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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

Time to amplification of TB DNA by LAMP and PCR.DNA was extracted from lysed surrogate sputum samples using the low-resource extraction technique and eluted into water. Eluent was amplified by LAMP (red triangle) and PCR (black circle); N = 6.
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pone.0130260.g003: Time to amplification of TB DNA by LAMP and PCR.DNA was extracted from lysed surrogate sputum samples using the low-resource extraction technique and eluted into water. Eluent was amplified by LAMP (red triangle) and PCR (black circle); N = 6.

Mentions: Surrogate sputum samples were chemically lysed, and DNA was manually extracted and eluted into water using the tube configuration shown in Fig 1A. The eluent was subsequently removed from the tubing and amplified by LAMP and PCR in a Rotor-Gene Q thermal cycler. PCR and LAMP amplified each of the three cell concentrations in similar reaction times (Fig 3). PCR amplified low (103 cells/mL), medium (104 cells/mL), and high (105 TB cells/mL of sputum) samples at 51.7 ± 1.0, 47.1 ± 0.6, and at 43.3 ± 0.6 minutes respectively (N = 6). LAMP amplified low, medium, and high samples at 53.5 ± 3.3, 46.3 ± 2.2, and 41.6 ± 1.9 minutes (N = 6). Neither LAMP nor PCR amplified any of the negative samples before 90 min.


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

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

Time to amplification of TB DNA by LAMP and PCR.DNA was extracted from lysed surrogate sputum samples using the low-resource extraction technique and eluted into water. Eluent was amplified by LAMP (red triangle) and PCR (black circle); N = 6.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130260.g003: Time to amplification of TB DNA by LAMP and PCR.DNA was extracted from lysed surrogate sputum samples using the low-resource extraction technique and eluted into water. Eluent was amplified by LAMP (red triangle) and PCR (black circle); N = 6.
Mentions: Surrogate sputum samples were chemically lysed, and DNA was manually extracted and eluted into water using the tube configuration shown in Fig 1A. The eluent was subsequently removed from the tubing and amplified by LAMP and PCR in a Rotor-Gene Q thermal cycler. PCR and LAMP amplified each of the three cell concentrations in similar reaction times (Fig 3). PCR amplified low (103 cells/mL), medium (104 cells/mL), and high (105 TB cells/mL of sputum) samples at 51.7 ± 1.0, 47.1 ± 0.6, and at 43.3 ± 0.6 minutes respectively (N = 6). LAMP amplified low, medium, and high samples at 53.5 ± 3.3, 46.3 ± 2.2, and 41.6 ± 1.9 minutes (N = 6). Neither LAMP nor PCR amplified any of the negative samples before 90 min.

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