Oct 02, 2025
REDI-NET FV-2 FLYING VECTOR PROCESSING
- REDI-NET Consortium1
- 1REDI-NET Consortium
- Remote Emerging Disease Intelligence - NETwork (REDI-NET)
Protocol Citation: REDI-NET Consortium 2025. REDI-NET FV-2 FLYING VECTOR PROCESSING. protocols.io https://dx.doi.org/10.17504/protocols.io.261gekwqwg47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 17, 2025
Last Modified: October 02, 2025
Protocol Integer ID: 227506
Keywords: zoonotic disease, extraction, mosquito processing, biting midge processing, sand fly processing, insect imaging, IDX imaging, Dinoscope imaging, vector processing for total nucleic acid extraction, flying vector processing, flying vector processing this protocol detail, total nucleic acid extraction, extraction, processing
Funders Acknowledgements:
USAMRAA
Grant ID: W81XWH-21-C-0001
USAMRAA
Grant ID: W81XWH-22-C-0093
USAMRAA
Grant ID: HT9425-23-C-0059
USAMRAA
Grant ID: HT9425-24-C-0072
Disclaimer
This work is supported by the US Army Medical Research and Development Command under Contract No.W81XWH-21-C-0001, W81XWH-22-C-0093, HT9425-23-C-0059, and HT9425-24-C-0072. The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army or Navy position, policy or decision unless so designated by other documentation.
Abstract
This protocol details flying vector processing for total nucleic acid extraction.
Guidelines
OBJECTIVE
To clearly document the correct process for effective standardized field collections of ticks, and recommended personal protection measures.
SUMMARY/SCOPE
The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on field tick collections in the vicinity of watering holes by dragging.
RESPONSIBLE PERSON
Principal Investigator, Study Coordinator, Entomology Component Lead, Managers
Note
NOTE: All study procedures must be conducted in compliance with national and local policies for prevention and control of COVID-19 infection.
MAINTENANCE OF EQUIPMENT
BEFORE EACH COLLECTION
- Clean forceps with 70%-ethanol.
- Freeze and clean ice-packs.
- Clean cool-boxes
- Fully charge all equipment (e.g., GPS unit, tablets/phone). Make sure the tablet has enough free-space for field sampling pictures.
AFTER EACH COLLECTION
- Clean all equipment thoroughly between sampling sites, including boots, cooler box (inside and outside), etc.
- Tick drags should be sealed into trash bags for transport and cleaned without detergents (only use tap water and then air-dry) before the next collection.
- Lint roller sheets should be disposed of in the waste bag after all ticks have been removed for testing.
- Store sterile equipment separate from used equipment and samples.
Caution on RNA handling
- RNases are very stable and difficult to inactivate, and only minute amounts are sufficient to destroy RNA.
- Care should be taken to avoid inadvertently introducing RNases into the samples during or after the purification procedure.
- Sample handling and extraction should be performed under an extraction hood and respecting Good Laboratory Practices.
- Use filter tips all the time.
Storage of the buffers from IndiMag Pathogen Kit
- Proteinase K is stable for at least 1 year after delivery when stored at Room temperature (15-25 °C ). To store for more than 1 year or if ambient temperature often exceeds 25 °C , storage at 2-8 °C is recommended. Do not add Proteinase K directly to the Buffer VXL mixture! This can cause clogs or precipitates.
- Precipitation may form after storage at low temperature or prolonged storage. To dissolve precipitate, incubate Buffer VXL or ACB for 00:30:00 at 37 °C , with occasional shaking.
- Reconstituted Buffer AW1 can be stored at Room temperature (15-25 °C ) for up to 1 year. Mix well after adding Ethanol.
- Buffer AVE is RNase-free upon delivery. It contains sodium azide, an antimicrobial agent that prevents growth of RNase-producing organisms. However, as this buffer does not contain any RNase degrading chemicals, it will not actively inhibit RNases introduced by inappropriate handling. When handling Buffer AVE, take extreme care to avoid contamination with RNases. Follow general precautions for working with RNA, such as frequent change of gloves and keeping tubes closed whenever possible.
QUALITY CONTROL
This SOP is reviewed by the applicable supervisor annually or as required in order to maintain its relevance
APPENDICES
APPENDIX 1 MEASURING SPOON FOR 0.1 mm BEATING BEADS
The spoon (Next Advance, MSP01-RNA) is used for 0.1 mm beating beads measurement. The step is described on 6.6.4 the preparation before tick homogenization. One spoon equals to 100 µL .
