Mar 04, 2024
Version 2
W-2 WATER PROCESSING V.2
- 1University of Notre Dame
Protocol Citation: REDI-NET Consortium 2024. W-2 WATER PROCESSING . protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1opwplr2/v2Version created by REDI-NET Consortium
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: February 22, 2024
Last Modified: March 15, 2024
Protocol Integer ID: 95610
Funders Acknowledgement:
USAMRAA
Grant ID: W81XWH-21-C-0001
USAMRAA
Grant ID: W81XWH-22-C-0093
Abstract
OBJECTIVE
To outline procedures for total nucleic acid extraction from water 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 water samples to allow downstream library preparation and sequencing for pathogen detection.
Guidelines
APPENDIX 3. Preparation of 40% PEG-8000 Solution
| A | B | |
| PEG-8000 | 400 g | |
| NaCl | 70 g | |
| Add 1x PBS to final volume | 1 L |
The final concentrations: 40% PEG-8000 and 1.2 Molarity (M) NaCl.
Procedure:
- Add stir bar, PEG-8000 and NaCl into an empty 1L sterile bottle (plastic or glass, autoclavable).
- Add sterile 1xPBS to final volume 1L.
- Autoclave the whole bottle with lid at 121 °C for 00:30:00 . After autoclave, place the hot solution on a stirring hot plate and stir the solution until it cools down to Room temperature .
- If autoclave is not available, stir the solution on a stirring hot plate until the crystals are fully dissolved and filter the solution through 0.45 µm Corning Disposable Vacuum Filter/Storage Systems.
- Store the 40% PEG-8000 solution at 4 °C .
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 step 40 the preparation before sample homogenization. One spoon equals to 100 µL .
APPENDIX 5. 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 3 µL of sample, add 197 µL of 1x HS dsDNA Qubit Assay.
RNA Quantification:
1. In a new microcentrifuge tube/falcon tube (depending on the number of samples processed), prepare a working solution of the Qubit HS RNA Assay:
| Reagents | Volume/sample | Volume for n+1 sample | |
| Qubit RNA HS Assay buffer | 199 µL | …. µL | |
| Qubit RNA HS Assay Dye | 1 µL | …. µL |
2. In a new 0.6 mL tube, mix 197 µL of Qubit HS RNA Assay working solution and 3 µL of the sample. Incubate for 00:01:00 at Room temperature before reading.
APPENDIX 6. Expected Outcomes
| Sample | Amount | Sample condition | Elution volume | DNA conc. (ng/ul) | RNA conc.(ng/ul) | |
| Tick | 1 unfed adult or 10 nymphs | Frozen/live | 75 | 20 - 30 | 10 - 20 | |
| Leech | 50 ul/ 3x3 mm/ 1 swab | Blood meal/ tissue/ swab | 75 | 5 - 100 | 5 - 100 | |
| Soil | 0.25 - 0.3 g | Frozen/Fresh | 75 | <0.025 - 20 | <0.01 - 20 | |
| Water | 750 ml | Half of the membrane | 75 | <0.025 - 20 | <0.01 - 20 |
- RESPONSIBLE PERSON
Principal Investigator, Study Coordinator, Entomology Component Lead, Managers
Note
All study procedures must be conducted in compliance with national and local policies for prevention and control of COVID-19 infection.
DATA MANAGEMENT
- Date of water filtration, Personnel-ID, and Lab-ID
- Date of extraction, Personnel-ID, and Lab-ID
- Project-ID and associated project code
- Sample-ID
- Volume of water sample used (in ml)
- Number of membrane (10, 5, 0.45 µm) used to complete the water filtration.
- Number of half membrane subjected to the extraction.
- Extraction kit and extraction instrument
- Volume of elution of total nucleic acids
- Well-ID
- Concentration of total nucleic acids in ng/μL
- Personnel signature
- Any comments during the water filtration process or the TNA extraction
Water sample number is defined as follows:
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.
Cleaning filter compartments
- Wipe magnetic filter funnel with 70% ethanol between each sample. If dirt accumulated, clean the unit with soapy water then completely rinse off the soap with deionized water. Magnetic filter funnel and 1L glass bottle are autoclavable (as well as tubing if it was made by silicon); autoclave them after filtering 2-3 batches of water samples. If autoclave is not available, immerse the compartments in 1% bleach for 00:15:00 then rinse off the bleach and air dry the whole unit. Make sure the water inlet tube is completely dried before processing a new batch filtration.
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.
QUALITY CONTROL
- This SOP is reviewed by the applicable supervisor annually or as required in order to maintain its relevance.
