Dec 16, 2024
REDI-NET FF-2 FILTH FLY PROCESSING
- 1REDI-NET Consortium
- Remote Emerging Disease Intelligence - NETwork (REDI-NET)
Protocol Citation: REDI-NET Consortium 2024. REDI-NET FF-2 FILTH FLY PROCESSING. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8e6nv5b/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 12, 2024
Last Modified: December 16, 2024
Protocol Integer ID: 107535
Keywords: Filth fly, Musca sp, Musca domestica, Haematobia irritans, Sarcophagidae, Horn fly, House fly, Stable fly, Face fly, extraction, TNA, homogenization, fly wash, KingFisher
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
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 filth fly total nucleic acid extraction to allow downstream library preparation and sequencing for pathogen detection.
Guidelines
APPENDIX 1. BASIC FLY IDENTIFICATION
APPENDIX 3. POOLING STRATEGY
APPENDIX 4. 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 Fly homogenization. One spoon equals to 100
APPENDIX 5. QUBIT
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.
MAINTENANCE OF EQUIPMENT
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 °C —25 °C ). To store for more than 1 year or if ambient temperature often exceeds 25 °C , storage at 2 °C —8 °C is recommended. Do not add Proteinase K directly to the Buffer VXL mixture! This can cause clogs or precipitates.
- Precipitates 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 °C —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.
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 | |
| Dino-Lite Driver Software | REDI-NET Program | |
| BioQuip Portable Chill Table | BioQuip, 1429 | |
| AC power converted to US standard | 120 V and 60Hz AC | |
| KingFisher Flex Magnetic Particle Processor with 96 Deep-Well Head or 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 |
| 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 fly during imaging | Locally sourced | |
| Petri dishes, disposable | To hold flies under DinoScope (consumable) | Bioquip, 4787 | |
| Color lab tape | For labeling and sealing boxes of flies (consumable) | Thomas Scientific, 1184X64 | |
| ZymoBIOMICS Microbial Community Standard Material | For TNA extraction positive control | Zymo Research, D6300 | |
| AcroMetrix HIV-1 Controls | For TNA extraction positive control; BSL-2 | ThermoFisher, CLS430320-12EA | |
| 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 | |
| Orange RINO RNA lysis kit | Bead lysis kits (consumable) | Next Advance, ORANGER5-RNA | |
| Clear RINO brand microcentrifuge tubes | 1.5 mL, screw-cap (consumable) | Next Advance, TUBE1R5-S | |
| Thermo Scientific Screw Cap Micro Tubes | 1.5 mL Screw Cap Tube, NonKnurl, NonSkirted, Natural, E-Beam Sterile tube w/ attached cap | Fisher Scientific, 14-755-208 | |
| Zirconium oxide beads | 0.1 mm, 400 g (consumable) | 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 (consumable) | ThermoFisher, 4306311 | |
| Nonstick, RNase-Free Microfuge Tubes | 1.5 mL (consumable) | ThermoFisher, AM12450 | |
| Nonstick, RNase-Free Microfuge Tubes | 2.0 mL (consumable) | 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) (consumable) | Locally sourced | |
| Isopropanol | 100% (molecular biology grade) (consumable) | Locally sourced | |
| Nuclease-free Water | For negative control | Locally sourced | |
| Dry ice | To maintain cold chain during sample handling using Bullet Blender | Locally sourced |
Safety warnings
RISK 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, avoid contact with skin while preparing the workbench for nucleic acid extractions.
SORT FLY SAMPLES
SORT FLY SAMPLES
Flies can be sorted one of three ways:
Flies are separated into groups of biting flies and non-biting flies based on their mouthparts.
Flies can be identified and sorted by genera (e.g., Musca sp.).
Flies can be identified to species level using appropriate morphological keys. Morphological keys present in this SOP (Appendix 1) should aid identification.
Following sampling, flies can be stored at -20 °C for up to 2 months and at -80 °C for long term storage.
Flies can be pooled for processing by mouthpart type, genus, or species. Do not pool flies from different cartons. The maximum recommended pool sizes are noted in the table below.
| A | B | C | D | E | |
| Size | Example | SS Beads | ZrO2 Beads | Fly Wash | |
| Small Flies | Horn Fly (Haematobia irritans) | 15 | 10 | 20 | |
| Medium Flies | Housefly (Musca domestica) | 5 | 3 | 10 | |
| Large Flies | Flesh Fly (Sarcophagidae) | 3 | 2 | 5 |
STEPS BEFORE HOMOGENIZATION
STEPS BEFORE 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 4) to Thermo Scientific Screw Cap 1.5 mL Micro 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 (15-25 °C ).
MagAttract Suspension G from the IndiMag pathogen kit needs to be vortexed thoroughly for 3 mins (before first use) or 1 minute (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended.
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.
Select a processing strategy for the samples using the guidance in the figure below:
FLY HOMOGENIZATION - STAINLESS STEEL BEADS
FLY HOMOGENIZATION - STAINLESS STEEL BEADS
Clean forceps with 70% ethanol and Kimwipes before use and between samples
Label orange RINO® tubes on the caps to avoid the label rubbing off during homogenization.
Add the flies to the tubes and note the number of flies being pooled in each tube.
Add 400 µL ice cold 1x PBS to each tube.
Ensure the Bullet Blender is fully cooled down. Add more dry ice into the cooling compartment of the Bullet Blender, if necessary, then load the RINO® tubes with flies and PBS.
Set the controls for Speed 10 and Time 3. Press start.
Inspect the tubes and repeat Step 18 if the flies are not fully homogenized.
Remove the tubes and centrifuge the samples at 100 x g, 4°C, 00:01:00 to pellet debris.