APPENDIX 4 EXPECTED OUTCOMES
Materials
EQUIPMENT AND MATERIALS
Note
NOTE: If product number is listed, please ensure use of this or equivalent product.
| A | B | |
| Equipment | Mfg / Product # | |
| Dino-Lite 5MP Edge | AM7115MZT | |
| Adjustable DinoScope Stand | MS35B | |
| Magnetic light source (x2) | IMAGE Grill Lights Magnetic BBQ Grill Light | |
| AAA batteries | EBL Pack of 8 AAA Batteries 1,100mAh AAA | |
| Wifi or ethernet internet connexion | (WPA-2 security only. WPA-e not yet supported) | |
| Laptop or desktop computer with Google Chrome | (Setup requires Ethernet or laptop with Bluetooth LE capabilities); PC preferred for DinoScope | |
| Dino-Lite Driver Software | REDI-NET Program | |
| BioQuip Portable Chill Table | BioQuip, 1429 | |
| Multiplex imaging system Tick e-ID device | Vectech | |
| AC power converted to US standard | 120 V and 60Hz AC | |
| ● KingFisher™ Flex Magnetic Particle Processor with 96 Deep-Well Head ● KingFisher™ Duo Prime Magnetic Particle Processor | ThermoFisher, 5400630 (Flex) or ThermoFisher, 5400110 (Duo Prime) | |
| Bullet Blender 24 Gold | Next Advance, BB24-AU | |
| Adjustable micropipettes | Locally sourced | |
| Multi-channel micropipettes | Locally sourced | |
| Vortex | Locally sourced | |
| Tube centrifuge | Locally sourced | |
| Plate centrifuge | Locally sourced | |
| Qubit 4 Fluorometer | ThermoFisher, Q33238 | |
| Thermo Heater Mixer | Locally sourced |
Equipment
Dino-Lite 5MP Edge AM7115MZT
NAME
Microscope
TYPE
Dino-Lite
BRAND
AM7115MZT
SKU
LINK
magnification range of this microscope is 20x to 220x
SPECIFICATIONS
Equipment
Adjustable Vertical Mount stand
NAME
vertical stand
TYPE
Dino-Lite
BRAND
MS35B
SKU
LINK
Equipment
Bullet Blender 24 Gold (1.5 mL snap and screw cap tubes, 4°C cooling)
NAME
Blender
TYPE
Next Advance
BRAND
BB24-AU
SKU
LINK
Equipment
Qubit™ 4 Fluorometer, with WiFi
NAME
Fluorometer
TYPE
Invitrogen
BRAND
Q33238
SKU
LINK
Equipment
KingFisher™ Duo Prime Purification System
NAME
Purification System
TYPE
Thermo Scientific™
BRAND
5400110
SKU
LINK
| A | B | C | |
| Material | Description | Mfg / Product # | |
| Posi-Click™ 5 mL Microcentrifuge Tubes | For storage of individual specimen | Thomas Scientific, 1149Y05 | |
| 96 well Eppendorf tube racks | To hold specimen on chill plate during imaging | 1164M62 | |
| Porcelain chill table stage or white paper note cards | To better visualize tick during imaging | Locally sourced | |
| Petri dishes, disposable | To hold ticks under DinoScope | Bioquip, 4787 | |
| Color lab tape | For labeling and sealing boxes of ticks | Thomas Scientific, 1184X64 | |
| ZymoBIOMICS Microbial Community Standard Material | For TNA extraction positive control | Zymo Research, D6300 | |
| ATCC (DENV-1) positive control | For TNA extraction positive control; BSL-2 | ATCC, VR-1856 or locally sourced | |
| Human gammaherpesvirus (EBV) positive control | For TNA extraction positive control | Naval Medical Research Center | |
| IndiMag Pathogen Kit | w/o plastics, 384 reactions | Indical Bioscience, SP947257 | |
| Buffer ATL | 200 mL, Tissue Lysis Buffer | Qiagen, 19076 | |
| Reagent DX | 1 mL, Antifoaming Reagent | Qiagen, 19088 | |
| Measuring Spoon 100 µL | RNase Free, pack of 10, reusable | Next Advance, MSP01-RNA | |
| Navy RINO RNA lysis kit | Bead lysis kits | Next Advance, NAVYR5-RNA | |
| Clear RINO brand microcentrifuge tubes | 1.5 mL, screw-cap | Next Advance, TUBE1R5-S | |
| Zirconium oxidase beads | 0.1 mm, 400 g | Fisher Scientific, 50-154-2950 | |
| KingFisher™ Deepwell 96 Plate | KingFisher | ThermoFisher, 95040450 | |
| KingFisher™ 96 tip comb for DW magnets | KingFisher Flex ONLY | ThermoFisher, 97002534 | |
| KingFisher™ Duo Prime 12-tip comb | KingFisher Duo Prime ONLY | ThermoFisher, 97003500 | |
| Elution Strip | KingFisher Duo Prime ONLY | ThermoFisher, 97003520 | |
