TITLE: Stanford and Purigen Biosystems Microfluidics Team protocol XPRIZE_Hudson Alpha
AUTHORS: jnesvet 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Stanford University and Purigen Biosystems team's SARS-CoV-2 assay leverages on-chip microfluidics to eliminate laborious and time consuming steps associated with standard molecular diagnostics such as solid phase spin-column extraction and PCR amplification. Purification of nucleic acids from a variety of biological sources is achieved in a one-step, automated fashion using on-chip isotachophoresis (ITP). The purified nucleic acids are then amplified using reverse transcription (RT) loop-mediated isothermal amplification (LAMP) in 30 minutes, less than half of the time associated with standard qPCR. We then use CRISPR-Cas12 fluorescent detection to identify amplicons associated with the SARS-CoV-2 genome for enhanced specificity. </div></div>

[STEPS]
?. [Preparation for Purigen Ionic Instrument ]
Prepare your laboratory for the receipt of the Purigen Ionic Instrument.
NOTE: When handling nucleic acids and amplicon, it is critical to reduce contamination. Therefore, 3 separate preparation areas are required for this protocol: 1) a "patient sample preparation area" for viral RNA extraction with the Purigen Ionic Instrument and dispensing sample into LAMP reaction 8-strip tubes, 96, or 384 well plates, 2) a "LAMP reagent preparation area" for preparing the LAMP mastermix and dispensing it into 8-strip tubes, 96, or 384 well plates prior to viral RNA addition, and 3) a "CRISPR/amplicon preparation area" for preparing the CRISPR reaction mix and performing the CRISPR reactions.To aid in reducing contamination, spray surfaces, vortex, microcentrifuge, pipettes, and all reagent tubes with RNase Away, or 10% bleach and RNase Zap prior to use. Each of the three preparation areas should have dedicated pipettes, tips, etc. to reduce cross-contamination. Change gloves often and when necessary.
?. [Preparation for Purigen Ionic Instrument ]
Complete Purigen Ionic installation and training with Purigen's Field Application Support.
During the training run with the Field Application Support, you will process positive and negative control samples. The eluates can be stored at   for future downstream processing with LAMP and CRISPR, or stored on ice for same day downstream LAMP and CRISPR processing  to ensure the positive and negative controls are validated.
?. [Viral RNA Extraction from Patient Samples]
Purify viral RNA using the Purigen Ionic Viral RNA kit.This is a safe stopping point. If you are not proceeding directly to LAMP/CRISPR, store the resulting viral RNA samples on ice for same-day use, at  for long-terms storage.
-80 °C
?. [Program thermal cycler for LAMP]
Open a new template. Select "Presence/Absence" then "Presence Absence Post."Program the thermal cycler to hold   for . Save the protocol as "Stanford Purigen LAMP protocol" for future use.
LAMP can be performed on any thermal cycler. For the QuantStudio instruments, follow below instructions.
62 °C
?. [Program thermal cycler for LAMP]
If applicable, set the lid heater to a value greater than  . We recommend .
62 °C
105 °C
?. [Program thermal cycler for LAMP]
No fluorescence readouts are required for the LAMP amplification step. Adjust the program accordingly, if necessary.
?. [Preparation of LAMP Mastermix]
Decontaminate LAMP reagent preparation area with RNase Away or 10% bleach plus RNase Zap spray, including all surfaces and pipettes. Thaw all reagents on ice.
NOTE: To reduce cross-contamination, prepare the LAMP mastermix and dispense in 8-tube strips, 96, or 384 well plates in an area separate from the patient sample preparation area and the CRISPR/amplicon preparation area.
?. [Preparation of LAMP Mastermix]
After thawing, briefly vortex 10x isothermal amplification buffer, 25x primers, dNTPs, and MgSO4 while inverting Bst2.0 WarmStart DNA Polymerase and WarmStart RTx Reverse Transcriptase to mix. Collect reagents at the bottom of the tubes with a brief centrifuge on benchtop microcentrifuge. Keep all reagents on ice, decontaminate all reagent tubes prior to use.
