The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

The overall goal of this experiment is to develop a simple LAMP method using lyophilized reagents for the detection of C.burnetii in patient samples. The main advantage of this protocol is that no coaching is required for the storage of the reagents. This method is ideal for use in resource-limited settings where Q fever is endemic.

Demonstrating the procedure will be Tatiana, a technician from our laboratory. Beginning with DNA for the target gene IS1111a from C.burnetii RSA493, determine the plasmid concentration with a spectrophotometer at 260 nanometers. Convert the plasmid DNA concentration into copy number using the following calculations, where 6330 equals the length and base pairs of the plasmid, and 34.2 nanograms per microliter is the concentration of the plasmid, as just determined using the spectrophotometer.

Then prepare 8-to 10-fold serial dilutions of the plasmid to arrive at the following number of copies per microliter. To prepare the DNA template for the LAMP reaction, begin with 200 microliters of human plasma samples, and with the DNA miniprep kit, extract the DNA according to the manufacturer's protocol. Dilute the final sample into a 20-microliter volume.

Prepare 1 mililiter of 2x LAMP reaction buffer by combining the following reagents. Mix by pulse-vortexing for 10 seconds. To perform the standard LAMP reaction, in a 0.2 milliliter PCR tube, mix 12.5 microliters of 2x LAMP buffer, 1.2 microliters of primer mix, 1 microliter of Bst DNA polymerase, and 5.3 microliters of water.

Next, add 5 microliters of DNA to the 20 microliter reaction and mix well, then incubate at 60 degrees Celsius in a water bath or heating block for 60 minutes. After the incubation, terminate the reaction by adding 5 microliters of 10x gel loading buffer to the tube. Then load 5 microliters of the reaction products onto a 2%agarose gel stained with intercalating nucleic acid stain.

Run the gel in 1x TBE at 100 volts for 35 minutes, then use UV light to visualize the results. To perform the LAMP reaction with reconstituted reagents, prepare 1 milliliter of reconstitution buffer by combining the following reagents. Mix by pulse-vortexing for 10 seconds.

Next, remove the tubes that contained the lyophilized reagents from the sealed aluminium foil bag. For Step 4.2, it is very important to make sure the aluminium foil bag is properly sealed before opening it. Do not use the tubes if the aluminum bag is not sealed or is damaged.

Add 20 microliters of reconstitution buffer to each tube of reagents and mix by pipetting up and down five times. Ensure that the lyophilized reagents are completely re-suspended. Then, add 5 microliters of DNA template to the reconstituted reagents and mix well.

Incubate at 60 degrees Celsius in a water bath or heating block for 60 minutes. To terminate the reaction after the incubation, add 5 microliters of 10x gel loading buffer, then add 5 microliters of the reaction products onto a 2%agarose gel stained with intercalating nucleic acid stain. Run the gel in 1x TBE buffer at 100 volts for 35 minutes, then visualize the results by UV light.

To perform the LAMP reaction with hydroxynaphthol blue, or HNB, or intercalating dye, prepare 1 milliliter of reconstitution buffer by combining the following reagents that include HNB or a fluorescent intercalating dye. Mix by pulse-vortexing for 10 seconds. Carry out the LAMP reaction as just described and use UV light or the naked eye to visualize the results.

To perform the LAMP reaction with a tube scanner, add 5 microliters of DNA to the reconstituted reagents containing the fluorescent intercalating dye, then insert the closed tube into the tube scanner. Set the incubation temperature to 60 degrees Celsius and measure the fluorescence at 520 nanometers for 60 minutes. This figure shows the LAMP reaction results on agarose gels with freshly-prepared reagents and lyophilized reagents.

Observe the lyophilized reagent maintains its activity. LAMP reactions prepared by both reagents detected 25 copies of DNA template. In this figure, the detection of the reaction's products with HNB or fluorescent intercalating dye in the reaction is shown.

The addition of HNB to the reaction produces a visual color change, from purple to blue, with 25, 50, and 100 copies of DNA template present in the reactions. In addition, the inclusion of fluorescent intercalating dye in the reaction shows a strong fluorescent signal with UV light when similar DNA copy numbers are present. Shown here are the results from using the tube scanner to monitor the fluorescent signals in real-time with fluorescent intercalating dye.

The fluorescent signals are above baseline after 14, 18, 21, and 23 minutes, with 10^6, 10^4, 10^3, and 100 copies of DNA template in the reactions. Once mastered, this procedure can be done in 90 minutes. After its development, this assay paved the way for quick diagnosis of Coxiella burnetii infection in resource-limited areas.

After watching this video, you should have a good understanding of how to reconstitute lyophilized LAMP reagents. Don't forget that patient samples can be infectious and safety precautions should always be taken while performing this procedure.