| KingFisher™ Duo Cap for Elution Strip | KingFisher Duo Prime ONLY | ThermoFisher, 97003540 | |
| BRAND Self-adhesive Plate Sealing Film | Aluminum (consumable) | Fisher Scientific, 13-882-329 or equivalent | |
| MicroAmp™ Clear Adhesive Film | KingFisher | ThermoFisher, 4306311 | |
| Nonstick, RNase-Free Microfuge Tubes | 1.5 mL | ThermoFisher, AM12450 | |
| Nonstick, RNase-Free Microfuge Tubes | 2.0 mL | ThermoFisher, AM12475 | |
| Qubit assays tubes | For Qubit™ DNA/RNA measuring (consumable) | Thermo Fisher, Q32856 | |
| RNaseZap™ RNase Decontamination Solution | To remove RNase from the working area | ThermoFisher, AM9780 | |
| Qubit™ 1X dsDNA HS Assay Kit | (consumable) | ThermoFisher, Q33230 | |
| Qubit™ RNA HS Assay Kit | (consumable) | ThermoFisher, Q32852 | |
| Ethanol | 100% (molecular biology grade) | Locally sourced | |
| Isopropanol | 100% (molecular biology grade) | Locally sourced | |
| Nuclease-free Water | For negative control | Locally sourced | |
| Dry ice | To maintain cold chain during sample handling using Bullet Blender | Locally sourced | |
| Ice bucket and ice | To keep sample cold | Locally sourced | |
| Kimwipes | To dry material | Locally sourced | |
| Falcon tubes | 15 mL and 50 mL | Locally sourced | |
| Forceps | Fine point, straight tip, stainless, sterile; For sample handling | BioQuip, 4731 | |
| Sterile 1x PBS | To clean tick sample | Locally sourced | |
| Sterile petri dishes | To hold tick sample during cleaning | Locally sourced | |
| Data sheets | REDI-NET DCS T-2 | REDI-NET Data Portal |
Posi-Lock™ 5 mL Microcentrifuge TubesThomas ScientificCatalog #1149Y05
96 Place Reversible Racks with CoversThomas ScientificCatalog #1164M62
Color LAB-TAPE™ 0.94Thomas ScientificCatalog #1184X64
ZymoBIOMICS Microbial Community StandardZymo ResearchCatalog #D6300
IndiMag Pathogen Kit w/o plastics (384 reactions)INDICAL BIOSCIENCECatalog #SP947257
Buffer AL, Lysis bufferQiagenCatalog #19076
Reagent DXQiagenCatalog #19088
Measuring Spoon 100 uL RNase Free pack of 10Next AdvanceCatalog #MSP01-RNA
Sterile Microcentrifuge Tube 1.5 mL (RINO®) 500/caseNext AdvanceCatalog #TUBE1R5-S
Bertin Corp 0.1mm Zirconium oxide beads (450g) (qty 500)Fisher ScientificCatalog #50-154-2950
KingFisher™ Plastics for 96 deep-well formatThermo Fisher ScientificCatalog #95040450
KingFisher™ Flex™ Systems Consumables, KingFisher 96 tip comb for DW magnetsThermo FisherCatalog #97002534
KingFisher™ Duo and KingFisher™ Duo Prime Consumables, 12-tip comb, for Microtiter 96 Deepwell plateThermo FisherCatalog #97003500
KingFisher™ Duo and KingFisher™ Duo Prime Consumables, Elution stripThermo FisherCatalog #97003520
KingFisher™ Duo and KingFisher™ Duo Prime Consumables, KingFisher Duo Cap for elution stripThermo FisherCatalog #97003540
BRAND™ Self-adhesive Plate Sealing FilmFisher ScientificCatalog #13-882-329
MicroAmp Clear Adhesive FilmApplied Biosystems (ThermoFisher Scientific)Catalog #4306311
Nonstick, RNase-free Microfuge Tubes, 1.5 mLThermo FisherCatalog #AM12450
Navy RINO RNA Lysis Kit 250 pack (1.5 mL)Next AdvanceCatalog #NAVYR5-RNA
Nonstick, RNase-free Microfuge Tubes, 2.0 mLThermo FisherCatalog #AM12475
Qubit assay tubesThermo Fisher ScientificCatalog #Q32856
RNaseZap™ RNase Decontamination SolutionThermo Fisher ScientificCatalog #AM9780
Qubit 1X dsDNA High Sensitivity Assay KitThermo Fisher ScientificCatalog #Q33230
Qubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852
APPENDIX 12: Tray Cleaning Materials
- Vectech IDX tray
- Lens cleaner, glass cleaner or ethanol
- Kimwipe or microfiber cloth
- Phillips head screwdriver
APPENDIX 14. SET-UP INSTRUCTIONS FOR BARCODE PRINTING
Safety warnings
RISKS AND PERSONAL PROTECTION
- Caution should be taken while processing samples as some chemicals may be harmful. Please use a fume-hood when required to avoid inhaling harmful chemicals.
