Feb 02, 2024
F-2 FECES PROCESSING
- REDI-NET Consortium1
- 1REDI-NET Consortium
Protocol Citation: REDI-NET Consortium 2024. F-2 FECES PROCESSING. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx9ddog8j/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: April 28, 2023
Last Modified: March 15, 2024
Protocol Integer ID: 81132
Keywords: SAMPLE LYSIS, EXTRACTION, QUANTIFICATION AND STORAGE
Funders Acknowledgement:
USAMRAA
Grant ID: W81XWH-21-C-0001
USAMRAA
Grant ID: W81XWH-22-C-0093
USAMRAA
Grant ID: HT9425-23-C-0059
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 and HT9425-23-C-0059. 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 feces processing.
Guidelines
OBJECTIVE
To outline procedures for total nucleic acid extraction from fecal samples.
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 procedures for total nucleic acid extraction from fecal samples to provide materials for downstream library preparation and sequencing for pathogen detection.
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
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.
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. One scoop equals to 100 µL .
| Sample | Amount | Sample condition | Elution volume | DNA conc. (ng/ul) | RNA conc.(ng/ul) | |
| Fecal | 100 mg | Frozen/Fresh | 75 | 5-50 | 5 - 100 |
Materials
EQUIPMENT AND MATERIALS
Note
NOTE: If product number is listed, please ensure use of this or equivalent product.
| A | B | |
| Equipment | Mfg / Product # | |
| KingFisher™ Flex Magnetic Particle Processor with 96 Deep-Well Head or KingFisher™ Duo Prime Magnetic Particle Processor | ThermoFisher, 5400630 or ThermoFisher, 5400110 | |
| Bullet Blender 24 Gold | Next Advance, BB24-AU | |
| Qubit 4 Fluorometer | ThermoFisher, Q33238 | |
| Adjustable micropipettes | Locally sourced | |
| Multi-channel micropipettes | Locally sourced | |
| Vortex | Locally sourced | |
| Tube centrifuge | Locally sourced | |
| Plate centrifuge | Locally sourced | |
| Digital scale/balance | Locally sourced | |
| Thermo Heater Mixer | Locally sourced |
| A | B | C | |
| Material | Description | Mfg / Product # | |
| IndiMag Pathogen Kit | w/o plastics, 384 reactions | Indical Bioscience, SP947257 | |
| Buffer ASL | 140 mL, Stool Lysis Buffer | Qiagen, 19082 | |
| Measuring Spoon 100 µL | RNase Free, pack of 10. reusable | Next Advance, MSP01-RNA | |
| KingFisher™ Deepwell 96 Plate | KingFisher | ThermoFisher, 95040450 | |
| KingFisher™ 96 KF microplate | KingFisher Flex ONLY | ThermoFisher, 97002540 | |
| 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 | |
| MicroAmp™ Clear Adhesive Film | KingFisher | ThermoFisher, 4306311 | |
| RNase-Free Microfuge Tubes | Nonstick, 1.5 mL | ThermoFisher, AM12450 | |
| 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 | Fisher Scientific, 50-154-2950 | |
| Qubit™ 1X dsDNA HS Assay Kit | (consumable) | ThermoFisher, Q33230 | |
| Qubit™ RNA HS Assay Kit | (consumable) | ThermoFisher, Q32852 | |
| Qubit Assay Tubes | For Qubit DNA/RNA measurement (consumable) | Thermo Fisher, Q32856 | |
| RNaseZap™ RNase Decontamination Solution | To remove RNase from working area | ThermoFisher, AM9780 | |
| ZymoBIOMICS Microbial Community Standard Material | For positive controls | Zymo Research, D6300 | |
| Zika virus (ZIKV) positive control | For TNA extraction positive control | NMRC made | |
| Human gammaherpesvirus (EBV) positive control | For TNA extraction positive control | NMRC made | |
| Forceps | For use with samples | Locally sourced | |
| Spatula | For use with samples | Locally sourced | |
| Ethanol | 100% (molecular biology grade) | Locally sourced | |
| Isopropanol | 100% (molecular biology grade) | Locally sourced | |
| Nuclease-free Water | To elute total nucleic acids | Locally sourced | |
| Dry ice | To maintain cold chain during sample handling | Locally sourced | |
| Ice bucket | To contain the dry ice | Locally sourced | |
| Kimwipes | To dry material | Locally sourced | |
| Falcon tubes | 15 mL and 50 mL | Locally sourced | |
| Data sheets | REDI-NET DCS SP-1 Sample Processing Form | REDI-NET Data Portal |
Equipment
KingFisher™ Flex Purification System, KingFisher with 96 Deep-well Head
NAME
Purification System
TYPE
Thermo Scientific™
BRAND
5400630
SKU
LINK
Equipment
KingFisher™ Duo Prime Purification System
NAME
Purification System
TYPE
Thermo Scientific™
BRAND
5400110
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 Fluorometer
NAME
Fluorometer
TYPE
Invitrogen
BRAND
Q33238
SKU
LINK
IndiMag Pathogen Kit w/o plastics (384 reactions)INDICAL BIOSCIENCECatalog #SP947257
Buffer ASL (560 ml)QiagenCatalog #19082
Measuring Spoon 100 uL RNase Free pack of 10Next AdvanceCatalog #MSP01-RNA
KingFisher™ Flex™ Systems Consumables, KingFisher Flex Microtiter Deepwell 96 plate, V-bottomThermo FisherCatalog #95040450
KingFisher MicroplateThermo Fisher ScientificCatalog #97002540
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
MicroAmp™ Clear Adhesive FilmThermo FisherCatalog #4306311
Nonstick, RNase-free Microfuge Tubes, 1.5 mLThermo FisherCatalog #AM12450
Thermo Scientific™ Screw Cap Micro TubesFisher ScientificCatalog #14-755-208
Bertin Corp 0.1mm Zirconium oxide beads (450g) (qty 500)Fisher ScientificCatalog #50-154-2950
Qubit™ 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Assay KitsThermo Fisher ScientificCatalog #Q33230
Qubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852
Qubit™ Assay TubesInvitrogen - Thermo FisherCatalog #Q32856
RNaseZap™ RNase Decontamination SolutionThermo Fisher ScientificCatalog #AM9780
ZymoBIOMICS Microbial Community StandardZymo ResearchCatalog #D6300
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.