Materials
UIPMENT AND MATERIALS
Note
If product number is listed, please ensure use of this or equivalent product.
| A | B | C | |
| Equipment | Mfg / Product # | NSN | |
| KingFisher™ Flex Magnetic Particle Processor with 96 Deep-Well Head or KingFisher™ Duo Prime Magnetic Particle Processor | ThermoFisher, 5400630 or ThermoFisher, 5400110 | Not found | |
| Bullet Blender 24 Gold | Next Advance, BB24-AU | Not found | |
| Adjustable micropipettes | Locally sourced | N/A | |
| Multi-channel micropipettes | Locally sourced | N/A | |
| Sartorius Microsart™ Maxi Vacuum Pumps | Fisher Scientific, 14-555-788 or equivalent | Not found | |
| Vortex | Locally sourced | N/A | |
| Fisherbrand Reusable Glass Media Bottles with Cap, autoclavable | Fisher Scientific, FB8001000 or equivalent | Not found | |
| DWK Life Sciences DURAN™ Screw Cap GL 45 with 2-hose connector | Fisher Scientific, 05-719-310 | ||
| Magnetic Filter Funnel, 500 mL, 47mm | Fisher Scientific, 50-206-3019 | Not found | |
| Fisherbrand™ Heavy-Duty/Utility Funnels | |||
| Tube centrifuge | Locally sourced | N/A | |
| Plate centrifuge | Locally sourced | N/A | |
| Qubit 4 Fluorometer | ThermoFisher, Q33238 | Not found | |
| Mini Block Heater | VWR, 10818-597 or locally sourced | Not found | |
| Autoclave | Locally sourced | N/A | |
| Stir bar | Locally sourced | N/A | |
| RT2 Basic Hotplate Stirrer | ThermoFisher, 88880004 or locally sourced | Not found |
| A | B | C | D | |
| Material | Description | Mfg / Product # | NSN | |
| Water Samples | Collected using SOP W-1; stored in 250 ml sterile bottle at 4℃ for processing the same day or at -80℃ or -20℃ for long-term storage | From REDI-NET Program | N/A | |
| Sterile 1x PBS as negative control | 180 mL, sterile. | Thermo Fisher, 10010023 | Not found | |
| Sterile 1x PBS spike-in positive control | 180 ml sterile 1x PBS, spike in 37.5 µl of ZymoBIOMICS Microbial Community Standard and 100 µl HIV standard, 100 µl EBV standard. | Thermo Fisher, 10010023 | Not found | |
| ZymoBIOMICS Microbial Community Standard Material | For positive controls | Zymo Research, D6300 | Not found | |
| AcroMetrix HIV-1 Controls | For TNA extraction positive control; BSL-2 | ThermoFisher, CLS430320-12EA | Not found | |
| Dengue virus type 1 (DENV-1) positive control | For TNA extraction positive control | ATCC, VR-1856 | Not found | |
| Fisherbrand™ Heavy-Duty/Utility Funnels | 410 mL | Fisher Scientific, 10-500-9 | ||
| Gauze | Sterile, 4 x 4 inches, 8-ply | Fisher Scientific, 10-000-684 or equivalent | Not found | |
| Fisherbrand Reusable Glass Media Bottles | 1L, with Cap, autoclavable | Fisher Scientific, FB8001000 or equivalent | Not found | |
| DWK Life Sciences DURA Screw Cap | GL45 thread with 2-hose connector | Fisher Scientific, 05-719-310 | ||
| Thermo Scientific Nalgene 50 Platinum-Cured Silicone Tubing | 6.4 mm inner diameter, 50 ft. | Fisher Scientific, 14-176-332E or equivalent | ||
| Magnetic Filter Funnel | 500 mL, fits 47mm filter (consumable) | Fisher Scientific, 50-206-3019 | Not found | |
| Nylon Membrane Filter | 30.0 μm Pore Size, 47 mm, 100/pack (consumable) | Millipore Sigma, NY3004700 | ||
| Polypropylene Membrane Filter | 10.0 μm Pore Size, 47 mm, 100/pack (consumable) | Millipore Sigma, AN1H04700 | ||
| MCE Membrane Filter | 5.0 μm Pore Size, 47 mm, 100/pk (consumable) | Millipore Sigma, SMWP04700 | ||
| Fisher brand Sterile Sampling Bags with Flat-Wire Closures | Clear, 42 oz/1190 ml, 3.5 mil thickness (consumable) | Fisher Scientific, 14-955-188 or equivalent | Not found | |
| Fisherbrand Sterile Sampling Bags with Flat-Wire Closures | Clear, 18 oz. /540 ml, 3 mil thickness (consumable) | Fisher Scientific, 14-955-186 or equivalent | Not found | |