- Gloves should be worn all the time when handling samples.
- Decontaminants such as DNA/RNaZap could irritate the skin, please, try to avoid contact with skin while preparing workbench for nucleic acid extraction.
Before start
BEFORE START
- Pre-cool the Bullet Blender by adding dry ice into the cooling compartment and running the cooling program.
- Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% /molecular biology grade ethanol to remove additional contaminants.
- Transfer 0.1 mm zirconium oxide beads (2 spoons, Appendix 1) to Thermo Scientific Screw Cap Micro 1.5 ml Tubes.
- For the first time use of IndiMag pathogen kit, add 100% ethanol to Buffer AW1 and AW2, and add 100% isopropanol to ACB as indicated on the bottles.
- MagAttract Suspension G from IndiMag pathogen kit needs to be vortexed thoroughly for 00:03:00 (before first use) or 00:01:00 (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended.
- Aliquot nuclease-free water in big bottle into a few 15 mL tubes for preparing TNA elution in KingFisher Flex or KingFisher Duo Prime to avoid cross-contamination.
1. SORT FLYING VECTOR SAMPLES
Note
NOTE: 1) The sections 6.1 to 6.5 in this SOP are for imaging adult female mosquitoes. The sections 6.6 to 6.16 in this SOP are for total nucleic extraction from samples previously imaged and identified.
Sort flying vectors by species, collection date, and collection location. You may also consider sorting specimens by trap type (if using multiple at the same location).
Note
NOTE: Only female flying vectors should be processed for testing.
Following sampling, flying vectors can be stored at -20 °C for up to 2 months and at -80 °C for long term storage.
2. PREPARING MOSQUITOES e-ID (IDX): Device setup using Bluetooth
Note
NOTE: If the Tick e-ID is not recognizing the device, please, logout from google and your institution, and reboot the computer. Then login to the Tick e-ID platform using google account. Current device is not supporting larval ticks, which will only be imaged using DinoScope in section 5 and 6.
Plug in the device. On a phone or computer which has both Google Chrome and Bluetooth capabilities, visit https://mos-id.vectech.io (if on mobile, use the desktop site option) and log in. For first time use, click the “Sign Up” tab, enter in a username (email), and a password. We will approve your account, and then you can continue.
If set-up with ethernet is desired, skip to Section 3.
Click the drop-down arrow at the top-center of the page (see figure). Click “Add New Device,” click on the bluetooth icon button. When a device with a name “Agamotto” appears, select the device and click “Pair”. When given the opportunity, select and enter your Wi-Fi credentials [user name, passcode] (the website may throw an error,but ignore this error). Return to the “Data” tab.
Click the drop-down arrow at the top-center of the page, and you should see a device with status [booting] or [Idle]. If [booting], wait for status to change to [idle] and stay. Then select “Device.” Setup is complete. If your device soon changes to [Idle] under the device drop down menu, continue to 4. Imaging Protocol.
3. PREPARING MOSQUITOES e-ID (IDX): Device setup using Ethernet
If setting up with ethernet instead of Bluetooth, follow these steps. Select the user menu at the bottom right corner of the screen. Select “Connect Device.”
Select “Device.”If the device name is not known, contact Vectech for support. Then select your Wi-Fi name from the “Wi-Fi SSD” drop down menu. If you do not see your Wi-Fi in the drop down, verify the network is functioning on other devices. Then enter your Wi-Fi password. Select connect.
If a successful Wi-Fi connection is established, disconnect the device from power and ethernet, then reconnect power. If your device soon changes to [Idle] under the device drop down menu, continue to Section 4. Imaging Using Mosquitoes e-Id.
If you have trouble connecting to the device via Wi-Fi, try to connect an Ethernet cable directly from your device to your modem. You should soon see your device status change to [Idle] under the device drop down menu.
4. IMAGING USING MOSQUITOES E-ID
A single representative individual mosquito from a pool will suffice for documentation.
On https://mos-id.vectech.io, navigate to the “Trays” tab. Verify that the device is plugged in. The device should flash lights when booting up.
