TITLE: L-2 LEECH PROCESSING
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol details leech processing.

[BEFORE_START]

 

Pre-cool the Bullet Blender by adding dry ice into the cooling compartment and running the cooling program. 
Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% molecular biology grade ethanol to remove additional contaminants.
Transfer 0.1 mm zirconium oxide beads (2 spoons, Appendix 2) 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 (Optional if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit).
Buffer ATL may form precipitates upon storage. If necessary, warm to Temperature56 °C   until the precipitates have fully dissolved. Prepare buffer ATL-DX: add Amount100 µL   Reagent DX to Amount15 mL   Buffer ATL. If smaller amounts are needed, transfer Amount1.5 mL   of Buffer ATL into a sterile 2 ml vial and add Amount10 µL   Reagent DX. Mix well, after addition of Reagent DX. After preparation, the mixture is stable for 6 months at TemperatureRoom temperature   (15-25°C)** 
MagAttract Suspension G from IndiMag pathogen kit needs to be vortexed thoroughly for 3 min (before first use) or 1 min  (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended.
When processing viable leeches, freeze at -20 °C for 60 min or in dry ice for 20 min to inactivate them and process them right away.
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]
OBJECTIVE

To outline procedures to process leech samples for total nucleic acid extraction.

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 leech total nucleic acid extraction to allow downstream library preparation and sequencing for pathogen detection.

RESPONSIBLE PERSON

Principal Investigator, Study Coordinator, Entomology Component Lead, Managers
 

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.
Precipitate may form after storage at low temperature or prolonged storage. To dissolve precipitate, incubate Buffer VXL or ACB for 30 min 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. PHOTOS OF BLOOD MEAL AND COTTON SWAB

Figure 1: Blood meal                         Figure 2: Cotton swab
 


APPENDIX 2. 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 44 the preparation before tick homogenization. One spoon equals to 100 µL. 
 


APPENDIX 3. EXPECTED OUTCOMES

 



[STEPS]
SECTION: 2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
3. 
 

Add 400 µL 1x PBS and 100 µL ATL-DX buffer in a Thermo Scientific Screw Cap 1.5 mL Micro tube containing 0.1 mm beads from the step 3 in Before You Start.

SECTION: 2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
4. Clean forceps with 70% ethanol and Kimwipes before use and between samples.
SECTION: 2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
5. Use ice-cold 1x PBS to wash leech three times sequentially in petri dishes on ice to remove external contaminants.
SECTION: 2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
8. Use a pair of scissors to cut across the leech in the middle.
SECTION: 2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
9.        
Drip the blood meal into the petri dish as much as possible.
SECTION: 2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
11. Place the leech back into the petri dish, use a scalpel to cut open the entire leech longitudinally.
SECTION: 2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
12. Open the leech with forceps, use a sterile cotton swab to wipe the opened inner organs thoroughly.

SECTION: 2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
13. Put the cotton tip of the swab in a 1.5 ml tube prepared in step 3, cut off the plastic handle from the cotton tip.

SECTION: 3. SMALL LEECH TISSUE HOMOGENIZATION
22. Set the controls for Speed 10 and Time 3 min. Press Start.

SECTION: 3. SMALL LEECH TISSUE HOMOGENIZATION
24. After the run, remove the tubes from the instrument and visually inspect the samples. If homogenization is incomplete, repeat the homogenization at speed 10 and Time 3 min.

SECTION: 3. SMALL LEECH TISSUE HOMOGENIZATION
25. Centrifuge the suspension at 100 x g, 1 min to pellet debris.

SECTION: 4. MICROBE LYSIS
28. 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 Screw Cap 1.5 mL MicroTube containing 0.1 mm beating beads. Add 162.5 µL 1x PBS and 100 µL ATL-DX buffer.

SECTION: 4. MICROBE LYSIS
29. Include a negative control for each batch of samples: add 400 µL sterile 1xPBS and 100 µL ATL-DX Buffer to a Screw Cap 1.5 mL MicroTube containing 0.1 mm beating beads.

SECTION: 4. MICROBE LYSIS
30. Load the tubes with blood meal and/or cotton tip or leech tissue lysate from step 13 and/or 14 and/or 27 into the fully cooled Bullet Blender (including samples and controls). Refill the dry ice compartment if necessary.
SECTION: 4. MICROBE LYSIS
31. Set the speed at 12 and time at 5 min. Press Start.

SECTION: 4. MICROBE LYSIS
32. Let the samples settle for 1 min  and then repeat step 31.
 



SECTION: 5. INSTRUMENT SET UP (KingFisher Flex only, if using KingFisher Duo Prime, go to step 9
33. Confirm 96 deep-well magnetic heads and 96 well deep-well heat blocks are being used.
SECTION: 5. INSTRUMENT SET UP (KingFisher Flex only, if using KingFisher Duo Prime, go to step 9
34. Ensure the program IndiMag_Pathogen_KF_Flex_4wash has been downloaded and loaded onto the KingFisher Flex instrument.
SECTION: 6. SET UP THE PROCESSING PLATES
35. 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: 7. EXTRACTION
36. Centrifuge the 1.5 mL tubes with lysate from step 32 for 12000 x g, 5 min. 

SECTION: 7. EXTRACTION
37. Add 20 µL of Proteinase K into wells (based on number of samples) of a new Deep-well plate.