?. [Preparation of LAMP Mastermix]
Determine number of reactions (N) to prepare for each batch of LAMP amplifications. Each patient sample should have duplicate LAMP reactions performed. Additionally, be sure to include one LAMP Positive Control (PC) and one No Template Control (NTC) in each batch. Mastermix should be prepared in slight excess to account for pipette error, therefore the total number of reactions (N) = (2.2 x [# patient samples]) + 1 PC + 1 NTC. So for example, if you were to amplify all 8 patient samples from a single chip, N = (2.2 x 8) + 1PC + 1 NTC = 19.6
?. [Preparation of LAMP Mastermix]
Calculate the volume of each reagent required for N total reactions according to the table below.10x Isothermal Amplification BufferN x 2.5 uL25x primersN x 1.0 uLWarmStart Bst2.0 DNA PolymeraseN x 1.0 uLWarmStart RTx Reverse TranscriptaseN x 0.5 uLdNTP mixN x 3.5 uLMgSO4N x 1.5 uLTotal Mastermix VolumeN x 10 uL
10x Isothermal Amplification BufferN x 2.5 uL25x primersN x 1.0 uLWarmStart Bst2.0 DNA PolymeraseN x 1.0 uLWarmStart RTx Reverse TranscriptaseN x 0.5 uLdNTP mixN x 3.5 uLMgSO4N x 1.5 uLTotal Mastermix VolumeN x 10 uL
?. [Preparation of LAMP Mastermix]
Organize the 8-tube strips, 96, or 384 well plate for the given N number of reactions. As an example, Patient Samples 1-8 (P1-P8) duplicates plus a PC and NTC are shown below. Please scale accordingly; note for 384 well plates use every other row and column to reduce cross-contamination between samples and isolate NTC and PC wells. Label the strips with the corresponding patient sample number. 123456789101112AP1 AP1 BNTCBCP2 AP2 BDEP3 AP3 BFGP4 AP4 BHIP5 AP5 BJKP6 AP6 BLMP7 AP7 BNOP8 AP8 BPPC
123456789101112AP1 AP1 BNTCBCP2 AP2 BDEP3 AP3 BFGP4 AP4 BHIP5 AP5 BJKP6 AP6 BLMP7 AP7 BNOP8 AP8 BPPC
*Note that because no fluorescent data is collected in this step, the top of the tube can be labeled if using 8-tube strips.
?. [Preparation of LAMP Mastermix]
Dispense mastermix reagents in a clean 1.5 mL microcentrifuge tube. Vortex to thoroughly mix mastermix. Briefly centrifuge to collect reagents at bottom of tube.
?. [Preparation of LAMP Mastermix]
Set up 8-tube strips, 96, or 384 well plate over ice or cold rack.
?. [Preparation of LAMP Mastermix]
Dispense  of mastermix into each tube or well.
10 µl
?. [Preparation of LAMP Mastermix]
Prepare NTC reactions in reagent preparation area. Decontaminate Nuclease Free Water with RNase Away or RNase Zap, then pipette   into appropriately labeled tubes and securely cap them, if applicable. Additionally,  dispense  of Nuclease Free Water into the Positive Control well. Now, you can move the strips loaded with the mastermix to the patient sample preparation area.
15 µl
13 µl
?. [Patient Sample Template Addition]
If sample extracts are stored in individual Lo-Bind microcentrifuge tubes, vortex prior to dispensing extract into LAMP reaction. If extracts were stored in 8-tube strips or 96-well plates, mix by gently pipetting up and down prior to dispensing extract into LAMP reaction. Centrifuge to collect contents at bottom of the tube. Keep samples in cold rack or on ice.
?. [Patient Sample Template Addition]
Carefully dispense   of patient sample eluates into the corresponding wells containing the  of mastermix . Pipette the sample up and down 5 times to mix, DO NOT VORTEX. Change tips after each addition to reduce contamination
15 µl
10 µl
?. [Patient Sample Template Addition]
Carefully dispense  of the LAMP positive control (LAMP Pos Ctrl) to appropriate tube or well.
2 µl
?. [Patient Sample Template Addition]
Securely cap or seal wells after addition, briefly centrifuge to collect samples at bottom.
?. [Perform 30 Minute LAMP Reaction on Thermal Cycler]
Load "Stanford Purigen LAMP protocol" on thermal cycler. Add sealed tubes or well plate to thermal cycler and perform LAMP for  .
?. [Perform 30 Minute LAMP Reaction on Thermal Cycler]
After LAMP reaction is complete, briefly centrifuge tubes or well plate and keep on ice in CRISPR/amplicon handling area.