Organize the specimens to be multi-imaged in sample tubes laid out in tubes in a 96-well sample rack (8x12). MAINTAIN THE COLD CHAIN. Keep samples in dry ice or in polystyrene boxes filled with cool beans until ready to use. Place a glass tray on the chill table as you fill the rows before imaging and immediately return to storage tubes once imaged. Sequentially lay out the sequential sample across the row, with each row of 12 equaling one tray of multiplex photos.
Note
Tip: If you run samples in sequential order from top left to bottom right through the tray (A1- A4; A5- 8, A9-12), the system will auto-fill your sample numbers in the associated data fields.
Figure 1: Placement of samples in the tray for identification.
Mosquitoes will need to be collected, stored, and moved dry. Any liquid contact will degrade important identifying features.
Repeat step 12, until all 12 specimens are placed in the tray, dorsal side facing up.
Close the tray by placing the lid on the well-side of the tray until you hear the gentle click as the magnets connect to keep the tray closed.
Figure 2: Insertion of the tray.
Insert tray into device tray slot. Tray will “click” into place when fully inserted.
To capture the dorsal view, go to the “Trays” tab in the website, click, “Capture New Tray.”
Figure 3: Screenshot of the “Capture New Tray” window.
MmosquitoesImages auto upload.
Note
NOTE:
- Writing the tray number and the well-ID on the side of the tube after the mosquitoes e-ID imaging process could be helpful for double-checking and labeling Dinoscope images when done several days apart and for TNA extraction information.
Figure 4: https://www.vectech.io/idx-mosquito-control.
Enter the following information:
Genus and species (if known).
Note
This field will autofill by clicking the arrow to view the next specimen. Autofill for this field is: previous specimen=current specimen. Once a field has data saved, autofill is no longer active.
Plate and well number.
Note
This field will autofill by clicking the arrow to view the next specimen. Autofill for this field is: for well, A1-H12, consecutive; for plate, previous specimen=current specimen. Once a field has data saved, autofill is no longer active.
Remove tray, flip upside down (i.e. dorsal to ventral), and insert tray again to capture the ventral image of the mosquito.
Click “Capture Other Side”.
Click “Finish,” and return specimens to original storage. Make a note of the tray number that corresponds to your sample row to facilitate data updates at a later date.
5. PREPARING DINOSCOPE
Place a portable chill table on the adjustable stand.
Place white porcelain stage directly on top of the chill table surface. If a porcelain stage is not available, cut a 5 cm x 5 cm square from a paper note card and use color lab tape to attach to the bottom of a petri dish to make a stage.
Once in place, secure the stage by taping either porcelain or petri dish stands to sides of the chill table.
Adjust the DinoScope holster on the stand so it is 4.5 cm above the surface of the stage.
Note
Note: make sure to secure the rear set screw just below the holster to secure placement on the vertical post. (See Appendix 4 for more information).
Turn on the chill table (skip if not maintaining a cold chain) and wait until the surface temperature monitor reads -20 °C .
Ensure all specimens slated for imaging are arranged in individual Eppendorf tubes and stored in 96-well racks labeled with a plate number.
Use a Kimwipe and 75% ethanol to wipe clean the stage surface. If using a petri dish stage, replace paper cards.
6. IMAGING USING DINOSCOPE
Note
Note: Flying vectors should be pooled to maximize sample processing efficiency (see pooling strategy QRG). A single, representative specimen from a pool of like specimens should be imaged (e.g., one female Aedes aegypti in a pool of twenty).
Remove a specimen from tube with forceps and place in the center of the stage with the dorsal side facing up.
Using the DinoScope interface and stand fine adjustment, bring the specimen into focus. Specimens should be centered in the field of view. Make sure all important characters are visible.
Note
NOTE: Additional adjustments may need to be made to the camera settings, possibly camera holster placement depending on size of specimen. (See Appendix 1) for more information on DinoScope camera settings profile).
Annotate with the file naming convention: Sample ID-View (D = Dorsal, V = Ventral) Example: RUSFV-00001D.
Ensure the correct magnification is entered into the Magnification field and that all metadata is included in the image output (magnification, magnification profile, scale, username and annotation) but does not overlap with specimen in the image.
Note
NOTE: Please do not use abbreviations in the annotations.
Just before taking an image readjust the white balance for each individual image to standardize the background color for all images.
Take a single image and review the photo to ensure quality (focus, frame, taxonomic characters). If the image is not satisfactory delete and repeat steps 31 to 35.
Use forceps to flip the specimen so that the ventral side is facing up.
Repeat steps 31 to 35 for ventral view.
