TITLE: W-2 WATER PROCESSING
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol details about water processing.

[BEFORE_START]
BEFORE START


 


Make sure the feeding tube and PTFE sinker for SolVac Filter are properly clean by using 70% ethanol and allowed to air dry. 
For the samples stored at 4 °C, pour three 250 mL samples from the same sampling location into a sterile 1190 mL sample bag.
If using a frozen water sample, fully thaw them at room temperature. For the frozen sample in a plastic sample bag, wipe the bag surface with 70% ethanol to remove dusts and sanitize the surface. Source 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 defrosting. 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. 
Prepare 40% PEG-8000 solution for microbe aggregation. Check Appendix 3 for the recipe.
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 4) to Clear RINO brand 1.5 ml screw-cap microcentrifuge 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.
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 (15-25°C).
MagAttract Suspension G from the IndiMag pathogen kit needs to be vortexed thoroughly for 3 min (before first use) or 1 minute (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended.
Binding beads need to be vortexed thoroughly before each use. 
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.


[GUIDELINES]
A
AOBJECTIVE

To outline steps for properly collecting sediment samples from waterholes to evaluate the risk of zoonotic disease transmission by the detection of pathogens from environmental DNA (eDNA).

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 the protocol for the correct sediment sampling at CONUS/OCONUS sites to evaluate and predict the risk of zoonotic disease transmission.

RESPONSIBLE PERSON

Principal Investigator, Study Coordinator, Entomology Component Lead, Managers
 

MAINTENANCE OF EQUIPMENT

BEFORE EACH COLLECTION

Clean corer and scoops
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.
Store sterile equipment separate from used equipment and samples.

QUALITY CONTROL

This SOP is reviewed by the applicable supervisor annually or as required in order to maintain its relevance.

 

APPENDICES

APPENDIX 1: SolVAC Filter Holder Compartments
 
 APPENDIX 2: Microfunnel ST Filter Funnel Compartments
 
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 6.1.7 the preparation before sample homogenization. One spoon equals to 100 uL.
 
APPENDIX 6: Expected Outcomes

 
 

 
 


 
 


[STEPS]
SECTION: 1. VACUUM PUMP SET UP
1. Wipe the surfaces with 70% ethanol to remove contaminants.
SECTION: 1. VACUUM PUMP SET UP
2. Use tubing to connect a 3 liter Medi-Vac Canister with vacuum pump through the vacuum outlet on the lid. 
 

SECTION: 1. VACUUM PUMP SET UP
3. Connect tubing with the 3 liter Medi-Vac Canister through the inlet on the lid. Close unused inlets. Turn on the pump to test the vacuum suction by feeling the airflow.
SECTION: 2. WATER SAMPLE PRE-FILTRATION
4. 
 
Assemble seal gasket, housing base, and vacuum port adaptor of SolVac filter holder on the top of a new 1L sterile glass or plastic bottle. Connect the tubing from the Medi-Vac Canister to the vacuum port adaptor on the housing base. 
 

SECTION: 2. WATER SAMPLE PRE-FILTRATION
5. Wipe the housing base with 70% ethanol and let ethanol air dry (Vacuum pump can be turned on to help drying).
SECTION: 2. WATER SAMPLE PRE-FILTRATION
6. Clip a thumb clamp on the feeding tubing.
SECTION: 2. WATER SAMPLE PRE-FILTRATION
7. Connect upper housing, feeding tubing, and PTFE sinker together.
SECTION: 2. WATER SAMPLE PRE-FILTRATION
8. Place a 5 µm Versapor membrane disc filter on the housing base.
SECTION: 2. WATER SAMPLE PRE-FILTRATION
9. Seal the membrane with a membrane seal gasket and the upper housing.
SECTION: 2. WATER SAMPLE PRE-FILTRATION
10. Place the feeding tubing with PTFE sinker in the 750 mL sample (see steps 1 and 2 from Before start section).

SECTION: 2. WATER SAMPLE PRE-FILTRATION
11. Turn on the pump (<15 psi). Release the thumb clamp of the feeding tube to allow the water sample to pass through. The water sample will be filtered and collected in the 1L  sterile bottle. Turn off the vacuum pump after the water sample runs out. 
 

SECTION: 2. WATER SAMPLE PRE-FILTRATION
12. Include a positive control for each batch of samples: transfer 37.5 µL ZymoBIOMICS Microbial Community Standard and 100 µL EBV, and 100 µL HIV standard into 180 mL sterile 1x PBS.

SECTION: 2. WATER SAMPLE PRE-FILTRATION
13. Include a negative control for each batch of samples: 180 mL sterile 1x PBS.

SECTION: 2. WATER SAMPLE PRE-FILTRATION
14. Add 250 mL of PEG-8000  solution to the pre-filtered 750 mL water sample  and 60 mL of PEG-8000  solution in each control  (the final solution of PEG-8000 is 10%). Mix well by shaking.