?. [Program thermal cycler for CRISPR Reaction]
Program the QuantStudio thermal cycler for the CRISPR reaction. Open a new template and select "Presence/Absence"Ensure Pre-PCR Read and Post-PCR Read are selected.Set cover to  . Save experiment method as "Stanford Purigen CRISPR protocol."
50 °C
Note the reporters are FAM labeled, so ensure the reading is either in the FAM or SYBR green fluorescent channel.
?. [Preparation of CRISPR Reaction Mix]
Using the same number of N determined for LAMP reactions in Step 7, prepare the CRISPR reaction mix
Note that because tubes containing amplicon will be handled, all steps in this section should be performed in an area dedicated for amplicon handling, separate from both the reagent preparation and patient sample preparation area. Decontaminate surfaces and pipettes with RNase Away or  10% bleach RNase Zap.
Note that the CRISPR reaction proceeds immediately after LAMP amplicons are added. Therefore, this reaction is very time sensitive and the order of reagent addition is extremely important. Because of the time sensitive nature, each laboratory personnel should perform no more than 48 CRISPR reactions (6 columns) per run.
?. [Preparation of CRISPR Reaction Mix]
Calculate the volume of each reagent required for N total reactions according to the table below.RNP mixN x 17 uLreportersN x 1 uLTotal volumeN x 18 uL
RNP mixN x 17 uLreportersN x 1 uLTotal volumeN x 18 uL
?. [Preparation of CRISPR Reaction Mix]
Organize clean 8-tube strips, 96, or 384 well plate for the given N number of reactions in the same format as in Step 8 for the CRISPR reaction. Load this plate format on the QuantStudio touch screen. The patient samples should be classified as "Unknown" under the sample type, while the NTC and LAMP Positive Control are "Negative Control" and "Positive Control," respectively.123456789101112AP1 AP1 BNTCBCP2 AP2 BDEP3 AP3 BFGP4 AP4 BHIP5 AP5 BJKP6 AP6 BLMP7 AP7 BNOP8 AP8 BPPC
123456789101112AP1 AP1 BNTCBCP2 AP2 BDEP3 AP3 BFGP4 AP4 BHIP5 AP5 BJKP6 AP6 BLMP7 AP7 BNOP8 AP8 BPPC
Note that fluorescent measurements will be taken during CRISPR reaction, so do not label the top of the tubes or wells.
?. [Preparation of CRISPR Reaction Mix]
Dispense reagents in a clean 1.5 mL microcentrifuge tube to prepare CRISPR reaction mix. Vortex to thoroughly mix. Briefly centrifuge to collect reagents at bottom of tube, then keep on ice.
?. [Preparation of CRISPR Reaction Mix]
Add  from LAMP reaction tube into the new corresponding tube or 96 well plate. Store the remaining solution in the freezer.
2 µl
Note that the amplicons must be added BEFORE the CRISPR reaction mix to ensure speedy pipetting in the following step.If using a sealed well plate, ensure to carefully remove the seal to avoid sample cross-contamination.
?. [Preparation of CRISPR Reaction Mix]
Quickly add  of the CRISPR reaction mix to each tube, changing pipette tips after each addition. It is important to add the CRISPR reaction mix to the PC and NTC tubes prior to the patient samples to ensure the timing is accurate.
18 µl
Note: do not pipette-mix or vortex for this step. At most, 48 CRISPR reactions should be performed at a time, and it should take approximately 3 minutes to fully dispense the CRISPR reaction mix into the 48 individual tubes.
?. [Preparation of CRISPR Reaction Mix]
Load the "Stanford Purigen CRISPR protocol" on the thermal cycler. Add tube strips or well plate and run CRISPR reaction for 15 minutes.
?. [CRISPR Data Analysis - Threshold Calibration]
Data analysis of the CRISPR data will be a simple presence/absence call. However, it is necessary to determine the fluorescence threshold for presence/absence on each unique QuantStudio instrument. Therefore, calibration of the fluorescence threshold will be necessary during the first run on each QuantStudio instrument, which will occur during the Purigen instrument training using known samples. Calibration of each instrument is necessary prior to processing patient samples.If the QuantStudio has already been calibrated and the fluorescence threshold established, continue onto step 30. If the QuantStudio has not been calibrated, continue onto the next step.
