TITLE: COVID19 RTLAMP Assay_Nov_2020
AUTHORS: Arun Manoharan Arunprimediscoveriescom, Eugene Joseph

[STEPS]
?. [Sample Lysis / Inactivation]
Thaw the lysis Buffer on ice 1 to 3 hours before starting the experiment. It is recommended to aliquot the lysis buffer in small volumes and use it as needed to avoid excessive freeze-thawing cycles. If using the Lysis Bufferthaw the  Buffer on ice 1 to 3 hours before starting the experiment. It is recommended to aliquot the lysis buffer in small volumes and use it as needed to avoid excessive freeze-thawing cycles. Proceed to step 2.If using the QIAmp Viral RNA Mini Kit(Qiagen cat# 52906) for lysis and RNA extraction, vortex the sample to pellet debris and start with 140 ul of input material. Proceed following the manufacturer's instructions and elute the sample in 60 ul of AVE buffer. Proceed to step 26
?. [Sample Lysis / Inactivation]
Thaw samples on ice if necessary and briefly vortex. Centrifuge the samples at 1000xg for 2 minutes to pellet solid debris. Alternatively, when completely thawed, samples can be left on ice for 30 minutes. Solid debris should be visible at the bottom of the tube. or
?. [Sample Lysis / Inactivation]
If using a Heat Block or water bath proceed to step 4. If using a thermal cycler proceed to step 9. If following the full method/sample purification proceed to step 12.
?. [Sample Lysis / Inactivation]
SAMPLES PREPARATION FOR INCUBATION IN A HEAT BLOCK OR WATER BATHTransfer 100 μL of the sample Input Material (or clarified supernatant if working with fresh saliva) to a new microcentrifuge tube (1.5 mL capacity) containing an equal volume of lysis buffer (e.g. for 100 μL of sample add 100 μL of lysis buffer). Mix the obtained solution a few times by pipetting up and down. Label the tube with sample ID and other relevant information.
[Lysis Buffer]
[Clarified Sample]
?. [Sample Lysis / Inactivation]
Lock the microcentrifuge lid using a lid lock (alternatively, an Eppendorf Safe-Lock tube can be used) to prevent tubes from popping open when exposed to high temperature.
?. [Sample Lysis / Inactivation]
Set the temperature of a heat block or water bath at 95°C. When the temperature has reached 95°C, place the tubes inthe heat block or water bath and incubate for 5 minutes.
?. [Sample Lysis / Inactivation]
Carefully transfer the tubes to ice or a 4°C cold block for 30 sec, or let sit at Room Temperature for 5 min. 4 °COr Room temperature
?. [Sample Lysis / Inactivation]
Centrifuge the tubes for 3 sec at 2000xg to spin down any condensation using a benchtop mini centrifuge. Samples are ready for the RT-LAMP reaction.  Proceed to step 26.
?. [Sample Lysis / Inactivation]
SAMPLES PREPARATION FOR INCUBATION IN A THERMAL CYCLERTransfer 20ul of the sample input material (or clarified supernatant if Fresh Saliva) to a new 200 μL tube containing an equal volume of lysis buffer (e.g. for 20 μL of sample add 20 μL of lysis buffer). Mix the obtained solution a few times by pipetting up and down. Label the tube with sample ID and other relevant information.
[Lysis Buffer]
[Clarified Sample]
?. [Sample Lysis / Inactivation]
Create a Method on the Thermal Cycler using the following steps:Step 1:   for Step 2: Hold at Lid temperature: When ready run the program.
95 °C
4 °C
105 °C
?. [Sample Lysis / Inactivation]
Centrifuge the tubes for 3 sec at 2000xg to spin down any condensation using a benchtop mini centrifuge. Samples are ready for the RT-LAMP reaction. Proceed to step 26.
?. [Sample Purification / Concentration]
Preheat the heat block or water bath as instructed in step 6.
?. [Sample Purification / Concentration]
Vortex to mix the binding solution so there is no sediment.