SECTION: 2. WATER SAMPLE PRE-FILTRATION
15. Store the sample at 4 °C  .

SECTION: 3. MICROORGANISM COLLECTION
16. 
 
Disconnect the SolVac filter holder from the Medi-Vac Canister tubing. 
SECTION: 3. MICROORGANISM COLLECTION
17. Place a 300 ml MicroFunnel ST Filter funnel on a wire rack.  
 

SECTION: 3. MICROORGANISM COLLECTION
18. Connect the tubing from the Medi-Vac Canister to the base of a 300 ml MicroFunnel ST Filter Funnel. 
 

SECTION: 3. MICROORGANISM COLLECTION
19. Turn on the pump (< ). Pour the PEG-8000 treated water sample into the filter funnel (Stir the water sample while pouring).

SECTION: 3. MICROORGANISM COLLECTION
20. If possible, filter the entire 750 mL water sample using one filter funnel (the filtrate will be collected in the Medi-Vac Canister as waste). When clogging happens, use a new funnel to filter the rest.

SECTION: 3. MICROORGANISM COLLECTION
21. Disassemble the filter funnel. Carefully use an autoclaved forceps (or wipe with 70% ethanol and let dry) to move the GN-6 membrane on the supporting pad to a sterile 60 mm Petri dish.
SECTION: 3. MICROORGANISM COLLECTION
22. If not processing the membrane immediately, evenly distribute 250 µL DNA/RNA Shield Reagent on the membrane in the Petri dish, seal the Petri dish with parafilm and store at -20 °C for short-term and -80 °C for long-term until DNA/RNA extraction.

SECTION: 4. SAMPLE LYSIS
23. Pre-cool the Bullet Blender by adding dry ice into the cooling compartment and running the cooling program. 
SECTION: 4. SAMPLE LYSIS
24. Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% molecular biology grade ethanol to remove additional contaminants.
SECTION: 4. SAMPLE LYSIS
25. Transfer 0.1 mm zirconium oxide beads (2 spoons, Appendix 4, see Guidelines & Warnings tab) and four 3.5 mm UFO beads to Clear RINO brand 1.5 ml screw-cap microcentrifuge tubes.
SECTION: 4. SAMPLE LYSIS
26. Add 500 µL of ATL-DX buffer and 135 µL VXL buffer to the Clear RINO brand 1.5 ml screw-cap micro-centrifuge tubes containing 0.1 mm and 3.5 mm UFO beating beads. 
 

SECTION: 4. SAMPLE LYSIS
27. Cut the membrane with a new razor blade into 2 halves.
SECTION: 4. SAMPLE LYSIS
28. Place a half of the filter membrane in a new Petri dish and store the unused half membrane in the Petri dish at -20 °C for future use.

SECTION: 4. SAMPLE LYSIS
29. Use 70% ethanol to wipe forceps and surgical scissors. Use the forceps to fold the half membrane into a smaller size sector and directly cut the sector (into tiny pieces, smaller than 1 mm x 3 mm) into the 1.5 mL RINO tube with lysis buffer and beads.
SECTION: 4. SAMPLE LYSIS
30. Add 20 µL Proteinase K from IndiMag kit and incubate the tube at 56 °C in the heat block shaker set up at   . 
 

SECTION: 4. SAMPLE LYSIS
31. Load the sample/bead tubes in the Bullet Blender.
SECTION: 4. SAMPLE LYSIS
32. Set the speed at 12 and time at 3. Press Start.
SECTION: 4. SAMPLE LYSIS
33. Let the samples settle for 1 min and then repeat step 32. 
 

SECTION: 5. INSTRUMENT SET UP
34. 
 

Confirm 96 deep-well magnetic head and 96 well deep-well heat block are being used.
SECTION: 5. INSTRUMENT SET UP
35. Ensure the program IndiMag_Pathogen_KF_Flex_4wash has been downloaded and loaded onto the KingFisher Flex instrument.
SECTION: 5.1 SET UP THE PROCESSING PLATES
36. Set up the Wash, Elution, and Tip Comb Plates outside the instrument according to the following table.  
 
 
 
     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   

SECTION: 5.2 EXTRACTION
37. Centrifuge the bead tubes with lysate from step 33 for 12000 x g, 5 min. 
SECTION: 5.2 EXTRACTION
38. Transfer 425 µL supernatant without any particle carryover to the wells of the Deep-well plate. This plate becomes the Sample Plate.
SECTION: 5.2 EXTRACTION
39. 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 2 min). Add 560 µL mixture to each sample.

SECTION: 5.2 EXTRACTION
40. Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.
SECTION: 5.2 EXTRACTION
41. Start the run, then load the prepared plates into the positions when prompted by the instrument.
SECTION: 5.3 QUANTIFICATION AND STORAGE
42. 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. 
SECTION: 5.3 QUANTIFICATION AND STORAGE
43. 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. 
 
