We're developing point-of-care test to detect genetics material of infectious diseases. This detect use isothermal nucleic acid amplification and CRISPR-based detection. With highly osmotic accuracy, similar to PTR, they can detect small sequence differences, including single nucleotide polymorphism, and can be multi-page.
CRISPR the osmotic reactions contain multiple components and effects and are difficult to perform at the point of care, As CRISPR-based osmotics are highly sensitive. there is a risk of carryover contamination, which can lead to false results. Fortunately, this problem can be mitigated if we simplify the procedures of the reactions.
We offer an easy-to-use method to detect disease pathogens and genetic markers using the combination isothermal amplification and CRISPR-based reagents. These agents are simple to store and use. We also offer tips to fix common issues when using these methods and the agents, making it easier for people to use them.
The protocol here uses freeze-dried reagents, which facilitate the transportation and storage. The freeze-dried reagents can easily be rehydrated at the points of use and retained high amplification and detection efficiencies. We hope to apply CRISPR diagnostic technologies to detect endemic and emerging infectious disease.
If this test can be routinely and correctly performed at the point of care, that could inform antiviral and antibiotic treatment options, potentially save lives, reduce drug overuse and the development of resistance. To begin, incubate the triglycine solution at 60 degrees Celsius. Remove three RPA pellet strip tubes from the freezer.
Tap the strip tubes gently against the lab bench to dislodge the white pellets. Transfer all three pellets into a 1.5 milliliter microcentrifuge tube A.Pipette 25 microliters of nuclease-free water to rinse any remaining RPA powder in the strip tubes. Transfer the rinse solution to a new 1.5 milliliter microcentrifuge tube B.then add 10 microliters of the 0.57 molar triglycine solution and 10 microliters of primer mix to the master mix tube B.After vortexing and spinning down the tube, place it on ice.
Next, add 28.4 microliters of the five times RPA buffer to the master mix tube B.Mix thoroughly by strong tapping or shaking multiple times. Transfer pellets collected in tube A into the master mix tube B and invert the tube or tap the bottom of the tube with your finger several times to mix. Observe whether the solution appears homogeneous and monophasic.
Place the tube on ice, then add 0.71 microliters of reverse transcriptase and 2.84 microliters RNase H to the master mix tube. Gently tap or invert the master mix tube up and down many times. Briefly spin down the tube before placing it on ice.
Now, aliquot 8.4 microliters of the master mix solution to 10 pre-cooled 1.5 milliliter tubes and place the tubes on ice. After aliquoting, place the tubes in a metal rack submerged in liquid nitrogen to prepare them for lyophilization. At the freeze dryer, check collector and shelf temperatures.
Now, cover open tubes in the metal rack with a sheet of cleaning white paper. Place the metal rack along with the open tubes in the freeze dryer shelf and close the shelf door. Then, select the desired program and press start.
After 30 minutes of the secondary drying step, press stop. Release the pressure by opening the vacuum release valve. After removing the rack from the shelf, cap the tubes immediately.
To begin, incubate the triglycine solution at 60 degrees Celsius. Remove three RPA pellet strip tubes from the freezer. Tap the strip tubes gently against the lab bench to dislodge the white pellets.
Transfer all three pellets into a 1.5 milliliter microcentrifuge tube A.Pipette 25 microliters of nuclease-free water to rinse any remaining RPA powder in the strip tubes. Transfer the rinse solution to a new 1.5 milliliter microcentrifuge tube B, then, add 10 microliters of the 0.57 molar triglycine solution and 10 microliters of primer mix to the master mix tube B.After vortexing and spinning down the tube, place it on ice. Next, add 28.4 microliters of the five times RPA buffer to the master mix tube B.Mix thoroughly by strong tapping or shaking multiple times.
Transfer pellets collected in tube A into the master mix tube B and invert the tube or tap the bottom of the tube with your finger several times to mix. Observe whether the solution appears homogeneous and monophasic. Place the tube on ice, then add 0.71 microliters of reverse transcriptase and 2.84 microliters RNase H to the master mix tube.
Gently tap or invert the master mix tube up and down many times. Briefly spin down the tube before placing it on ice. Now, aliquot 8.4 microliters of the master mix solution to 10 pre-cooled 1.5 milliliter tubes and place the tubes on ice.
After aliquoting, place the tubes in a metal rack submerged in liquid nitrogen to prepare them for lyophilization. At the freeze dryer, check collector and shelf temperatures. Now, cover open tubes in the metal rack with a sheet of cleaning white paper.
Place the metal rack along with the open tubes in the freeze dryer shelf and close the shelf door, then select the desired program and press start. After 30 minutes of the secondary drying step, press stop. Release the pressure by opening the vacuum release valve After removing the rack from the shelf, cap the tubes immediately.
To begin, prewarm a thermal incubator at 42 degrees Celsius. Add one microliter of magnesium acetate and potassium acetate solution mix to the side of the tube containing the lyophilized premixed RT-RPA pellet. Resuspend the pellet in 12.4 microliters of the RNA sample.
To initiate the RT-RPA reaction, briefly spin down the added solution using a spin-down mini centrifuge at room temperature for three seconds. Carefully tap the tube to mix the contents thoroughly and spin down again. Incubate the reaction at 42 degrees Celsius for 60 minutes in a preheated thermal shaking incubator.
After the reaction has ended, remove the reaction tubes and place them on ice before proceeding to the CRISPR/Cas detection. For nucleic acid detection, turn on a fluorescence microplate reader. Set and preheat the microplate reader to 37 degrees Celsius and select the program, then, place a 384-well plate on ice.
To resuspend the lyophilized CRISPR/Cas pre-mixed detection reaction, add 17 microliters of eight millimolar magnesium chloride solution. Mix by carefully tapping the tube. Briefly spin down for three seconds before placing the tube on ice.
Transfer 18 microliters of the resuspended reaction mix to each well of the pre-cooled 384-well plate on ice. Add two microliters of the RPA product prepared to each reaction well. Remove the plate from the ice.
Quickly place it into a preheated fluorescence microplate reader and press start. For lyophilized pre-mixed Cas13a-based reactions, either 6%trehalose or 2%sucrose, preserves the reaction efficiency well after lyophilization. The lyophilized pre-mixed reagents for RT-RPA and Cas13-based reactions are stable at minus 20 degrees Celsius for at least eight months.
