TITLE: DetectXRv Kit
AUTHORS: bkatchman 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The Detect</span><span style = "vertical-align:super;">X</span><span>-Rvtest is a test based on end-point reverse transcription polymerase chain reaction (RT-PCR) coupled to DNA microarray hybridization for the detection of multiple genes within SARS-CoV-1 and SARS-CoV-2 viruses.The DNA microarray contains:</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>Detect</span><span style = "vertical-align:super;">X</span><span>-Rv Kit – </span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>-Detect</span><span style = "vertical-align:super;">X</span><span>-Rv SARS-CoV-2 Multiplex Assay—contains 5 (five) SARS-CoV-2 primer sets; 4 (four) SARS-CoV-2probes targeting each N1, N2, and N3 genes</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>-Detect</span><span style = "vertical-align:super;">X</span><span>-Rv RNase P Control—internal process control for nucleic acid extraction 2 (two) RNase P primer and probe sets as an internal positive control. </span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Detect</span><span style = "vertical-align:super;">X</span><span>-Rv SARS-CoV-2 Control – RNA control that contains targets specific to the SARS-CoV-2 genomic regions that are targeted by the assay</span></li></ul></div><div class = "text-block"><span>-Detect</span><span style = "vertical-align:super;">X</span><span>-Rv SARS-CoV-2 Multiplex Assay—contains 5 (five) SARS-CoV-2 primer sets; 4 (four) SARS-CoV-2probes targeting each N1, N2, and N3 genes</span></div><div class = "text-block"><span>-Detect</span><span style = "vertical-align:super;">X</span><span>-Rv RNase P Control—internal process control for nucleic acid extraction 2 (two) RNase P primer and probe sets as an internal positive control. </span></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div></div>

[STEPS]
?. [RNA Extraction]
-	Extract RNA with the Zymo Research Quick-DNA/RNA Viral MagBead Kit (R2140 or R2141): For a complete description view the Zymo Research Quick-DNA/RNA Viral MagBead Kit product insert. Follow the manufacturer’s guidelines for the proper use and procedure for this product.
?. [RNA Extraction]
-	Place the nasopharyngeal swab into 400 µL – 2mL 1x VTM for storage prior to RNA extraction.
?. [RNA Extraction]
-	If using a swab transported in VTM, vortex for 10 seconds to release viral particles and remove 400 µL for Sample Preparation and DNA/RNA Purification procedures per the Zymo Research Quick-DNA/RNA Viral MagBead Kit.
?. [RNA Extraction]
-	Store any remaining samples at -20°C for up to two weeks or -80°C for long term storage.
?. [RNA Extraction]
-	Once complete the extracted RNA can be used immediately or stored frozen at -20°C for up to two weeks or -80°C for long term storage.
?. [One Step RT PCR]
Mix and briefly centrifuge each component before use. Combine the following into a master mix, multiply per reaction as shown in Table 1:(Volume per reaction)  -   Component - Final Concentration - Access Quick Mastermix, 2X -  1X - RT-PCR Primer Set 1- -  AMV RT (5u/microliter)  - 0.1u/microliter - Purified RNA Sample  - N/A - Nuclease Free Water
25 µl
2 µl
1 µl
5 µl
17 µl
[Total Volume per Reaction]
?. [One Step RT PCR]
-	Determine the number of samples in the reaction and multiply each reagent to prepare the master mix, leaving out the Purified RNA Template from the master mix and add individually to each designated well
?. [One Step RT PCR]
-	Mix by pipetting and add 45 µl of the master mix per well
?. [One Step RT PCR]
-	Add 5 µl of the RNA template to each designated well making sure to change tips between samples
?. [One Step RT PCR]
Cover the plate with clear adhesive film and seal.
?. [One Step RT PCR]
Centrifuge the plate in a for 30 seconds.
?. [One Step RT PCR]
Place the plate in the thermal cycler and cover with a heat pad before closing the thermal cycler cover.
?. [One Step RT PCR]
Input the Reverse Transcriptase and PCR cycling program as shown in Table  ABCDE1     Step    Temperature (oC)    Time     Cycle Number  2  First Strand cDNA Synthesis    Reverse Transcription  45  min1x3  AMV RT Inactivation and RNA/cDNA/primer denaturation    94    2 min    1x  4  PCR Amplification  5Denature9430 sec40x6  Annealing    55    30 sec  7  Extension    68    30 sec  8  Final Extension    68    7 min    1x
ABCDE1     Step    Temperature (oC)    Time     Cycle Number  2  First Strand cDNA Synthesis    Reverse Transcription  45  min1x3  AMV RT Inactivation and RNA/cDNA/primer denaturation    94    2 min    1x  4  PCR Amplification  5Denature9430 sec40x6  Annealing    55    30 sec  7  Extension    68    30 sec  8  Final Extension    68    7 min    1x
?. [DNA Hybridization]
General guidelines to follow for hybridization: When pipetting with the multichannel onto the microarray slide, only dispense to the first stop. DO NOT depress the multichannel to the second stop, or full evacuation of the tips to avoid cross contamination.