SECTION: 7. EXTRACTION
38. Transfer 270 µL supernatant of step 36 without any particle carryover to the wells of the Deep-well plate containing proteinase K. This plate becomes the Sample Plate.

SECTION: 7. EXTRACTION
39. Add 135 µL Buffer VXL, 540 µLBuffer 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 (can be mixed on Hula mixer for 2 min). Add 700 µL mixture to each sample.

SECTION: 7. EXTRACTION
40. Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.
SECTION: 7. EXTRACTION
41. Start the run, then load the prepared plates into position when prompted by the instrument.
SECTION: 8. 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: 8. QUANTIFICATION AND STORAGE
43. 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.


SECTION: 8. QUANTIFICATION AND STORAGE
44. Proceed with sample testing following the REDI-NET SOP L-4 Leech Testing or store at -20 °C for less than 2 weeks.
 

SECTION: 9. INSTRUMENT SET UP (KingFisher Duo Prime only, if using KingFisher Flex, go to section 5)
45. Confirm 12-tip magnetic head and 12 well deep-well heat blocks are being used.
SECTION: 9. INSTRUMENT SET UP (KingFisher Duo Prime only, if using KingFisher Flex, go to section 5)
46. Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.
SECTION: 10. SET UP THE 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       F      Tip comb       700 µL               G         Empty               H   
 
SECTION: 10. SET UP THE 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: 11. EXTRACTION
49. Centrifuge the bead tubes with lysate from step 32 for 12000 x g, 5 min. 

SECTION: 11. EXTRACTION
50. Add 20 µL of Proteinase K into wells (based on number of samples) of a sample row.

SECTION: 11. EXTRACTION
51. Transfer 270 µL supernatant without any particle carryover to the wells of the sample row containing proteinase K. This plate becomes the Sample Plate.

SECTION: 11. EXTRACTION
52. 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. Add 695 µL mixture to each sample.

SECTION: 11. EXTRACTION
53. Select program IndiMag_Pathogen_KF_Duo_4wash on the instrument.
SECTION: 11. EXTRACTION
54. Start the run, then load the prepared plate/strip into position when prompted by the instrument.
SECTION: 12. QUANTIFICATION AND STORAGE
55. 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.
SECTION: 12. QUANTIFICATION AND STORAGE
56. Use 1 µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer.
 


SECTION: 12. QUANTIFICATION AND STORAGE
57. Proceed with sample testing following the REDI-NET SOP L-4 Leech Testing or store at -20 °C for less than 2 weeks. 
 

SECTION: 1. LEECH IDENTIFICATION
1.        
Leeches from each vial (either freshly from the field/animals or stored at –80°C) should be morphologically examined to identify its species. Only parasitic Euhirudinea species will be kept and other species will be discarded.
SECTION: 1. LEECH IDENTIFICATION
2. Leeches can be stored at -20℃ for up to 2 weeks and at -80℃ for up to 1 month. Long term storage at 4℃ is not recommended.
SECTION: 2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
7. Place the rinsed leech on Kimwipes to absorb the ethanol residuals. Place the leech in a petri dish on ice.
SECTION: 2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
6. Rinse leech with 70% ethanol.
SECTION: 2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
10. Transfer the blood meal to a 1.5 ml tube by pipetting.
SECTION: 2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
14.        
For processing bloodmeal, add 50 µL bloodmeal to a tube prepared in step 3.

 

SECTION: 2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
15.        
The cotton swabs and blood meal in lysis buffer can be stored at -20 °C for a few days before processing.

SECTION: 3. SMALL LEECH TISSUE HOMOGENIZATION
16. Clean forceps with 70% ethanol and Kimwipes before use and between samples.
SECTION: 3. SMALL LEECH TISSUE HOMOGENIZATION
19. Keep an empty Petri dish on ice. Cut the leech from head to tail, and trim the leech tissue into small pieces around 3mm x 3mm in the Petri dish.
SECTION: 3. SMALL LEECH TISSUE HOMOGENIZATION
20. Add one piece of 3mm x 3mm leech tissue into an orange RINO tube prepared in step 17.
SECTION: 3. SMALL LEECH TISSUE HOMOGENIZATION
21. Load the RINO tubes with leech tissue into the Bullet Blender. Add more dry ice into the cooling compartment of the Bullet Blender, if necessary.
SECTION: 3. SMALL LEECH TISSUE HOMOGENIZATION
23. Repeat step 22. 
SECTION: 3. SMALL LEECH TISSUE HOMOGENIZATION
27. Add 80 µL ATL-DTX buffer into the tube.

SECTION: 3. SMALL LEECH TISSUE HOMOGENIZATION
18. Use ice-cold1x PBS to wash leech three times sequentially in Petri dishes on ice to remove external contaminants.
SECTION: 3. SMALL LEECH TISSUE HOMOGENIZATION
17. Label orange RINO RNA lysis tubes on the cap, and add 60 µL cold, sterile 1x PBS into each orange RINO tube (avoid labeling on the side of the tubes due to potential damage during the beads beating process)

SECTION: 3. SMALL LEECH TISSUE HOMOGENIZATION
26. Without disturbing the tubes, carefully transfer the top 320 µL supernatant into the 1.5 ml tubes of Before You Start section step 3.