Return specimen to tube and repeat steps 30 to 39 until a dorsal and ventral image are captured for all specimens.
Review both photos for quality and record mosquito characteristics.
When the imaging session is completed, copy photos from the dated folder and save.
7. BEFORE FLYING VECTOR HOMOGENIZATION
Note
NOTE: To prevent contamination samples nucleic acid extraction and amplification (PCR) should be performed in separate rooms.
Pre-cool the Bullet Blender by adding dry ice into the cooling compartment and running the cooling program.
Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% molecular biology grade ethanol to remove additional contaminants.
Transfer 0.1 mm zirconium oxide beads (two spoons, Appendix 5) to Clear RINO brand 1.5 mL screw-cap microcentrifuge tubes.
For the 1st time use of the IndiMag pathogen kit, add 100% ethanol to Buffer AW1 and AW2, and add 100% Isopropanol to ACB as indicated on the bottles.
Buffer ATL may form precipitates upon storage. If necessary, warm to 56 °C until the precipitates have fully dissolved. Prepare buffer ATL-DX: add 100 µL Reagent DX to 15 mL Buffer ATL. If smaller amounts are needed, transfer 1.5 mL of Buffer ATL into a sterile 2 ml vial and add 10 µL Reagent DX. Mix well, after addition of Reagent DX. After preparation, the mixture is stable for 6 months at Room temperature (15 °C -25 °C ).
MagAttract Suspension G from the IndiMag pathogen kit needs to be vortexed thoroughly for 00:03:00 (before first use) or 00:01:00 (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended.
4m
Prepare a few 15 mL or 50 mL conical centrifuge tubes with nuclease-free water for preparing TNA elution in KingFisher Flex or KingFisher Duo Prime to avoid cross-contamination.
8. FLYING VECTOR HOMOGENIZATION
Clean forceps with 70% ethanol and Kimwipes before use and between samples.
Label navy RINO RNA lysis tubes on the cap.
Note
Avoid labeling on the side of tubes due to potential damage during beads beating process.
Transfer ≤20 individuals into the labeled RINO tube and place tubes on ice.
Note
NOTE: Pooling strategies will vary between labs. Consult a Gold Lab for feedback when designing a testing strategy.
Add 400 µL cold sterile 1xPBS into each navy RINO tube with flying vectors.
Ensure the Bullet Blender is fully cooled down. Add more dry ice into the cooling compartment of Bullet Blender, if necessary, then load the RINO tubes with the samples.
Set the controls for Speed 10 and Time 3. Press Start.
Centrifuge the suspension at 100 x g, 4°C, 00:01:00 to pellet debris.
1m
Without disturbing the tubes, carefully transfer the top 320 µL supernatant into 1.5 mL tubes.
9. MICROBE LYSIS
Note
NOTE: Check section 7 for the buffer preparation and storage details, before setting up microbe lysis and KingFisher instrument.
Add 80 µL of ATL-DX Lysis Buffer to the 1.5 mL tubes containing 0.1 mm beads from step 44 and label the sample or pool ID on the tubes. Also, prepare two tubes for positive and negative controls.
Transfer 320 µL homogenate (individual or pooled) to each of the pre-labeled bead tubes.
Include a positive control for each batch of samples: transfer 37.5 µL ZymoBIOMICS Microbial Community Standard Material into a tube from step 57. Add 282.5 µL 1x PBS to make the final control mix volume 320 μL.
Include a negative control for each batch of samples: a bead tube with 320 µL cold sterile 1xPBS only.
Add more dry ice into the cooling compartment of Bullet Blender, if necessary and then load the all bead tubes (samples and controls).
Set the speed at 12 and time at 3. Press Start.
Let the samples settle for 00:01:00 and then repeat step 62.
1m
Centrifuge the tube at 120 x g, 4°C, 00:05:00 .
5m
Carefully transfer the 350 µL supernatant from the RINO tube to a new snap-cap 1.5 mL microcentrifuge tube, avoiding bead carryover (slight bead contamination is tolerated).
Note
STOPPING POINT: lysed samples can be stored at 4 °C Overnight .
10. INSTRUMENT SET UP
Note
NOTE: KingFisher Flex only, if using KingFisher Duo Prime, go to section 11
Confirm 96 deep-well magnetic heads and 96 well deep-well heat blocks are being used.
Ensure the program IndiMag_Pathogen_KF_Flex_4wash has been downloaded and loaded onto the KingFisher Flex instrument.