?. [CRISPR Data Analysis - Threshold Calibration]
The CRISPR fluorescent data can be analyzed on the cloud-based platform Thermo Fisher Connect.Register for an account here: https://www.thermofisher.com/account-center/cloud-signin-identifier.htmlThe Thermo Fisher Connect dashboard can be found here: https://apps.thermofisher.com/apps/spa/#/dashboardUpload the .eds file from the Purigen training run to the Thermo Fisher Connect platform and open the file.
?. [CRISPR Data Analysis - Threshold Calibration]
Go to the "Plate Setup" menu and change the Passive Reference to "None." Additionally, ensure that wells are correctly labeled as "Unknown," "Negative Control," or "Positive Control" according to the plate format.
?. [CRISPR Data Analysis - Threshold Calibration]
Go to the "Quality Check" menu. The Amplification Plot should be displayed, as shown below.
?. [CRISPR Data Analysis - Threshold Calibration]
Select the "Actions" dropdown menu in the top right of the screen and select "Primary Analysis Setting." Set "Algorithm Settings" to "Baseline Threshold." Change the Baseline Start to 1 and the Baseline End to 2. Click "Save."
?. [Interpretation of Results]
Ensure that the NTC sample is classified as "absence" for every run. If the NTC sample does appear positive, this is likely due to contamination from the LAMP reaction setup and the results are invalid. If possible, repeat the protocol from the sample extraction on remaining raw sample using the Purigen system (Step 3).Ensure that the LAMP Positive Control sample is classified as "presence" for every run. If the sample does not appear positive, this could be due to the time sensitive nature of CRISPR, and it may have taken too long to add the CRISPR reaction mix to all 48 samples. To troubleshoot, try running another CRISPR reaction on just the LAMP PC. If it now appears positive, it likely took too long to load the CRISPR reaction mix into the 48 samples. Redo the CRISPR reaction starting from Step 23, instead doing 16 samples at a time. If this troubleshooting did not solve the issue, repeat the protocol from the sample extraction on remaining raw sample using the Purigen system (Step 3).
?. [Interpretation of Results]
Once the results have been validated by the controls, the patient samples can be interpreted as follows:If either 1 of 2 or 2 of 2 of the patient sample replicates is classified as "presence" this can be interpreted as positive for SARS-CoV-2.If both patient sample replicates are classified as "absence" this can be interpreted as negative for SARS-CoV-2.
?. [CRISPR Data Analysis - Threshold Calibration]
Go to the Settings menu, and change Y value from ΔRn to Rn. Ensure the CRISPR fluorescence data qualitatively passes quality control, the Purigen Negative Controls should show no CRISPR fluorescent signal, and while the Purigen Positive Controls should show an increase in CRISPR fluorescent signal, as shown below. Note any negative controls that show an increase in CRISPR fluorescence signal and exclude them in analysis for calculating threshold.
?. [CRISPR Data Analysis - Threshold Calibration]
To calculate the fluorescence threshold, go to the "Presence Absence" menu. Click "..." and choose Export. This will create and download a csv file, which should contain the ΔRn for each well.
?. [CRISPR Data Analysis - Threshold Calibration]
Identify the ΔRn for each Purigen Negative Control (8 samples) sample and the No Template Control (1 sample). Calculate the average ΔRn of these 9 samples and the standard deviation (σ). The threshold is equal to:Threshold = avgΔRn  + 2σ
Record the value of the threshold, as it will be the same for all subsequent runs on this QuantStudio instrument.
?. [CRISPR Data Analysis]
Return to the "Primary Analysis Setting" from the "Actions" dropdown menu and enter the threshold unique to the QuantStudio, then click "Save." In the "Actions" dropdown menu, go to the "Presence Absence Analysis Setting," set the Default Cq Cutoff to 20 and select Real-time Rn Data, then click "Apply."
?. [CRISPR Data Analysis]
Click "Analyze." The sample wells should now show a call of either presence or absence.Export the data as a csv, which will contain the well, presence/absence call, and ΔRn. Results can be analyzed on the exported csv, or they can be analyzed on the cloud platform.
Note: During the Purigen training run, ensure that the threshold produces the correct presence/absence assignments to the respective positive and negative samples.