 

SECTION: 5.3 QUANTIFICATION AND STORAGE
44. Proceed with sample testing following the REDI-NET SOP W-4 Water Testing or store at -20 °C for less than 2 weeks. 
 

SECTION: 6. INSTRUMENT SET UP
45. 
 

Confirm 12-tip magnetic head and 12 deep-well heat blocks are being used.
SECTION: 6. INSTRUMENT SET UP
46. Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.
SECTION: 6.1 SET UP SAMPLE PLATE AND ELUTION STRIP
47. Set up the Sample Plate according to the table below:  
     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   

SECTION: 6.1 SET UP SAMPLE PLATE AND ELUTION STRIP
48. Set up the Elution Strip according to the table below: 
 

 
     Row ID      Plate Row      Reagent      Volume per well        Elution       A      Nuclease-free water      75 µL   

SECTION: 6.2 EXTRACTION
49. Centrifuge the bead tubes with lysate from step 33 for 12000 x g, 5 min. 

SECTION: 6.2 EXTRACTION
50. Transfer 425 µL supernatant without any particle carryover to the wells of the Deep-well plate. This plate becomes the Sample Plate.
SECTION: 6.2 EXTRACTION
51. 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.

SECTION: 6.2 EXTRACTION
52. Select the program IndiMag_Pathogen_KF_Duo_4wash on the instrument.
SECTION: 6.2 EXTRACTION
53. Start the run, then load the prepared plates into position when prompted by the instrument.
SECTION: 6.2 EXTRACTION
54. 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.
SECTION: 6.3 QUANTIFICATION AND STORAGE
55. 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. 
SECTION: 6.3 QUANTIFICATION AND STORAGE
56. 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. (see Appendix 5 and Appendix 6).
 


SECTION: 6.3 QUANTIFICATION AND STORAGE
57. Proceed with sample testing following the REDI-NET SOP W-4 Water Testing or store at -20 °C for less than 2 weeks). 
 

SECTION: APPENDIX 3. Preparation of 40% PEG-8000 Solution
None.  
     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.

SECTION: APPENDIX 3. Preparation of 40% PEG-8000 Solution
None. Add stir bar, PEG-8000 and NaCl into an empty 1L sterile bottle (plastic or glass, autoclavable).
SECTION: APPENDIX 3. Preparation of 40% PEG-8000 Solution
None. Add sterile 1xPBS to final volume 1 L.

SECTION: APPENDIX 3. Preparation of 40% PEG-8000 Solution
None. Autoclave the whole bottle with lid at 121 °C for 30 min. After autoclave, place the hot solution on a stirring hot plate and stir the solution until it cools down to Room temperature.

SECTION: APPENDIX 3. Preparation of 40% PEG-8000 Solution
None. 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.
SECTION: APPENDIX 3. Preparation of 40% PEG-8000 Solution
None. Store the 40% PEG-8000 solution at 4 °C. 

SECTION: APPENDIX 5. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
None. 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. Vortex for 5 -10 seconds, then incubate for 2 min at Room temperature before reading.

SECTION: APPENDIX 5. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
None. RNA Quantification: 
SECTION: APPENDIX 5. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
None. 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   
 
SECTION: APPENDIX 5. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
None. In a new 0.6 ml tube, mix 197 µL of Qubit HS RNA Assay working solution and 3 µL of the sample. Vortex for 5 s, then incubate for 2 min at Room temperature before reading.

SECTION: APPENDIX 1. SolVAC Filter Holder Compartments
None. 
SECTION: APPENDIX 3. PEG-8000 Preparation
58. Preparation of 40% PEG-8000 Solution 
 
  PEG-8000400 gNaCl70 gAdd 1x PBS to final volume 1 L
 
SECTION: APPENDIX 3. PEG-8000 Preparation
59. Add stir bar, PEG-8000 and NaCl into an empty 1L sterile bottle (plastic or glass, autoclavable).
SECTION: APPENDIX 3. PEG-8000 Preparation
60. Add sterile 1xPBS to final volume 1L.
SECTION: APPENDIX 3. PEG-8000 Preparation
61. Autoclave the whole bottle with lid at 121℃ for 30 min. After autoclave, place the hot solution on a stirring hot plate and stir the solution until it cools down to room temperature. 
SECTION: APPENDIX 3. PEG-8000 Preparation
62. 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.
SECTION: APPENDIX 3. PEG-8000 Preparation
63. Store the 40% PEG-8000 solution at 4℃.    
SECTION: APPENDIX 5. DNA and RNA Measurement using Qubit Fluorometer 4.0
64. 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. 
 
SECTION: APPENDIX 5. DNA and RNA Measurement using Qubit Fluorometer 4.0
65. 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: 



SECTION: APPENDIX 5. DNA and RNA Measurement using Qubit Fluorometer 4.0
66. 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 1 minute at room temperature before reading.