10.1 SET UP THE PROCESSING PLATES
Set up the Wash, Elution, and Tip Comb Plates outside the instrument according to the following table:
Note
NOTE: DO NOT use the elution buffer provided by the kit for TNA elution. The ingredients in the elution buffer inhibit the downstream DNA sequencing efficiency.
| A | B | C | D | E | |
| Plate ID | Plate position | Plate type | Reagent | Volume per well | |
| Tip comb | 7 | Place a 96 Deep-well Tip comb in a deep-well plate | |||
| Elution | 6 | Deep-Well | Nuclease-free water | 75 µL | |
| Wash 4 | 5 | Deep-Well | 100% ethanol | 750 µL | |
| Wash 3 | 4 | Deep-Well | 80% ethanol | 750 µL | |
| Wash 2 | 3 | Deep-Well | Buffer AW2 | 700 µL | |
| Wash 1 | 2 | Deep-Well | Buffer AW1 | 700 µL | |
| Sample | 1 | Sample Lysate | Lysate and lysis buffer | 985 µL | |
10.2 EXTRACTION
Add 20 µL of Proteinase K into wells (based on number of samples) of a new Deep-well plate.
Transfer 270 µL supernatant of step 65 without any particle carryover to the wells of the Deep-well plate containing proteinase K. This plate becomes the Sample Plate.
Add 135 µL Buffer VXL, 540 µL Buffer ACB, and 20 µL MagAttract Suspension G to each sample in the sample plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix, avoid foaming (can be mixed on Hula mixer for 2 min). Add 695 µL mixture to each sample.
Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.
Start the run, then load the prepared plates into position when prompted by the instrument.
10.3. QUANTIFICATION AND STORAGE
After the running protocol is completed (~35 minutes), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage.
In a 0.6 mL microcentrifuge tube, use 1 µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions.
Note
Kits needed: Qubit 1X dsDNA HS Assay Kit and Qubit RNA HS Assay Kit. (see Appendix 6)
Proceed with sample testing following the REDI-NET SOP FV-4 Flying vector Testing or store at -20 °C for less than 2 weeks.
Note
For long-term storage the sample needs to be stored at -80 °C following the REDI-NET SOP FV-3 Flying Vector Storage.
11. INSTRUMENT SET UP
Note
NOTE: KingFisher Duo Prime only, if using KingFisher Flex, go to section 10.
Confirm 12-tip magnetic head and 12 well deep-well heat blocks are being used.
Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.
11.1 SET UP THE SAMPLE PLATE AND ELUTION STRIP
Set up the Sample Plate according to the table below:
| A | B | C | D | |
| Row ID | Plate Row | Reagent | Volume per well | |
| Sample row | A | Lysate and lysis buffer | 985 µL | |
| Wash 1 | B | Buffer AW1 | 700 µL | |
| Wash 2 | C | Buffer AW2 | 700 µL | |
| Wash 3 | D | 80 % ethanol | 750 µL | |
| Wash 4 | E | 100 % ethanol | 750 µL | |
| Tip Comb | F | Tip comb | 700 µL | |
| G | Empty | |||
| H | ||||
Set up the Elution Strip according to the table below:
Note
Note: DO NOT use the elution buffer provided by the kit for TNA elution. The ingredients in the elution buffer inhibit the downstream DNA sequencing efficiency.
| A | B | C | D | |
| Row ID | Plate Row | Reagent | Volume per well | |
| Elution | A | Nuclease-free water | 75 µL |
11.2. EXTRACTION
Add 20 µL of Proteinase K into wells (based on number of samples) of a sample row.
Transfer 270 µL supernatant from step 69 without any particle carryover to the wells of the sample row containing proteinase K.
Add 135 µL Buffer VXL, 540 µL Buffer ACB, and 20 µL MagAttract Suspension G to each sample in the sample row. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix, avoid foaming (can be mixed on Hula mixer for 2 min). Add 695 µL mixture to each sample.
Select program IndiMag_Pathogen_KF_Duo_4wash on the instrument.
Start the run, then load the prepared plate/strip into position when prompted by the instrument.
Note
Keep the door open while extraction is in process. The chamber of the KingFisher Duo Prime is small. Closing the door makes the ethanol vapor restrained inside the chamber and increases the ethanol contamination.
11.3 QUANTIFICATION AND STORAGE
After the protocol is completed (~35 minutes), immediately remove the elution strip from the instrument and transfer the eluate to the final tube or plate of choice for final storage.
Use 1 µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer.
Note
Kits needed: Qubit 1X dsDNA HS Assay Kit and Qubit RNA HS Assay Kit. (see Appendix 6).
Proceed with sample testing following the REDI-NET SOP FV-4 Flying Vector Testing or store at -20 °C for less than 2 weeks.
