There was no significant change in DNA recovery with increased adsorption (white circles) or elution (black circles) times (mean ± s.d., n = 3).

<p>In each experiment, the adsorption time was held at 30 s while the elution time was varied (black circles), and the elution time was held at 30 s while the adsorption time was varied (white circles). To more clearly illustrate the error bars on each data point, the adsorption data was plotted two seconds to the right at each time point.</p

The DNA recovery for the magnetic bead-based extraction method and a commercially available laboratory-based kit are comparable.

<p>Though both are significantly higher than the unextracted sample, which is undetectable by PCR, there is no statistical difference between the two extraction methods (mean ± s.d., n = 9); * denotes statistically higher recovery than unextracted sample.</p

DNA recovery is significantly increased with increasing DNA sequence length (mean ± s.d., n = 3).

<p>* denotes statistically different than 75 bp sequence.</p

The LOD of the PCR reaction is shown at 8800 copies (dotted line).

<p>The lowest initial concentration of IS6110 DNA detectable at the LOD was determined to be 77 copies/µL at the point at which this intersects the PCR-concentration curve (red circle) (mean ± s.d., n = 3).</p

The final concentration of IS6110 DNA in the eluate following magnetic bead-based extraction of 3 and 5 mL spiked urine samples is significantly higher than the IS6110 DNA concentration in the eluate following the extraction of 1 mL samples (mean ± s.d., n = 3); * denotes statistically different from 1 mL sample.

<p>The final concentration of IS6110 DNA in the eluate following magnetic bead-based extraction of 3 and 5 mL spiked urine samples is significantly higher than the IS6110 DNA concentration in the eluate following the extraction of 1 mL samples (mean ± s.d., n = 3); * denotes statistically different from 1 mL sample.</p

There is no significant difference in DNA recovery using the lyophilized urine collection pipette stored for 0, 4, 8, or 12 weeks (black circles) compared to freshly prepared adsorption reagents (white circle) (mean ± s.d., n = 3,).

<p>There is no significant difference in DNA recovery using the lyophilized urine collection pipette stored for 0, 4, 8, or 12 weeks (black circles) compared to freshly prepared adsorption reagents (white circle) (mean ± s.d., n = 3,).</p

The percentage of DNA recovered from 1 mL urine samples increases as the concentration of magnetic beads increases to 6×10⁸ beads/mL (mean ± s.d., n = 3).

<p>The percentage of DNA recovered from 1 mL urine samples increases as the concentration of magnetic beads increases to 6×10⁸ beads/mL (mean ± s.d., n = 3).</p

Time to amplification of TB DNA by LAMP and PCR.

<p>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.</p

Tuberculosis Biomarker Extraction and Isothermal Amplification in an Integrated Diagnostic Device

<div><p>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.</p></div

Integrated DNA extraction and amplification device.

<p>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.</p