?. [DNA Hybridization]
Before starting, thaw Buffer 2 at room temperature.  You will need Buffer 1 as well
?. [DNA Hybridization]
Cut paper towel to size to fit the bottom of the hybridization chamber provided.
?. [DNA Hybridization]
Place the plate to be used in the Hybridization Chamber.
?. [DNA Hybridization]
Apply 200 μL of Molecular Biology Grade Water to each well of the 96-well while being careful to avoid contact with the array.
?. [DNA Hybridization]
Aspirate and then again, dispense 200 μL of Molecular Biology Grade Water to each well of the 96-well and allow to sit covered in the Hybridization Chamber for 5 minutes before aspirating water from the slides.
?. [DNA Hybridization]
Prepare the Pre-hybridization Buffer and Hybridization Buffers in clean tubes for the number of microarrays that will be hybridized as per Tables 5 and 6.  Vortex briefly to mix.
?. [DNA Hybridization]
Table 5:  Reagent Volumes for preparation of PRE-Hybridization Buffer  AB196-Well Plate  Volumes corresponding to a full 96 well
  plate being hybridized  2  Molecular biology grade water (µL)    16761  3  Buffer 1 (µL)    4976  4  Buffer 2 (µL)    2618  5
AB196-Well Plate  Volumes corresponding to a full 96 well
  plate being hybridized  2  Molecular biology grade water (µL)    16761  3  Buffer 1 (µL)    4976  4  Buffer 2 (µL)    2618  5
?. [DNA Hybridization]
Table 6:  Reagent Volumes for preparation of Hybridization Buffer  AB1  96-Well Plate    Volumes corresponding to a full 96 well
  plate being hybridized  2  Buffer 1 (µL)    1390  3  Buffer 2 (µL)    700  4
AB1  96-Well Plate    Volumes corresponding to a full 96 well
  plate being hybridized  2  Buffer 1 (µL)    1390  3  Buffer 2 (µL)    700  4
?. [DNA Hybridization]
Aspirate the water wash and add 200 μL of Pre-hybridization Buffer to each well of the  96-well slides without touching the pipette tip to the array surface. Close the Hybridization Chamber box lid.
?. [DNA Hybridization]
Allow Pre-hybridization Buffer to stay on the arrays for 5 minutes; do not remove slides from the Hybridization Chamber.
?. [DNA Hybridization]
Briefly centrifuge the tubes or plate containing the Labeling PCR product.
?. [DNA Hybridization]
Add 18μL of Hybridization Buffer to each well of the Labeling PCR product within the 96-well PCR plate or tubes, pipette up and down to mix. It is important that no cross- contamination occurs during this step. The PCR product and the Hybridization Buffer mix constitute the Hybridization Cocktail.
?. [DNA Hybridization]
Aspirate Pre-hybridization Buffer from the arrays. Caution: Do not allow the arrays to air dry. Avoid contact with the array surface
?. [DNA Hybridization]
Immediately add 68μL (68 μL – Total Volume of PCR Reaction + Hyb Buffer) of the Hybridization Cocktail to each array of the 96-well being careful not to touch the array surface with the pipette tip. Ensure that the sample ID and location are recorded.
?. [DNA Hybridization]
Close the Hybridization Chamber lid.
?. [DNA Hybridization]
Allow to hybridize for 30 minutes at room temperature in the Hybridization Chamber.  Caution:Avoid moving the Hybridization Chamber while hybridizing to avoid sample merging.
?. [Post Hybridization Plate Processing]
Prepare Wash Buffer according to the number of slides to be used. (Table 7). Washing must be performed according to the protocol to ensure detectable signal and adequate washing to prevent elevated backgroundsignals.