| Beaker | 1L | Fisher Scientific, 02-591-32 or equivalent | Not found | |
| Easy Grip Polystyrene Storage Bottles | 1L (consumable) | Corning, 430518 or equivalent | Not found | |
| Easy Grip Polystyrene Storage Bottles | 250 ml (consumable) | Corning, 430281 or equivalent | Not found | |
| Cardinal Health™ Medi-Vac™ Guardian™ Suction Canisters | 3L (consumable) | Fisher Scientific, 19-162-321 or equivalent | Not found | |
| IndiMag Pathogen Kit | w/o plastics, 384 reactions | Indical Bioscience, SP947257 | Not found | |
| Buffer ATL | 200 mL, Tissue Lysis Buffer | Qiagen, 19076 | Not found | |
| Reagent DX | 1 mL, Antifoaming Reagent | Qiagen, 19088 | Not found | |
| Measuring Spoon 100µL | RNase Free, pack of 10 | Next Advance, MSP01-RNA | Not found | |
| 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 | Not found | |
| Stainless Steel UFO Beads | 3.5 mm, RNase free | Next Advance, SSUFO35-RNA | Not found | |
| Zirconium oxidase beads | 0.1 mm, 400 g | Fisher Scientific, 50-154-2950 | Not found | |
| KingFisher™ Deepwell 96 Plate | (consumable) | ThermoFisher, 95040450 | Not found | |
| KingFisher™ 96 KF microplate | KingFisher Flex ONLY (consumable) | ThermoFisher, 97002540 | Not found | |
| KingFisher™ 96 tip comb for DW Magnets | KingFisher Flex ONLY (consumable) | ThermoFisher, 97002534 | Not found | |
| KingFisher™ Duo Prime 12-tip comb | KingFisher Duo Prime ONLY (consumable) | ThermoFisher, 97003500 | Not found | |
| Elution Strip | KingFisher Duo Prime ONLY (consumable) | ThermoFisher, 97003520 | Not found | |
| KingFisher™ Duo Cap for Elution Strip | KingFisher Duo Prime ONLY (consumable) | ThermoFisher, 97003540 | Not found | |
| MicroAmp™ Clear Adhesive Film | KingFisher (consumable) | ThermoFisher, 4306311 | Not found | |
| Nonstick, RNase-Free Microfuge Tubes | 1.5 mL (consumable) | ThermoFisher, AM12450 | Not found | |
| Nonstick, RNase-Free Microfuge Tubes | 2.0 mL (consumable) | ThermoFisher, AM12475 | Not found | |
| RNaseZap™ RNase Decontamination Solution | To remove RNase from working area (consumable) | ThermoFisher, AM9780 | Not found | |
| PEG-8000 | Poly(ethylene glycol) BioUltra, 8,000 (consumable) | Millipore Sigma, 89510-1KG-F | Not found | |
| VacuCap 90 Vacuum Filtration Devices | 0.2 µm, 90 mm, gamma-irradiated, for PEG-8000 preparation(consumable) | PALL, TA4622 | Not found | |
| NaCl | For PEG-8000 buffer preparation | Sigma Aldrich, S9888-1KG | Not found | |
| Qubit™ 1X dsDNA HS Assay Kit | (consumable) | ThermoFisher, Q33230 | Not found | |
| Qubit™ RNA HS Assay Kit | (consumable) | ThermoFisher, Q32852 | Not found | |
| Qubit Assay tubes | For Qubit DNA/RNA measurement (consumable) | Thermo Fisher, Q32856 | Not found | |
| Ethanol | 96-100%, molecular biology grade (consumable) | Locally Sourced | N/A | |
| Isopropanol | 100%, molecular biology grade (consumable) | Locally Sourced | N/A | |
| Forceps | Stainless, sterile (consumable) | PALL, 51147 or equivalent | Not found | |
| Tubing | Plastic (consumable) | Locally sourced | N/A | |
| Razor blades | (consumable) | Fisher Scientific, 12-640 or equivalent | Not found | |
| Petri dishes | 60 mm disposable (consumable) | Fisher Scientific, FB0875713A or equivalent | 6640-00-051-9495 | |
| Wire racks | (consumable) | Fisher Scientific, FB147916A or equivalent | Not found | |
| Excelta Medical-Grade Scissors | For filter membrane cutting | Fisher Scientific, 17-456-005 or equivalent | Not found | |
| Parafilm M | Used for petri dish sealing (consumable) | Fisher Scientific, 13-374-12 or equivalent | 6640-01-185-3289 | |