?. [Sample Purification / Concentration]
To each sample tube, add 800 ul of Binding Solution
[Binding Solution]
?. [Sample Purification / Concentration]
Let the Tubes sit at Room Temperature for 10 min, every 2 min invert the tube to suspend the Binding Solution.  at
0 Room temperature
?. [Sample Purification / Concentration]
Centrifuge for 3 sec at 2000xg to pellet the Binding Reagent in the Binding Solution.Note: A white smear at the bottom of the tube should be present. The Binding Reagent should adhere to the bottom of the tube with a short spin on a simple benchtop centrifuge. Depending on tube type and input sample viscosity, a longer spin may be required until it is sufficiently pelleted.
?. [Sample Purification / Concentration]
Remove the supernatant with a 1ml Pipette taking care not to disturb the pellet.
[Supernatant]
?. [Sample Purification / Concentration]
Add 1ml of Wash Solution, wash the pellet by closing and inverting the tube several times.
[Wash Solution]
?. [Sample Purification / Concentration]
Centrifuge for 3 sec at 2000xg to pellet the Binding Reagent in the Binding Solution
?. [Sample Purification / Concentration]
Remove the supernatant with a 1ml Pipette taking care not to disturb the pellet.
[Wash Supernatant]
?. [Sample Purification / Concentration]
Centrifuge for 3 sec at 2000xg to collect any residual Wash Solution.
?. [Sample Purification / Concentration]
Remove the residual supernatant with a 200ul Pipette taking care not to disturb the pellet.
[Wash Supernatant]
?. [Sample Purification / Concentration]
Air dry in a clean ventilated area until the Binding Reagent is dry, it should appear opaque and not reflective, air drying should take 10-15 min, some sample types may take longer to dry, do not let drying take longer than 30 min. Note: If the lab is equipped, air drying should take place in a ventilated air cabinet to prevent cross-contamination.  To speed up the air drying, if available it is possible to use a Centrifuge with open tube lids, or a heated vacuum concentrator.  If using these it would take less than 15 minutes as it should be evaporating less than 5-10ul of Wash Solution.
?. [Sample Purification / Concentration]
To each tube add 10ul of Resuspension Buffer, pipette mix to resuspend the pellet.  Depending on the Sample Input, the pellet may resuspend easily or stay as clumps that will take more pipetting to break apart.
[Resuspension Buffer]
?. [Sample Purification / Concentration]
Vortex until the Binding Reagent is resuspended, you can briefly centrifuge for 3 sec at 2000xg to collect droplets from the sides of the tube. Note: As the sample contains extracted resuspended RNA, the RT-LAMP reaction should proceed the same day.  The extracted samples should not be stored or frozen as it would impact the RNA integrity.
?. [RT-LAMP REACTION]
Prepare the Real-time PCR instrument, and work on ice for the following steps.  Calculate the number of samples being run and make sure to include a positive control and the assay negative control (water only).
?. [RT-LAMP REACTION]
Prepare the Assay mix by adding Reaction Mix and Primer mix according to the number of samples being run.
[Reaction Mix per sample]
[Primer Mix per Sample]
?. [RT-LAMP REACTION]
Transfer 15ul of Assay mix into the Assay wells.
[Assay Mix]
?. [RT-LAMP REACTION]
Transfer 5ul of each sample into the appropriate Assay well, pipette mix.
[Sample]
?. [RT-LAMP REACTION]
Add the Positive Control to the Plate or Strip Tube.
[Positive Control]
?. [RT-LAMP REACTION]
Transfer 5ul of UltraPure Distilled water into the appropriate Negative Control well.
[UltraPure Distilled Water]
?. [RT-LAMP REACTION]
Seal the plate and run it in the Real-Time PCR instrument with the following settings :Channels and DyeFAM - No Quencer = Assay TargetCy3 - No Quencher = Control TargetPCR STAGE   for 80 CyclesReaction Volume 20 ulBe sure that the capture/collect image option is selected at the end of the temperature stage (usually a small icon showing a camera should be selected below the temperature settings).
68.5 °C
[Reaction Volume]