1m
Meanwhile, add 80 µL of ATL-DX Lysis Buffer to 1.5 mL tubes containing 0.1 mm ZrO2 beads from Step 7.
Without disturbing the orange RINO® tubes, carefully transfer the top (clear) 320 µL homogenate into 1.5 mL tubes from the preceding step.
Include a positive control for each batch of samples: transfer 37.5 µL ZymoBIOMICS Microbial Community Standard Material and 100 µL EBV, and 100 µL HIV standard into a tube from step 7. Add 82.5 µL 1x PBS.
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, then load the bead tubes.
Set the speed at 12 and time at 3. Press Start.
Let the samples settle for 1 minute and then repeat step 26.
Centrifuge the tube at 100 x g, 4°C, 00:01:00 .
1m
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.
FLY HOMOGENIZATION - ZIRCONIUM OXIDE BEADS
FLY HOMOGENIZATION - ZIRCONIUM OXIDE BEADS
Clean forceps with 70% ethanol and Kimwipes before use and between samples
Label clear RINO® tubes on the caps to avoid the label rubbing off during homogenization.
Add the flies to the tubes and note the number of flies being pooled in each tube.
Add 320 µL 1x PBS and 80 µL of ATL-DX to each tube containing flies. A master mix can be made ahead for this step.
Include a positive control for each batch of samples: transfer 37.5 µL ZymoBIOMICS Microbial Community Standard Material and 100 µL EBV, and 100 µL HIV standard into a tube from step 7. Add 82.5 µL 1x PBS.
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.
Set the Bullet Blender to Speed 12 and Time 3. Press Start.
Let the samples settle for 1 minute and then repeat step 37.
Centrifuge the tube at 100 x g, 4°C, 00:01:00 .
1m
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.
FLY WASH HOMOGENIZATION
FLY WASH HOMOGENIZATION
Clean forceps with 70% ethanol and Kimwipes before use and between samples.
Label 1.5 mL or 2.0 microcentrifuge tubes.
Add the flies to the tubes and note the number of flies being pooled in each tube.
Add 320 µL 1x PBS and 80 µL ATL-DX to each tube containing flies. A master mix can be made ahead for this step.
Close the lids and vortex the samples on high for at least 30 seconds or up to 1 minute.
Note
NOTE: This step will continue to be optimized as more fly types from various environments and pool sizes can be tested. An alternate approach may include a longer mixing step on the Hula mixer.
Briefly centrifuge the tubes to collect liquid and flies at the bottom of the tube.
Remove the liquid portion (< 400 µL ) from these tubes and transfer to the prepared clear RINO® tubes containing ZrO2 beads from step 7.
Include a positive control for each batch of samples: transfer 37.5 µL ZymoBIOMICS Microbial Community Standard Material and 100 µL EBV, and 100 µL HIV standard into a tube from step 7. Add 82.5 µL 1x PBS.
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.
Set the Bullet Blender to Speed 12 and Time 3. Press Start.
Let the samples settle for 1 minute and then repeat step 51.
Centrifuge the tube at 100 x g, 4°C, 00:01:00 .
1m
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.
INSTRUMENT SET UP (KingFisher Flex only, if using KingFisher Duo Prime, go to step 67)
INSTRUMENT SET UP (KingFisher Flex only, if using KingFisher Duo Prime, go to step 67)
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.
SET UP THE PROCESSING PLATES
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 | |
EXTRACTION
EXTRACTION
Centrifuge the 1.5 mL tubes with lysate from step 54 for 12000 x g, 4°C, 00:05:00 .
5m
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 58 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.
QUANTIFICATION AND STORAGE
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 (Kits needed: Qubit 1X dsDNA HS Assay Kit and Qubit RNA HS Assay Kit). (see Appendix 5)
Proceed with sample testing following the REDI-NET SOP FF-4 Filth Fly Testing or store at -20 °C for less than 2 weeks (for long-term storage the sample needs to be stored at -80 °C following the REDI-NET SOP FF-3 Filth Fly Storage).
INSTRUMENT SET UP (KingFisher Duo Prime only, if using KingFisher Flex, go to step 55)
INSTRUMENT SET UP (KingFisher Duo Prime only, if using KingFisher Flex, go to step 55)
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.
SET UP THE SAMPLE PLATE AND ELUTION STRIP
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 |
EXTRACTION
EXTRACTION
Centrifuge the bead tubes with lysate from step 54 for12000 x g, 4°C, 00:05:00
5m
Add20 µL of Proteinase K into wells (based on number of samples) of a sample row.
Transfer 270 µL supernatant from step 71 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.
Keep the door open while extraction. 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.
QUANTIFICATION AND STORAGE
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 (Kits needed: Qubit 1X dsDNA HS Assay Kit and Qubit RNA HS Assay Kit). (see Appendix 5)
Proceed with sample testing following the REDI-NET SOP FF-4 Filth Fly Testing or store at -20 °C for less than 2 weeks (for long-term storage the sample needs to be stored at -80 °C following the REDI-NET SOP FF-4 Filth Fly Storage).
Protocol references
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- User Guide: MagMAX Microbiome Ultra Nucleic Acid Isolation kit, Applied Biosystems, Pub. No. MAN0018070 Rev. C.0
- User Guide: Indical IndiMag Pathogen Kit user’s manual
- H.R. Dodge, DIptera: Pictorial Key to Principal Families of Public Health Importance.
- Couri, Marcia S., “Key to the Afrotropical genera of Muscidae (Diptera), 2007
- Borror and DeLong’s Introduction to the Study of Insects, 2004
- Axtel, Richard C., Fly Control In Confined Livestock and Poultry Production