| Kimwipes | To dry material | Locally sourced | 6515-01-509-2474 | |
| Conical centrifuge tubes | 50 mL (consumable) | Fisher Scientific, 14-432-22 or equivalent | 6640270477303 | |
| DNA/RNA Shield Reagent | 50 ml | Zymo Research, R1100-50 | Not found | |
| Conical centrifuge tubes | 15 mL (consumable) | Fisher Scientific, 14-959-53A or equivalent | 6640270477300 | |
| Nuclease-free water | (consumable) | Locally sourced | N/A | |
| Dry ice | To maintain cold chain during sample handling (consumable) | Locally sourced | N/A | |
| Data Sheets | REDI-NET DCS SP-1 Sample Processing Form | REDI-NET Data Portal | N/A |
Equipment
RT2 Basic Hotplate Stirrer
NAME
Hot Plates & Stirrers
TYPE
Thermo Scientific™
BRAND
88880004
SKU
LINK
Equipment
Mini Block Heater, Greiner Bio-One
NAME
Mini Block Heater
TYPE
Greiner Bio-One
BRAND
10818-597
SKU
LINK
Equipment
Qubit 4
NAME
Fluorometer
TYPE
Invitrogen
BRAND
Q33238
SKU
LINK
Equipment
Cytiva Magnetic Filter Funnel, 500 mL, 47 mm
NAME
Magnetic Filter Funnel
TYPE
Fisher Scientific
BRAND
50-206-3019
SKU
LINK
Equipment
DWK Life Sciences DURAN™ Screw Cap GL 45 with 2-hose connector
NAME
Fisher Scientific
BRAND
05-719-310
SKU
LINK
Equipment
Fisherbrand™ Reusable Glass Media Bottles with Cap
NAME
Reusable Glass Media Bottles
TYPE
Fisher Scientific
BRAND
FB8001000
SKU
LINK
Equipment
Bullet Blender 24 Gold
NAME
Bullet Blenders
TYPE
Nextadvance
BRAND
BB24AU
SKU
LINK
Equipment
KingFisher Duo Prime
NAME
Automated Nucleic Acid Purification
TYPE
Thermo Scientific™
BRAND
5400110
SKU
LINK
Equipment
Kingfisher Flex
NAME
Automated Extraction System
TYPE
ThermoFisher
BRAND
5400630
SKU
PBS buffer Thermo Fisher ScientificCatalog #10010023
ZymoBIOMICS Microbial Community StandardZymo ResearchCatalog #D6300
Corning tube top vacuum filtration systemScientific Laboratory Supplies LtdCatalog #CLS430320-12EA
Equipment
Fisherbrand™ Heavy-Duty/Utility Funnels
NAME
Fisher Scientific
BRAND
10-500-9
SKU
LINK
Equipment
Stoelting™ Sterile Gauze
NAME
Gauze
TYPE
Fisher Scientific
BRAND
10-000-684
SKU
LINK
Equipment
Thermo Scientific™ Nalgene™ 50 Platinum-Cured Silicone Tubing
NAME
Thermo Scientific™
BRAND
14-176-332E
SKU
LINK
Equipment
Nylon Net Filter
NAME
Hydrophilic, 30 µm, 47 mm, 100
TYPE
Millipore
BRAND
NY3004700
SKU
LINK
Equipment
Polypropylene Prefilter
NAME
Hydrophobic, 10 µm, 47 mm
TYPE
Millipore
BRAND
AN1H04700
SKU
LINK
Equipment
MF-Millipore™ Membrane Filter, 5 µm pore size
NAME
47 mm diameter, mixed cellulose esters (MCE) membrane, hydrophilic, white, 100 discs
TYPE
Millipore
BRAND
SMWP04700
SKU
LINK
Equipment
Whatman® Mixed Cellulose Ester filters
NAME
Membrane filters
TYPE
Millipore Sigma
BRAND
WHA7141104
SKU
LINK
Equipment
Fisherbrand™ Sterile Sampling Bags with Flat-Wire Closures
NAME
Fisher Scientific
BRAND
14-955-188
SKU
LINK
Equipment
Fisherbrand™ Sterile Sampling Bags
NAME
An economical and efficient way to collect, contain and carry samples
TYPE
Fisher Scientific
BRAND
14-955-186
SKU
LINK
Equipment
Thermo Scientific™ Nalgene™ Polypropylene Griffin Low-Form Plastic Beakers
NAME
Fisher Scientific
BRAND
02-591-10G
SKU
LINK
Equipment
Cardinal Health™ Medi-Vac™ Guardian™ Suction Canisters
NAME
Suction Canisters
TYPE
Fisher Scientific
BRAND
19-162-321
SKU
LINK
Indical Bioscience, SP947257INDICAL BIOSCIENCECatalog #SP947257
Buffer ATL (tissue lysis buffer)QiagenCatalog #19076
Reagent DXQiagenCatalog #19088
Equipment
Measuring Spoon
NAME
100 uL RNase Free
TYPE
Next Advance
BRAND
MSP01-RNA
SKU
LINK
Equipment
Thermo Scientific™ Screw Cap Micro Tubes
NAME
Screw Cap Micro Tubes
TYPE
Fisher Scientific
BRAND
14-755-208
SKU
LINK
Equipment
Stainless Steel UFO Beads 3.5 mm RNase Free