Before start
BEFORE START
- Pre-cool the Bullet Blender by adding dry ice into the cooling compartment and running the cooling program.
- Pre-heat the heater mixer at 95 °C .
- 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. SAMPLE LYSIS
1. SAMPLE LYSIS
Add 700 µL of ASL buffer to the bead tubes prepared in step 4 from Before Start under the Guidelines & Warning tab.
Use a clean spatula or forceps to weigh about 100 mg fecal sample (roughly 5 drops of mouse feces) and place it into each prepared bead tube (If fecal sample is frozen, thaw and keep it On ice ). Record the weight.
Include a positive control for each batch of samples: transfer 37.5 µL ZymoBIOMICS Microbial Community Standard Material, 100 µL EBV, and 100 µL ZIKV standard into a tube from step 4 from Before Start. Add 250 µL ASL buffer.
Include a negative control for each batch of samples: a bead tube from step 4 from Before Start with 500 µL ASL buffer only.
Refill the dry ice compartment of the Bullet Blender if necessary. Load all samples into the Bullet Blender.
Set the speed at 12 and the time at 3. Press Start.
After beating, incubate samples at 95 °C for 00:15:00 .
15m
After incubation, beat the samples in the Bullet Blender (Refill the dry ice compartment if necessary) at speed 12 and time at 3.
Let the samples settle for 00:01:00 in the Bullet Blender and then repeat step 8.
1m
2. INSTRUMENT SET UP
2. INSTRUMENT SET UP
Note
KingFisher Flex only, if using KingFisher Duo Prime, go to 6. INSTRUMENT SET UP.
Confirm 96 deep-well magnetic heads and 96 well deep-well heat blocks are being used.
Ensure the program IndiMag_Pathogen_KF_Flex_4wash or the program has been downloaded and loaded onto the KingFisher Flex instrument.
3. SET UP THE PROCESSING PLATES
3. SET UP THE PROCESSING PLATES
Set up the Tip Comb, Wash, and Elution 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 | |
4. EXTRACTION
4. EXTRACTION
Centrifuge the bead tubes with lysate from step 9 for 12000 x g, 00:05:00 .
5m
Add 20 µL of Proteinase K into wells of a Deep-well plate (based on the number of samples).
Transfer 270 µL supernatant 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 (it can be mixed on a Hula mixer for 00:02:00 ). Add 695 µL mixture to each sample. Each well including sample lysate should be 985 µL .
2m
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.
5. QUANTIFICATION AND STORAGE
5. 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 2 and Appendix 3).
NOTE: If the DNA or RNA concentration measurement is too high, dilute 2 µL of sample in a new 1.5-mL tube with 18 µL nuclease-free water. Use 3 µL of the diluted sample for DNA or RNA quantification.
Proceed with sample testing following the REDI-NET SOP F-4 Fecal 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 F-3 Fecal Storage.
6. INSTRUMENT SET UP
6. INSTRUMENT SET UP
Note
KingFisher Duo Prime only, if using KingFisher Flex, go to 2. INSTRUMENT SET UP.
Confirm 12-tip magnetic heads 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.
7. SET UP THE SAMPLE PLATE AND ELUTION STRIP
7. 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 | |
| F | Tip comb | |||
| 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 | |
| Strip ID | Row | Reagent | Volume per well | |
| Elution | A | Nuclease-free water | 75 µL |
8. EXTRACTION
8. EXTRACTION
Centrifuge the bead tubes with lysate from step 9 for 12000 x g, 00:05:00 .
5m
Add 20 µL of Proteinase K into wells of Row A (based on number of samples) of a new Deep-well plate.
Transfer 270 µL supernatant without any particle carryover to the wells of the Deep-well Row A containing proteinase K. This row becomes the Sample Row.
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. Add 695 µL mixture to each sample. Each well including sample lysate should be 985 µL .
Select the program IndiMag_Pathogen_KF_Duo_4wash on the instrument.
Start the run, then load the prepared plate and Elution 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.
9. QUANTIFICATION AND STORAGE
9. 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 2 and Appendix 3.
NOTE: If the DNA or RNA concentration measurement is too high, dilute 2 µL of sample in a new 1.5 mL tube with 18 µL nuclease-free water. Use 3 µL of the diluted sample for DNA or RNA quantification.
Proceed with sample testing following the REDI-NET SOP F-4 Fecal 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 F-3 Fecal Storage.
APPENDIX 2. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
APPENDIX 2. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
1m
DNA quantification:
According to the volume of sample used, add the 1x HS 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). Vortex for 5 - 10 seconds, then incubate for 00:02:00 at Room temperature before reading.
2m
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. Vortex for 5 - 10 seconds, then incubate for 00:02:00 at Room temperature before reading.
2m
Protocol references
REFERENCES
1. User Guide: Indical IndiMag Pathogen Kit user’s manual
2. Indical Sample Pretreatment F2 (HB-2514-EN-001): For isolation of bacterial and viral DNA from Feces.
3. International Human Microbiome Standards Consortium, IHMS ‐ quality protocol sop for fecal samples DNA extraction protocol Q, Code: IHMS_SOP 06 V2.