Note
For long-term storage the sample needs to be stored at -80 °C following the REDI-NET SOP FV-3 Flying Vector Storage.
APPENDIX 1. DINOLITE DIGITAL MICROSCOPE CAMERA SETTINGS
CAMERA SETTINGS
AUTO EXPOSURE SETTINGS
LED BRIGHTNESS
CALIBRATION
Note
NOTE: Use the automatic calibration target included with the DinoScope to calibrate the camera each time the camera position is changed. For more information on how to calibrate the DinoScope, view this video: https://www.youtube.com/watch?v=mivZvOLtEBA
APPENDIX 2. SAMPLE IMAGE
APPENDIX 3. DINO-LITE DIGITAL MICROSCOPE
APPENDIX 4. MS35BE ELECTROSTATIC DISCHARGE PROTECTION MODEL
APPENDIX 5. MEASURING SPOON FOR 0.1 mm BEATING BEADS
The spoon (Next Advance, MSP01-RNA) is used for 0.1 mm beating beads measurement. The step is described on 6.6.4 the preparation before homogenization. One spoon equals to 100 uL.
APPENDIX 6. DNA and RNA Measurement Using QUBIT FLUOROMETER 4.0
DNA quantification:
According to the volume of sample used, add the 1xHS dsDNA Qubit Assay for a final volume of 200 µL (i.e., if using 1 µL of sample, add 199 µL of 1x HS dsDNA Qubit Assay.
RNA Quantification:
In a new microcentrifuge tube/falcon tube (depending on the number of samples processed), prepare a working solution of the Qubit HS RNA Assay:
| A | B | C | |
| Reagents | Volume/sample | Volume for n+1 sample | |
| Qubit RNA HS Assay buffer | 199 µL | …. µL | |
| Qubit RNA HS Assay Dye | 1 µL | …. µL |
In a new 0.6 mL tube, mix 199 µL of Qubit HS RNA Assay working solution and 1 µL of the sample. Incubate for 1 minute at room temperature before reading.
APPENDIX 7. IDX TRAY STANDARD OPERATING PROCEDURES
Naming Conventions
Guidelines
Use gloves while handling the tray.
The tray should be cleaned prior to every imaging session according to the steps provided in this SOP.
Specimens should be as free of ethanol as possible before being placed in the tray.
The tray’s cleanliness should be checked every 10 captures. If debris or smudges are observed, clean with lens cleaner and wipes, removing the tray walls to clean as necessary.
Tray Cleaning: Materials
Vectech IDX tray
Lens cleaner, glass cleaner or ethanol
Kimwipe or microfiber cloth
Phillips head screwdriver
Tray Cleaning: Steps
Remove the screws that secure the tray walls to the base of the tray and set the tray walls aside.
Apply a lens cleaner to the glass and wipe the area with a kimwipe or microfiber cloth. Rub the area lightly until the area is dry and free of debris. Repeat for both the top and bottom of the tray base and cover.
Hold up the tray to a light and look through the glass to check for remaining smudges or debris. Clean remaining areas as necessary.
Replace the tray walls onto the base of the tray (the half with the gold inserts). The indentations in the tray wall piece should be facing the gold inserts in the tray base. Replace the screws.
Protocol references
REFERENCES
1. Andrade Justi, S., Soghigian, J., Pecor, D. B., Caicedo-Quiroga, L., Rutvisuttinunt, W., Li, T., ... & Linton, Y. M. (2021). From e-voucher to genomic data: Preservingarchive specimens as demonstrated with medically important mosquitoes (Diptera:Culicidae) and kissing bugs (Hemiptera: Reduviidae). Plos one, 16(2), e0247068.
2. Hall RJ, Wang J, Todd AK, Bissielo AB, Yen S, Strydom H, Moore NE, Ren X, Huang QS, Carter PE, Peacey M. Evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery. J Virol Methods. 2014 Jan;195:194-204. doi: 10.1016/j.jviromet.2013.08.035. Epub 2013 Sep 13. PMID: 24036074; PMCID: PMC7113663.
3. Temmam S, Monteil-Bouchard S, Robert C, Pascalis H, Michelle C, Jardot P, Charrel R, Raoult D, Desnues C. Host-Associated Metagenomics: A Guide to Generating Infectious RNA Viromes. PLoS One. 2015 Oct 2;10(10):e0139810. doi: 10.1371/journal.pone.0139810. PMID: 26431175; PMCID: PMC4592258.
4. User Guide: MagMAX Microbiome Ultra Nucleic Acid Isolation kit, Applied Biosystems, Pub. No. MAN0018070 Rev. C.0.
5. User Guide: Indical IndiMag Pathogen Kit user’s manual.