NAME
Beads
TYPE
Next Advance
BRAND
SSUFO35-RNA
SKU
LINK
Equipment
Bertin Corp 0.1mm Zirconium oxide beads
NAME
Beads
TYPE
Fisher Scientific
BRAND
50-154-2950
SKU
LINK
Equipment
KingFisher™ Plastics for 96 deep-well format
NAME
Automated Nucleic Acid Purification
TYPE
ThermoFisher
BRAND
95040450
SKU
LINK
Equipment
KingFisher™ Plastics for 96 standard and PCR formats Copy Icon Thermo Scientific™ KingFisher™ Plastics for 96 standard and PCR formats
NAME
Automated Nucleic Acid Purification
TYPE
Thermo Fisher Scientific
BRAND
97002540
SKU
LINK
Equipment
KingFisher™ Plastics for 96 deep-well format
NAME
Automated Nucleic Acid Purification
TYPE
Thermo Fisher Scientific
BRAND
97002534
SKU
LINK
Equipment
KingFisher™ Plastics for 96 deep-well format
NAME
Automated Nucleic Acid Purification
TYPE
ThermoFisher Scientific
BRAND
97003540
SKU
LINK
Equipment
MicroAmp™ Clear Adhesive Film
NAME
Adhesive Film
TYPE
ThermoFisher Scientific
BRAND
4306311
SKU
LINK
Equipment
RNase-free Microfuge Tubes
NAME
RNA Extraction
TYPE
Invitrogen
BRAND
AM12450
SKU
LINK
Equipment
Nonstick, RNase-free Microfuge Tubes, 2.0 mL
NAME
Microcentrifuge tubes with a non-stick, low-binding surface
TYPE
Invitrogen
BRAND
AM12475
SKU
LINK
RNaseZap®Thermo ScientificCatalog #AM9780
Polyethylenglycol (MW=8000)Merck MilliporeSigma (Sigma-Aldrich)Catalog #89510-1KG-F
Equipment
PALL TA4622 VacuCap 90 Vacuum Filtration Devices
NAME
0.2 µm, 90 mm (supplied with individually attached tubing for each filter device), gamma-irradiated
TYPE
Pall
BRAND
TA4622
SKU
LINK
Sodium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9888
Qubit 1X dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q33230
Qubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852
DNA/RNA ShieldZymo ResearchCatalog #R1100-50
Equipment
Qubit™ Assay Tubes
NAME
Invitrogen
BRAND
Q32856
SKU
LINK
Equipment
Forceps
NAME
PALL
BRAND
51147
SKU
LINK
Equipment
Razor Blades
NAME
Fisherbrand™
BRAND
12-640
SKU
LINK
Equipment
Fisherbrand™ Petri Dishes with Clear Lid or equivalent
NAME
Petri Dishes
TYPE
Fisherbrand
BRAND
FB0875713A
SKU
LINK
Round, Raised Ridge, 60mm, 15mm
SPECIFICATIONS
Equipment
HDPE Coated Wire Racks
NAME
Fisherbrand™
BRAND
FB147916A
SKU
LINK
Equipment
Laboratory Wrapping Film
NAME
Bemis™
BRAND
13-374-12
SKU
LINK
Equipment
50 mL High Clarity Conical Centrifuge Tubes
NAME
Falcon™
BRAND
14-432-22
SKU
LINK
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
1. Water samples can be stored at 4 °C for 1 week, -20 °C for 1 month, and -80 °C for longer periods of time.
2. Make sure the water inlet tube and Magnetic Filter Funnel and glass bottles are properly clean and dry. If autoclave is not available, the parts in the filter system that directly contacted to the water samples needs to be fully rinsed by 10% bleach, followed by water and 70% ethanol then dry.
3. If the water samples were collected at a high temperature sampling site (≥25 °C ) with visible floating plants, microalgae, and sediments, use 250 mL water sample for downstream filtration, otherwise, use 750 mL water sample for filtration.
4. If water sample is frozen, fully thaw it at Room temperature then process it when it is still cold. When water sample frozen in a plastic sample bag, wipe the bag surface with 70% ethanol to remove dusts and sanitize the surface. Place three bags of 250 mL water samples from the same sampling location in a new 1190 ml sterile sample bag, then put the bagged samples in a suitable-sized container for thawing. After samples are fully defrosted, pour the water samples into the 1190 ml outer bag, and discard the original 250 ml sample bags. Hold the whole bag in a 1 L beaker.
