Mycobacterial DNA Extraction using Bead Beating in Custom Buffer Followed by NGS Workflow

Our diagnostics research focuses on improving the detection of drug resistance in tuberculosis in limited resource settings, with a focus on speed and pragmatism. In TB diagnostics, there are more comprehensive genotypic assays, and even a number of new rapid phenotypic assays, and there are consistent pushes to get these tests closer to the point of care. But there are a number of pragmatic barriers that still exist.

Resource challenges from reagents to infrastructure to logistics continue to be significant barriers. With anything beyond the technical sophistication of a Cepheid Xpert assay, protocol standardization is difficult in different settings. So, MTB DNA extraction as detailed in this work here, is a prime example of that.

There is a critical need to expand sequencing-based TB diagnostics, but many labs still lack a reliable way to extract high quality DNA. Our goal is to share a method that's simple, practical, and suitable for low-resource point-of-care setting. Here we present a simple low cost protocol designed for use in resource-limited settings.

It has been validated for NGS applications, and is compatible with automated liquid handling systems. To begin, combine sodium chloride solution Tris hydrochloride buffer, Triton X 100, and EDTA to make the Triton buffer. Mix by stirring and add ultrapure water to reach a final volume of 100 milliliters.

Filter sterilize the buffer before use. Next, prepare 100 milliliters of low-EDTA Tris-EDTA buffer by combining one milliliter of one-molar Tris-HCl and 20 microliters of 0.5-molar EDTA. Mix and fill with ultrapure water to reach a final volume of 100 milliliters.

Then filter sterilize the buffer. To prepare the lysis tubes, use a scalpel blade to carefully cut the bottom off a 1.5-milliliter screw-cap tube just below the inflection point. Cut the tip off a P1000 pipette tip and make a V-shaped wedge near the end.

Wedge the cut bottom of the screw-cap tube into the V-shaped pipette tip to form a scoop. Fill a sterile container with 0.1-millimeter zirconia silicate beads. Using the prepared scoop, transfer approximately 200 milligrams of beads into 1.5-milliliter screw-cap tubes.

For the preparation of the bacterial cell culture, transfer five milliliters of mycobacterium tuberculosis culture into a 15-milliliter conical centrifuge tube. Centrifuge it at maximum speed for 10 minutes. Now use a 10-milliliter serological pipette to carefully remove all but approximately 500 microliters of the supernatant without disturbing the pellet.

Use a P1000 pipette to remove the remaining supernatant. Resuspend the pellet in 350 microliters of custom triton buffer, then mix well by pipetting up and down. Heat the sample in a dry heat bath for 30 minutes at 95 degrees Celsius.

To prepare the sputum transfer one to five milliliters of sputum sample into a sterile 50-milliliter centrifuge tube. To perform Dithiothreitol liquefaction, add four volumes of 10-millimolar Dithiothreitol to the sputum sample, then vortex thoroughly for 30 seconds. Centrifuge the sample at maximum speed for 10 minutes.

Then use a 10-milliliter serological pipette to discard all but 500 microliters of the supernatant. Remove the rest of the supernatant with a P1000 pipette without disturbing the pellet. Resuspend the pellet in 350 microliters of custom triton buffer.

To perform the NALC sodium hydroxide liquefaction, add four volumes of the NALC sodium hydroxide solution to the sputum sample. Vortex the mixture for 30 seconds. Then incubate the tube for seven minutes at room temperature.

Now add PBS up to the 50 milliliter mark. Vortex the contents of the tube to mix well, then centrifuge the tube at maximum speed for 10 minutes. After centrifugation, with a 50-milliliter serological pipette, discard the supernatant as demonstrated earlier.

Then resuspend the pellet in 350 microliters of custom triton buffer. First, transfer 350 microliters of inactivated sample into a labeled 1.5-milliliter screw-cap tube containing 250 microliters of 0.1-millimeters zirconia silicate beads. Bead beat the lysate at 6.5 meters per second for 45 seconds with two minutes'rest between cycles.

Centrifuge the sample at maximum speed for two minutes, then transfer 150 microliters of the supernatant to a new labeled tube. Next vortex cleanup magnetic beads that have been equated at room temperature for 30 minutes, to resuspend them. Transfer 180 microliters of magnetic beads to the DNA sample.

Pipet up and down 10 times to mix. After a two minute incubation at room temperature, place the tube on a magnetic rack and wait two minutes for the solution to clear. Then use a 200-microliter pipette to discard the supernatant.

With the tube still on the magnetic rack, add 500 microliters of freshly prepared 70%ethanol along the opposite wall, and wait for 30 seconds. At the end of the last wash, remove residual ethanol with a 10-microliter pipette and air dry for two minutes. As soon as beads turn opaque, remove the tube from the magnetic rack.

Resuspend in 20 microliters of low EDTA Tris buffer. Mix by pipetting or vortexing to ensure all the beads are in solution before a five-minute incubation at room temperature. Next, place the tube on a magnetic rack for two minutes until clear.

Then transfer less than 20 microliters of the eluated DNA to a new labeled tube to avoid bead carryover. To quantify mycobacterial DNA using a quantitative PCR targeting 99 nucleotides of the mycobacterial atpE, assemble a QPCR master mix on ice. Adjust master mix based on the amount of samples and standards, including a 10%overage to account for pipetting loss.

Run the thermal cycler with 95 degrees Celsius for 60 seconds, followed by 35 cycles of 95 degrees Celsius for 10 seconds, and 60 degrees Celsius for 30 seconds with ramp rate of 2.11 degrees Celsius per second. Run all samples, standards and controls in triplicate. Here, mycobacterium tuberculosis cultures yielded the highest DNA concentrations among all tested conditions.

Spiked sputum samples showed progressively lower DNA yields, and increased variation with decreasing bacterial input, especially in the 10, 000 bacteria group.