How to Identify and Quantify Bacteria with Bacticount
The methods described in the corresponding EBioMedicine Paper give the full details for running LAMP reactions and quantifying them using Bacticount. For more details about LAMP, refer to Tomita et al., 2008 (1). Listed below are the general steps:
Prepare reaction solutions: The test uses a reaction called LAMP, which requires a special reaction solution. Specimens also need to be lysed with another solution to release DNA.
LAMP Master Mix: This mix contains a fluorescent dye, an enzyme, the specific LAMP primers for the pathogen, and other important components. Prepare and store on ice.
Lysis Mix: The lysis mix contains NaOH and a detergent.
Neutralization Solution: This neutralizes the lysed bacteria, and is 1 M Tris (pH 7.5).
Collect patient specimens: Collect patient specimens using your institutionally-approved methods.
Lyse specimens: Add lysis mix to the specimens and heat for 10 minutes at 100 degrees (Celsius).
For Urine and Feces: Cool samples, neutralize with Neutralization Solution, and mix well.
For Blood: Cool samples, centrifuge, and collect the supernatant (solution on top). Then add Neutralization Solution to this, and centrifuge again.
Mix samples with LAMP Master Mix: Add lysed specimens (made in step 3 above) to LAMP master mix in reaction tubes.
Prepare smaRT-LAMP apparatus: Prepare a reaction apparatus using a cardboard box, LED lights, and a hotplate as described. Bring the hotplate to 65 degrees (Celsius) before continuing.
Run the reaction: Place samples (from step 4) on the hotplate. Put the box with LEDs above, and place the smartphone camera directly over the samples. Start the app, and allow to record for 50 minutes.
Quantify the reaction: When the run is finished, use a previously generated standard curve file to quantify the sample results. Results are shown on screen for each of 36 sample positions, and saved as a report in a text file.
To make a new standard curve, complete steps 1-6 above using spiked samples at the correct concentrations.
(1) Tomita, N., Mori, Y., Kanda, H. and Notomi, T., 2008. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nature protocols, 3(5), p.877.
Disclaimer: Bacticount is a proof of concept for the scientific community, and has not been approved to diagnose or treat any disease.