Note
NOTE: Plastic sample bags holding 250 mL water samples can leak after freeze/thaw. The 1190 mL /42 oz sterile bag can prevent sample loss/contamination.
6. Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% molecular biology grade ethanol to remove additional contaminants.
7. Transfer 0.1 mm zirconium oxide beads (2 spoons, Appendix 4) and four 3.5 mm UFO beads to Thermo Scientific Screw Cap 1.5 mL Micro Tubes.
8. For the first time use of IndiMag pathogen kit, add 96—100% ethanol to Buffer AW1 and AW2, and add 100% Isopropanol to ACB as indicated on the bottles.
9. 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 the addition of Reagent DX. After preparation, the mixture is stable for 6 months at Room temperature (15 °C —25 °C ).
10. 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.
11. Binding beads need to be vortexed thoroughly before each use.
12. 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.
VACUUM PUMP SET UP
VACUUM PUMP SET UP
Note
- To prevent cross contamination, nucleic acid extraction and amplification (PCR) should be performed in separate rooms.
- Processing can be done prior to freezing samples to save freezer space. Each location/site (edge/1m from edge) would account for 4 filter paper water samples for each sampling site.
Wipe the surfaces with 70% ethanol to remove contaminants.
Use tubing to connect a 3-liter Medi-Vac Canister with vacuum pump through the vacuum outlet on the lid. (If possible, the canister should be set up inside a biosafety cabinet).
Connect tubing with the 3-liter Medi-Vac Canister through the port for air-in (indicated as patient) on the lid. Close unused inlets. Turn on the pump to test the vacuum suction by feeling the airflow.
WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
Note
When water sample is very dirty, filter the water sample with a sterile 8-ply gauze on a funnel using gravity to remove floating plants, mud, and microalgae, it could be done multiple times.
Assemble Magnetic Filter funnel, tubing, GL45 Screw Cap with 2-hose connector, 1L dry glass bottle (autoclaved or bleach rinsed) and the Medi-Vac Canister as Appendix 1.
Note
See Appendix 1 for the water filtration system setting. Place the Magnetic filter Funnel at a position higher than the 1L clean glass bottle, any way will do if the funnel is secure.
If the water samples have high turbidity, settle water at 4 °C for 01:00:00 .
1h
Wipe the filter holder of the magnetic funnel with 70% ethanol and let ethanol air dry.
Place a 30 µm filter membrane disc on the filter holder.
Attach the top funnel cup to the filter holder.
Pour the settled water sample to the magnetic funnel cup, avoid disturbing the precipitation as much as possible.
Turn on the pump (<15 psi) to allow the water sample to pass through the filter and be collected in the bottle (if the pump flow rate cannot be controlled, put a tube clip on the air outlet tube to control the flow rate avoiding the water splash in the bottle). Turn off the vacuum pump after the water sample runs out (If clogging happens, replace the membrane disc filter with a new one and collect all the filtrates in the same bottle).
Discard the 30 µm filter membrane disc.
Pour the filtrate to a sterile sampling bag with flat-wire closure or a clean bottle and connect the bottle back to the filtration system.
Place a new 10 µm filter membrane on the filter holder and filter the filtrate as step 9-10. Avoid using multiple 10 µm filter membranes to finish the filtration (the plastic bag can be reused to contain the same sample in the second round of the filtration).
Store the 10 µm filter membrane in a sterile 60 mm Petri dish and keep the dish On ice . Label the Petri dish with sample ID, filtration date, and membrane size.
Place a 5 µm filter membrane on the membrane holder then repeat step 9-10. Avoid using multiple 5 µm filter membranes to finish the filtration.
Store the 5 µm filter membrane in a sterile 60 mm Petri dish and keep the dish On ice . Label the Petri dish with sample ID, filtration date, and membrane size.
Evenly distribute 250 µL DNA/RNA Shield Reagent on the membranes in the Petri dishes, seal the Petri dishes with parafilm and store at -20 °C for short-term and -80 °C for long-term until DNA/RNA extraction.
Add 187 mL of PEG-8000 solution to a 750 mL water sample filtrate (or 62.5 mL PEG-8000 solution to a 250 mL filtrate, the final concentration of PEG-8000 is 8%). Mix well by shaking.
Note
If a clean 1L bottle for filtrate collection is not available, the filtrate can be transferred back to the plastic sampling bag with flat-wire closure for adding PEG-8000.
Rinse the magnetic funnel, water inlet tube, and 1L glass bottle with 10% bleach, then wash away the bleach with deionized water.
Rinse the inlet tube and 1L glass bottle with 70% ethanol, shake off the residuals and allow to air dry. Wipe dry the magnetic funnel with 70% ethanol.
Repeat steps from 4 to 19 for another sample.
