Combined Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Detecting Fusarium oxysporum f. sp. cubense Tropical Race 4

We are interested in the rapid and accurate detection of the Fusarium wilt of banana pathogen, known as Foc TR4. The combined use of recombinase polymerase amplification and CRISPR negates the use of expensive machines and improves the accuracy of current assays. To begin, transfer 250 milligrams of each piece of symptomatic pseudostem into a separate extraction bag.

Add two milliliters of 0.5 molar sodium hydroxide polyvinylpyrrolidone solution into each extraction bag containing plant material. Using a manual grinder, such as a pestle, grind the infected vessels thoroughly inside the extraction bag. Then, add 195 microliters of 100 millimolar Tris-HCl buffer at pH eight into a 1.5 milliliter tube.

Pipette five microliters of the plant extract from the extraction bag into the 1.5 milliliter tube containing Tris-HCl buffer to dilute the extract 40-fold. Vortex the tube for 15 to 30 seconds and store it at minus 20 degrees Celsius until further use. Using the RNA transcription kit, add the given components to a 0.2 milliliter PCR tube.

Incubate the prepared tube at 37 degrees Celsius in a water bath or PCR machine for at least four hours. Now, use a spectrophotometer to quantify the transcribed RNA. Dilute the RNA to a concentration of 250 nanograms per microliter in a final volume of 40 microliters using nuclease-free water.

Then, treat the RNA sample with DNase I provided in the RNA cleaning kit following the manufacturer's instructions. After that, clean the RNA sample using the RNA cleaning kit. Use a NanoDrop device to determine the quantity and quality of the cleaned CRISPR RNA.

Then, dilute the RNA to 100 nanograms per microliter with nuclease-free water. Now, heat the CRISPR RNA mixed with RNA loading dye to 70 degrees Celsius for 10 minutes in a water bath, and run the sample on a 1.5%agarose gel to visualize the CRISPR RNA through electrophoresis. Prepare 20 microliters of Cas12a reaction mixture in a separate 0.5 milliliter tube using the given components.

Next, prepare 9.5 microliters of RPA reaction mixture in a separate 0.5 milliliter tube with the components shown here. Place an eight-tube strip into a tube rack set on ice, and transfer the 20 microliters of Cas12a reaction mixture into the base of each tube in the strip. Then, transfer the 9.5 microliters of RPA reaction mixture into the lid of each tube in the strip.

Add 0.5 microliters of 280 nanomolar magnesium acetate to the RPA mix in the lid, and mix by pipetting up and down. Next, add 0.5 microliters of deionized water to the lid of the non-template control tube, and close it carefully to prevent the RPA and Cas12a reaction mixtures from mixing. Now, add 0.5 microliters of template DNA to the 10 microliters of RPA reaction mixture containing magnesium acetate in the tube strip lid.

Mix the contents by pipetting up and down. Carefully close all tube strip lids to ensure that the RPA and Cas12a reaction mixtures do not mix. Afterward, switch on the portable fluorimeter and create a run profile for the RPA Cas12a assay.

Open the saved run profile, and place the tube strip inside the fluorimeter to incubate for 30 minutes at 37 degrees Celsius. After 30 minutes, remove the tube strip from the fluorimeter, invert the tube six times to mix the RPA and Cas12a reactions, and briefly spin the strip down in a mini centrifuge for five seconds. Then, return the tube strip to the fluorimeter and incubate for another 30 minutes at 37 degrees Celsius using the saved run profile.

Interpret the average relative fluorescence units measured on the portable fluorimeter. Finally, visualize the reaction tubes under LED blue light at an excitation wavelength of 465 nanometers using a blue light transilluminator, and capture images of the fluorescent signal using a phone camera. Positive samples produced visible fluorescence in the Genie III portable fluorimeter following RPA Cas12a detection of Foc TR4 DNA, while negative samples yielded low endpoint fluorescence.

When viewed under LED blue light, the sample tubes containing DNA from Foc TR4 infected plant material displayed visible fluorescence, while tubes with DNA from uninfected tissue did not show any fluorescence. We have developed the first assay that employs CRISPR technology for the detection of Foc TR4. Once optimized, the assay offers faster, more accurate detection on inexpensive machines than existing commercial assays.

Our further work will explore a smartphone-linked fluorescent detector to be combined with the assay.