipetted into the tubing for extraction (Fig 1). The procedure was repeated for each concentration of TB in surrogate sputum: 0, 103, 104, and 105 cells/mL.

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