To speed up the water filtration, prepare multiple sets of tubes and clean 1L bottles to avoid the waiting time for the air dry. The magnetic funnel can be used right after the 70% ethanol wipe.
After finishing all the sample filtration for the day, prepare a positive control for the batch: add 100 µL EBV and 50 µL DENV-1 standard into 180 mL sterile 1x PBS then add 45 mL of PEG-8000 solution.
Negative control for the batch of sample filtration: 180 mL sterile 1x PBS then add 45 mL of PEG-8000 solution.
Store PEG-8000-added samples and controls at 4 °C for more than 04:00:00 or Overnight for the next filtration round (DO NOT store the PEG-8000-added samples at 4 °C more than 24:00:00 that will compromise the sample stability).
1d 8h
POTENTIAL VIRAL PARTICLE COLLECTION
POTENTIAL VIRAL PARTICLE COLLECTION
After overnight incubation with PEG-8000, water samples are ready for viral particle capturing filtration.
Assemble the filtration system following the steps described in steps 4-6.
Place a new 5 µm filter membrane on the filter holder and filter the filtrate as steps 9-10. Avoid using multiple 5 µm filter membranes to finish the filtration.
Store the 5 µm filter membrane in a sterile 60 mm Petri dish and keep the dish On ice . Label the Petri dish with sample ID, filtration date, and membrane size.
Pour the filtrate to a sterile sampling bag with flat-wire closure or a clean bottle and connect the bottle back to the filtration system.
Place a new 0.45 µm filter membrane on the membrane holder and filter the filtrate as steps 9-10. Avoid using multiple 0.45 µm filter membranes to finish the filtration.
Store the 0.45 µm filter membrane in a sterile 60 mm Petri dish and keep the dish on ice. Label the Petri dish with sample ID, filtration date, and membrane size.
Evenly distribute 250 µL DNA/RNA Shield Reagent on the membranes in the Petri dishes, seal the Petri dishes with parafilm and store at -20 °C for short-term and -80 °C for long-term until DNA/RNA extraction.
Discard the filtrate.
Rinse the magnetic funnel, water inlet tube, and 1L glass bottle with 10% bleach, then wash away the bleach by running under deionized water.
Rinse the inlet tube and 1L glass bottle with 70% ethanol, shake off the residuals, and allow to air dry.
Wipe dry the magnetic funnel with 70% ethanol. Repeat steps from 26 to 34 for another sample.
SAMPLE LYSIS
SAMPLE LYSIS
Pre-cool the Bullet Blender by adding dry On 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 4) and four 3.5 mm UFO beads to Thermo Scientific Screw Cap 1.5 mL Micro Tubes. Each water sample needs two bead tubes. Can be prepared in advance as described in Before Start.
Add 500 µL of ATL-DX buffer and 135 µL VXL buffer to the Thermo Scientific Screw Cap 1.5 mL Micro Tubes containing 0.1 mm and 3.5 mm UFO beating beads.
Note
For the preparation of the ATL-DX buffer, see step "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." in before start section.
Each water sample has 4 filter membranes from different filtrations (before PEG-8000 treatment: membrane of pore size 10 and 5, one of each; after PEG-8000 treatment: membrane of pore size 5 and 0.45, one of each)
Use 70% ethanol to wipe forceps and surgical scissors (or use new razor blade).
Trim the outer circle of the membrane that had no water sample flowing through off, discard the outer circle (see Figure 1).
Cut the membranes into 2 halves.
Place 4 halves of the filter membranes from different filtrations of the same water sample in a new Petri dish and store the unused half membranes in the original Petri dish at -20 °C for future use (see Figure 1).
Use the forceps to stack the 2 half membranes, then fold the stacked halves into a smaller sector and cut it ( smaller than 1 mm x 3 mm, see example in Figure 1) into the a tube prepared in step 40 (Suggest collecting the 2 half membranes before PEG-8000 in tube A and the 2 half membranes from after PEG-8000 treatment in tube B).
Add 20 µL Proteinase K from IndiMag kit and incubate the tube at 56 °C in the heat block shaker set up at 1400 rpm, 00:20:00 (if heat block shaker is not available, vortex the tube every 00:05:00 ).
25m
Load the sample/bead tubes in the Bullet Blender.
Set the speed at 12 and time at 3. Press Start.
Let the samples settle for 00:01:00 and then repeat step 50.
STOPPING POINT: lysed samples can be stored at 4 °C Overnight .
Figure 1. The examples of membrane trimming and cutting.