?. [Post Hybridization Plate Processing]
Table 7  AB1       Volumes corresponding to a full 96 well
  plate being hybridized  2  Buffer 1 (µL)    777.6 µL<b style="mso-bidi-font-weight:
  normal">  3  Molecular biology grade water  (mL)*<b style="mso-bidi-font-weight:
  normal">    102.9 mL  4  Total
  volume (mL)*    103.7 mL
AB1       Volumes corresponding to a full 96 well
  plate being hybridized  2  Buffer 1 (µL)    777.6 µL<b style="mso-bidi-font-weight:
  normal">  3  Molecular biology grade water  (mL)*<b style="mso-bidi-font-weight:
  normal">    102.9 mL  4  Total
  volume (mL)*    103.7 mL
?. [Post Hybridization Plate Processing]
Aspirate Hybridization Cocktail from the plates
?. [Post Hybridization Plate Processing]
Add 200 μL of Wash Buffer to each well of the 96-well, mix by pipetting 5 times, then aspirate.
?. [Post Hybridization Plate Processing]
Add  200 μL of Wash Buffer to each well of the  96-well, mix by pipetting 5 times, then aspirate.
?. [Post Hybridization Plate Processing]
Add  200 μL of Wash Buffer to each well of the 96-well, mix by pipetting 5 times, allow buffer to remain on the slides for 5 minutes, aspirate
?. [Post Hybridization Plate Processing]
Add 200 μL of Wash Buffer to each well of the  96-well, mix by pipetting 5 times, allow buffer to remain on the slides for 5 minutes, aspirate
?. [Post Hybridization Plate Processing]
Add 200 μL of Wash Buffer to each well of the 96-well, mix by pipetting 5 times, allow buffer to remain on the slides for 5 minutes, aspirate
?. [Post Hybridization Plate Processing]
The washing steps below (32-37) must occur quickly to ensure no drying occurs
?. [Post Hybridization Plate Processing]
Perform a final wash by dispensing and aspirating 200 μL of Wash Buffer 2 times to each well of the 96-well.
?. [Post Hybridization Plate Processing]
Following the last aspiration step, remove the slides from the Hybridization Chamber.  Do not allow drying to occur on slide surface. Therefore, if running more than plates, aspirate the two and move those to Step 37 - allow the final to dispense of Wash Buffer to remain on the additional slides until the Slide Spinner is available for use. This will ensure no drying occurs on the slide surface.
?. [Post Hybridization Plate Processing]
Load the slides into the plate centrifuge for 1 minute to completely dry the wells in the plate
?. [Post Hybridization Plate Processing]
PathogenDx plates should be placed back into a  moisture barrier bag with desiccant until scanning may be performed to protect the slides from light. Slides should be scanned within two weeks of hybridization.
?. [Scanning and Data Acquisition]
Access the Sensovation scanner desktop, select the application “Array Reader”.
?. [Scanning and Data Acquisition]
Open the tray, select “Open Tray”.
?. [Scanning and Data Acquisition]
Place the slides in the tray oriented with the barcode towards the technician and face down. a.	If scanning a 96-well plate, place the plate face up and aligned with A1 in the top left
?. [Scanning and Data Acquisition]
Close the tray, select “Close Tray”.
?. [Scanning and Data Acquisition]
Select “Scan”.
?. [Scanning and Data Acquisition]
From the dropdown menu select the 12-well slide – PDx, 96-well – PDx.
?. [Scanning and Data Acquisition]
Note: All other information on this screen is preprogrammed – do not alter.
?. [Scanning and Data Acquisition]
Select the Blue Arrow to begin the scanning process.
?. [Scanning and Data Acquisition]
While the slides are being scanned, select “Result Overview” to review the images of the wells.
?. [Scanning and Data Acquisition]
When the slides are finished scanning and the screen displays the digital image of a slide with all green wells, select the Red X to exit the scanning process.
?. [Scanning and Data Acquisition]
Open the tray, select “Open Tray”.
?. [Scanning and Data Acquisition]
Remove the slides and store in the slide case inside the moisture barrier bag with the desiccant packets.
?. [Scanning and Data Acquisition]
Close the tray, select “Close Tray”.
?. [Scanning and Data Acquisition]
Exit the Array Reader application, select “Exit”.
?. [Scanning and Data Acquisition]
On the Sensovation Scanner desktop, select the folder “Scan Results”.
?. [Scanning and Data Acquisition]
Locate the folder associated with your slide and rename the folder with the slide barcode number by scanning the barcode located either on the outside of the barrier bag or on the slide itself. (ex. rename: ScanJob-191108130334_1 to 7024001001)
?. [Scanning and Data Acquisition]
Submit the whole barcode labeled folder to the “Image Folder” within Dropbox.
?. [Scanning and Data Acquisition]
The folder will automatically begin uploading, the PathogenDx Augury© Software will analyze the data and directly deposit the reports into the “Reports” folder within Dropbox.