L-2 LEECH PROCESSING

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.

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.

Add 400µL 1x PBS and 100µL 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.

Clean forceps with 70% ethanol and Kimwipes before use and between samples.

Use ice-cold 1x PBS to wash leech three times sequentially in petri dishes on ice to remove external contaminants.

Rinse leech with 70% ethanol.

Place the rinsed leech on Kimwipes to absorb the ethanol residuals. Place the leech in a petri dish on ice.

Use a pair of scissors to cut across the leech in the middle.

Drip the blood meal into the petri dish as much as possible.

Transfer the blood meal to a 1.5 ml tube by pipetting.

Place the leech back into the petri dish, use a scalpel to cut open the entire leech longitudinally.

Open the leech with forceps, use a sterile cotton swab to wipe the opened inner organs thoroughly.

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.

For processing bloodmeal, add50µL bloodmeal to a tube prepared in step 3.

The cotton swabs and blood meal in lysis buffer can be stored at-20°C for a few days before processing.

Clean forceps with 70% ethanol and Kimwipes before use and between samples.

Label orange RINO RNA lysis tubes on the cap, and add 60µL 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)

Use ice-cold1x PBS to wash leech three times sequentially in Petri dishes on ice to remove external contaminants.

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.

Add one piece of 3mm x 3mm leech tissue into an orange RINO tube prepared in step 17.

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.

Set the controls for Speed 10 and Time 0h 3m 0s. Press Start.

Repeat step 22.

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 0h 3m 0s.

Centrifuge the suspension at 100x g to pellet debris.

Without disturbing the tubes, carefully transfer the top 320µL supernatant into the 1.5 ml tubes of Before You Start section step 3.

Add 80µL ATL-DTX buffer into the tube.

Include a positive control for each batch of samples: transfer 37.5µL ZymoBIOMICS Microbial Community Standard Material and 100µL EBV, and 100µL HIV standard into a Screw Cap 1.5 mL MicroTube containing 0.1 mm beating beads. Add 162.5µL 1x PBS and 100µL ATL-DX buffer.

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

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.

Set the speed at 12 and time at 0h 5m 0s. Press Start.

Let the samples settle for 0h 1m 0s and then repeat step 31.

Confirm 96 deep-well magnetic heads and 96 well deep-well heat blocks are being used.

Ensure the program IndiMag_Pathogen_KF_Flex_4wash has been downloaded and loaded onto the KingFisher Flex instrument.

Set up the Wash, Elution, and Tip Comb Plates outside the instrument according to the following table:

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	990 µL

Centrifuge the 1.5 mL tubes with lysate from step 32 for 12000x g.

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

Transfer 270µL 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.

Add 135µL Buffer VXL, 540µLBuffer ACB, and 25µL 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 700µL mixture to each sample.

Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.

Start the run, then load the prepared plates into position when prompted by the instrument.

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 1µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions.

Proceed with sample testing following the REDI-NET SOP L-4 Leech Testing or store at -20°C for less than 2 weeks.

Confirm 12-tip magnetic head and 12 well deep-well heat blocks are being used.

Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.

Set up the Sample Plate according to the table below:

A	B	C	D
Row ID	Plate Row	Reagent	Volume per well
Sample row	A	Lysate and lysis buffer	985 µL
Wash 1	B	Buffer AW1	700 µL
Wash 2	C	Buffer AW2	700 µL
Wash 3	D	80 % ethanol	750 µL
Wash 4	E	100 % ethanol	750 µL
Tip Comb	F	Tip comb
	G	Empty
	H

Set up the Elution Strip according to the table below:

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

Centrifuge the bead tubes with lysate from step 32 for 12000x g.

Add 20µL of Proteinase K into wells (based on number of samples) of a sample row.

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

Add 135µL Buffer VXL, 540µL Buffer ACB, and 20µL 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 695µL mixture to each sample.

Select program IndiMag_Pathogen_KF_Duo_4wash on the instrument.

Start the run, then load the prepared plate/strip into position when prompted by the instrument.

After the protocol is completed (~35 minutes), immediately remove the elution strip from the instrument and transfer the eluate to the final tube or plate of choice for final storage.

Use 1µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer.

Proceed with sample testing following the REDI-NET SOP L-4 Leech Testing or store at -20°C for less than 2 weeks.