On-site DNA Detection of Trypanosomatid Parasites and Nosema ceranae Through Alkaline Lysis Coupled to RPA/CRISPR/Cas12a System

Our main research interest focus on understanding the mechanism of transmission lifecycles, as well as improving diagnostic methods of the main bee pathogens, including trypanosomatid parasites. The advent of new hydrothermal nucleic acid techniques, which enable exponential amplification of nucleic acids at constant temperatures is making possible the detection of pathogens without the need for complex infrastructures or lab equipment. In our field of molecular target identification and selection for pathogen detection, we use isothermal RPA combined with CRISPR-Cas12a for rapid, sensitive, and portable molecular diagnostics.

Current challenges include optimizing a site's sensitivity and specificity, minimizing false positive, simplifying sample preparation, and developing roles, deployable, diagnostic, adaptable to diverse pathogen and conditions. We have addressed the need for a fast, simple, and field adaptable method to detect honeybee pathogens, overcoming limitations of traditional molecular diagnostics. To begin, obtain an anesthetized honeybee.

With a sterile scalpel, dissect the abdomen of the bee. Transfer the dissected tissue into a 1.5 milliliter microcentrifuge tube. Add 400 microliters of HotSHOT buffer to the tube, then use a disposable pestle to macerate the tissue thoroughly.

Vortex the tube to further homogenize the tissue. Next, incubate the tube in a heating block set to 95 degrees Celsius for 10 minutes to lyse the cells. After incubation, transfer the tube immediately onto ice to cool.

Neutralize the reaction with 400 microliters of 40 millimolar tris-HCL, resulting in a final concentration of 20 millimolar tris-HCL in the solution. For isothermal amplification by RPA, first prepare the master mix according to the number of reactions required. Use diethyl pyrocarbonate-treated water instead of amplification-ready cell lysis for the negative control and pathogen DNA for the positive control.

Transfer 46.5 microliters of the master mix into 0.2 milliliter PCR tubes. Add magnesium acetate and template DNA to the caps of each PCR tube according to specified volumes. Briefly centrifuge the tubes to mix and initiate the RPA reaction.

Then incubate the tubes in a thermocycler at 39 degrees Celsius for 40 minutes to carry out the RPA amplification reaction. Alternatively, incubate the samples using DNA-free Eppendorf tubes in a thermal block. First, prepare a 100 micromolar CRISPR RNA stock solution.

Reconstitute 10 nanomoles of lyophilized CRISPR RNA in 100 microliters of diethyl pyrocarbonate-treated water. To prepare a one micromolar working solution, mix one microliter of the 100 micromolar stock with 99 microliters of diethyl pyrocarbonate-treated water in a 0.2 milliliter tube. To prepare the Cas12a enzyme solution, dilute the 100 micromolar solution to a one micromolar working solution by mixing one microliter of the enzyme with 99 microliters of the diluent provided in the kit.

Now, prepare a 100 micromolar stock solution of the FAM probe by resuspending it in one microliter of diethyl pyrocarbonate-treated water per one nanomole of probe. For the 10 micromolar working solution, mix 90 microliters of DEPC-treated water with 10 microliters of the 100 micromolar stock. Next, prepare the CRISPR reaction mix, excluding the amplicons in a 1.5 milliliter microcentrifuge tube.

Vortex the suspension to mix well, then distribute it into PCR tubes. Now, pipette four microliters of RPA amplicon into each tube. Place the tubes in a thermocycler set to 37 degrees Celsius and incubate for 120 minutes with fluorescence recorded every minute.

Alternatively, incubate the samples using DNA-free Eppendorf tubes in a thermal block. At the end of the incubation, transfer the reactions into 0.2 milliliter tubes for visualization using a gel documentation system. To detect biotin probe in a lateral flow test, prepare the flow test components in a 1.5 milliliter tube.

After vortexing the solution, distribute it into a PCR plate. Then add four microliters of amplicon to the respective wells. Incubate the PCR tubes at 37 degrees Celsius for 120 minutes using a thermocycler or thermal block without fluorescence reading.

Then transfer the samples to 0.2 milliliter tubes. Then add 50 microliters of running buffer and 10 microliters of the reaction mixture to each tube. Insert ImmunoStrips and incubate for 15 minutes at room temperature.

Read results after 10 minutes of immersion. Recombinase polymerase amplification detected Lotmaria passim in 16 out of 32 honeybee samples, whereas quantitative polymerase chain reaction identified only 12 positives, demonstrating higher sensitivity of the RPA method. The detection limit of quantitative polymerase chain reaction was approximately 9.6 parasites as shown by the amplification curve with the lowest visible signal.

The detection limit of the recombinase polymerase amplification coupled to CRISPR-Cas12a matched that of QPCR, detecting as low as six picograms corresponding to 96 parasites.