1m
INSTRUMENT SET UP (KingFisher Flex only, if using KingFisher Duo Prime, go to section "SET UP SAMPLE PLATE AND ELUTION STRIP"
INSTRUMENT SET UP (KingFisher Flex only, if using KingFisher Duo Prime, go to section "SET UP SAMPLE PLATE AND ELUTION STRIP"
Confirm 96 deep-well magnetic head and 96 well deep-well heat block 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
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 bead tubes with lysate from step 51 for 12000 x g, 00:05:00 .
5m
Transfer 425 µL supernatant without any particle carryover to the wells of the Deep-well plate. This plate becomes the Sample Plate.
Add 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 00:02:00 ). Add 560 µL mixture to each sample.
2m
Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.
Start the run, then load the prepared plates into the positions when prompted by the instrument.
QUANTIFICATION AND STORAGE
QUANTIFICATION AND STORAGE
After the running protocol is completed (~00:35:00 ), 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.
Note
The elutes from the 2 bead tubes of the same water sample can be pooled in one final tube.
35m
In a 0.6 mL microcentrifuge tube, use 3 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 and Appendix 6).
Proceed with sample testing following the REDI-NET SOP W-4 Water 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 W-3 Water Storage).
INSTRUMENT SET UP (KingFisher Duo Prime only, if using KingFisher Flex, go to section "SET UP THE PROCESSING PLATES"
INSTRUMENT SET UP (KingFisher Duo Prime only, if using KingFisher Flex, go to section "SET UP THE PROCESSING PLATES"
Confirm 12-tip magnetic head and 12 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 SAMPLE PLATE AND ELUTION STRIP:
SET UP 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 Wash 2 | F | 12-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 | |
| 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 51 for 12000 x g, 00:05:00 .
5m
Transfer 425 µL supernatant without any particle carryover to the wells of the Deep-well plate. This plate becomes the Sample Plate.
Add 540 µL Buffer ACB, and 25 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 565 µL mixture to each sample.
Select the program IndiMag_Pathogen_KF_Duo_4wash on the instrument.
Start the run, then load the prepared plates 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 running protocol is completed (~00:35:00 ), 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.
Note
The elutes from the 2 bead tubes of the same water sample can be pooled in one final tube.
35m
In a 0.6 mL microcentrifuge tube, use 3 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 and Appendix 6).
Proceed with sample testing following the REDI-NET SOP W-4 Water 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 W-3 Water Storage).
APPENDIX 1. Example of the water filtration system assembly 0m
APPENDIX 1. Example of the water filtration system assembly 0m
Note
The reusable magnetic funnel cup (A) can be replaced by a disposable Microfunnel ST Filter funnel (B) or equivalent. Place the funnel at higher position can accelerate the filtration speed, it can be secured by any available resources as long as the setting is stable. The disposable funnel comes with a 0.45 µm membrane and secures the filter membrane by snap on the filter holding stage. The 0.45 µm can be replaced by a 30, 10, or 5 µm membrane, the support pad under the membrane needs to be kept during the filtration (D). A tube needs to be connected to the inner connector of the Screw Cap GL 45 with 2-hose connector for guide the filtrate to the bottom of the bottle to avoid the water entering the vacuum system (F). A tube clip can be secured to the air outlet tube to control the flowrate if it cannot be controlled through the vacuum pump.
APPENDIX 2. Reference of Water Filtration Speed
APPENDIX 2. Reference of Water Filtration Speed
APPENDIX 2. Reference of Water Filtration Speed
| A | B | C | D | E | F | G | H | |
| Before add PEG-8000 | After add PEG-8000 | |||||||
| Sample type | Water volume | 10 µm | 5 µm | Filtration time | 5 µm | 0.45 µm | Filtration time | |
| Summer pond water with floating plants, high turbidity | 250 ml | 16 s | 1 h 24 m | 1 hr 25 m | 35 s | 4 m 14 s | 5 m 49 s | |
| Winter pond water with little floating plants | 250 ml | 10 s | 14 s | 24 s | 29 s | 3 m 07 s | 3 m 36s | |
Note
The summer collected pond water was filtered through a gauze by gravity to remove all the floating plants. The water samples in this reference had been pre-filtered by 30 µm nylon membrane. A 30 µm nylon membrane can filter a 500 mL very dirty water sample within 1-00:05:00 .
Protocol references
- John, S.G., et al., A simple and efficient method for concentration of ocean viruses by chemical flocculation. Environ Microbiol Rep, 2011. 3(2): p. 195-202. https://www.ncbi.nlm.nih.gov/pubmed/21572525
- Chopyk, J., et al., Seasonal dynamics in taxonomy and function within bacterial and viral metagenomic assemblages recovered from a freshwater agricultural pond. Environ Microbiome, 2020. 15(1): p. 18. https://www.ncbi.nlm.nih.gov/pubmed/33902740
- User Guide: Indical IndiMag Pathogen Kit user’s manual