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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

Example raw data from integrated extraction and amplification assay.Fluorescence (orange) and temperature (black) were recorded during extraction of TB DNA from a high concentration PATH surrogate sputum sample and LAMP amplification. During the extraction phase of the assay, the temperature was transiently reduced by approximately 5°C as the room temperature tubing passed through the block. The peak in fluorescence at approximately 15 minutes indicated that the reaction chamber of the tubing was positioned correctly in the heat block as the amplification phase of the assay began.
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pone.0130260.g006: Example raw data from integrated extraction and amplification assay.Fluorescence (orange) and temperature (black) were recorded during extraction of TB DNA from a high concentration PATH surrogate sputum sample and LAMP amplification. During the extraction phase of the assay, the temperature was transiently reduced by approximately 5°C as the room temperature tubing passed through the block. The peak in fluorescence at approximately 15 minutes indicated that the reaction chamber of the tubing was positioned correctly in the heat block as the amplification phase of the assay began.

Mentions: Using the automated, integrated device, TB DNA was extracted from surrogate sputum samples and amplified by LAMP. The heat block in the device was brought to 65°C before the assay was started. During the 15 minute extraction phase of the assay, the temperature was transiently reduced by approximately 5°C as the room temperature tubing passed through the heat block (Fig 6, black). Small peaks in fluorescence were seen as the reaction chamber of the tube passed in front of the fluorescence reader during the sample preparation phase of the assay (Fig 6, orange). Once DNA extraction was completed, a characteristic peak in fluorescence was recorded at approximately 15 minutes indicating that the reaction chamber was correctly positioned in the heat block (Fig 6, orange). As the temperature in the tube increased from room temperature to 65°C, the fluorescence of the SYTO-82 intercalating dye decreased as the nonspecific DNA interaction present in the sample at room temperature was reduced. After approximately 30 min, the fluorescence curve characteristic of DNA amplification was observed.


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

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

Example raw data from integrated extraction and amplification assay.Fluorescence (orange) and temperature (black) were recorded during extraction of TB DNA from a high concentration PATH surrogate sputum sample and LAMP amplification. During the extraction phase of the assay, the temperature was transiently reduced by approximately 5°C as the room temperature tubing passed through the block. The peak in fluorescence at approximately 15 minutes indicated that the reaction chamber of the tubing was positioned correctly in the heat block as the amplification phase of the assay began.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130260.g006: Example raw data from integrated extraction and amplification assay.Fluorescence (orange) and temperature (black) were recorded during extraction of TB DNA from a high concentration PATH surrogate sputum sample and LAMP amplification. During the extraction phase of the assay, the temperature was transiently reduced by approximately 5°C as the room temperature tubing passed through the block. The peak in fluorescence at approximately 15 minutes indicated that the reaction chamber of the tubing was positioned correctly in the heat block as the amplification phase of the assay began.
Mentions: Using the automated, integrated device, TB DNA was extracted from surrogate sputum samples and amplified by LAMP. The heat block in the device was brought to 65°C before the assay was started. During the 15 minute extraction phase of the assay, the temperature was transiently reduced by approximately 5°C as the room temperature tubing passed through the heat block (Fig 6, black). Small peaks in fluorescence were seen as the reaction chamber of the tube passed in front of the fluorescence reader during the sample preparation phase of the assay (Fig 6, orange). Once DNA extraction was completed, a characteristic peak in fluorescence was recorded at approximately 15 minutes indicating that the reaction chamber was correctly positioned in the heat block (Fig 6, orange). As the temperature in the tube increased from room temperature to 65°C, the fluorescence of the SYTO-82 intercalating dye decreased as the nonspecific DNA interaction present in the sample at room temperature was reduced. After approximately 30 min, the fluorescence curve characteristic of DNA amplification was observed.

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