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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

Comparison of Bst 2.0 and GspM2.0 polymerases in LAMP reactions.LAMP reactions were performed with plasmid DNA with an insert of the IS6110 sequence. In a Rotor-Gene Q thermal cycler, 5X102, 5X104, and 5X106 copies per reaction as well as no template controls were compared. N = 4.
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pone.0130260.g005: Comparison of Bst 2.0 and GspM2.0 polymerases in LAMP reactions.LAMP reactions were performed with plasmid DNA with an insert of the IS6110 sequence. In a Rotor-Gene Q thermal cycler, 5X102, 5X104, and 5X106 copies per reaction as well as no template controls were compared. N = 4.

Mentions: One of the goals of the integrated device design was to shorten the overall time to result. A significant portion of the total assay time is isothermal amplification time. In order to decrease amplification time we explored the use of a faster polymerase. We compared the Bst 2.0 polymerase and buffer to the OptiGene Master Mix containing GspM 2.0 polymerase in a Rotor-Gene Q thermal cycler using reactions spiked with plasmid DNA containing the IS6110 gene. Amplification times of all target concentrations tested were significantly shorter with the GspM 2.0 enzyme (Fig 5). With GspM 2.0, 5X102 copies/reaction amplified in 21 ± 3 minutes, 5X104 in 17 ± 2 minutes, and 5X106 in 13 ± 2 minutes. With Bst 2.0, 5X102 copies/reaction amplified in 86 ± 30 minutes, 5X104 in 48 ± 11 minutes, and 5X106 in 46 ± 8 minutes. The GspM 2.0 no template control time (57 ± 3 minutes) was also significantly shorter than the Bst 2.0 no template control (143 ± 33 minutes). However, the GspM 2.0 amplification times were all still significantly lower than the no template control.


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

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

Comparison of Bst 2.0 and GspM2.0 polymerases in LAMP reactions.LAMP reactions were performed with plasmid DNA with an insert of the IS6110 sequence. In a Rotor-Gene Q thermal cycler, 5X102, 5X104, and 5X106 copies per reaction as well as no template controls were compared. N = 4.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130260.g005: Comparison of Bst 2.0 and GspM2.0 polymerases in LAMP reactions.LAMP reactions were performed with plasmid DNA with an insert of the IS6110 sequence. In a Rotor-Gene Q thermal cycler, 5X102, 5X104, and 5X106 copies per reaction as well as no template controls were compared. N = 4.
Mentions: One of the goals of the integrated device design was to shorten the overall time to result. A significant portion of the total assay time is isothermal amplification time. In order to decrease amplification time we explored the use of a faster polymerase. We compared the Bst 2.0 polymerase and buffer to the OptiGene Master Mix containing GspM 2.0 polymerase in a Rotor-Gene Q thermal cycler using reactions spiked with plasmid DNA containing the IS6110 gene. Amplification times of all target concentrations tested were significantly shorter with the GspM 2.0 enzyme (Fig 5). With GspM 2.0, 5X102 copies/reaction amplified in 21 ± 3 minutes, 5X104 in 17 ± 2 minutes, and 5X106 in 13 ± 2 minutes. With Bst 2.0, 5X102 copies/reaction amplified in 86 ± 30 minutes, 5X104 in 48 ± 11 minutes, and 5X106 in 46 ± 8 minutes. The GspM 2.0 no template control time (57 ± 3 minutes) was also significantly shorter than the Bst 2.0 no template control (143 ± 33 minutes). However, the GspM 2.0 amplification times were all still significantly lower than the no template control.

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