Dec 16, 2024
Version 2
REDI-NET L-2 LEECH PROCESSING V.2
- REDI-NET Consortium1
- 1REDI-NET Consortium
- Remote Emerging Disease Intelligence - NETwork (REDI-NET)
Protocol Citation: REDI-NET Consortium 2024. REDI-NET L-2 LEECH PROCESSING. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkop56v5r/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: January 11, 2024
Last Modified: December 16, 2024
Protocol Integer ID: 110529
Keywords: Leech TNA extraction, KingFisher DNA/RNA extraction, Pathogen nucleic acid extration, leech, total nucleic acid extraction, leech identification
Funders Acknowledgements:
USAMRAA
Grant ID: W81XWH-21-C-0001
USAMRAA
Grant ID: W81XWH-22-C-0093
USAMRAA
Grant ID: HT9425-23-C-0059
USAMRAA
Grant ID: HT9425-24-C-0072
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, HT9425-23-C-0059, and HT9425-24-C-0072. 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 leech processing for total nucleic acid extraction.
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
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.
- Precipitate 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. 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
Expected result
| A | B | C | D | E | F | |
| Sample | Amount | Sample condition | Elution volume | DNA conc. (ng/ul) | RNA conc. (ng/ul) | |
| Tick | 1 unfed adult or 10 nymphs or 60 larvae | 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 |
APPENDIX 4. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
DNA quantification:
According to the volume of sample used, add the 1xHS dsDNA QubitAssay for a final volume of 200 µL (i.e., if using 1 µL of sample, add 199 µL of 1x HS dsDNA Qubit Assay
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:
- In a new 0.6 mL tube, mix 199 µL of Qubit HS RNA Assay working solution and 1 µL of the sample. Incubate for 1 minute at room temperature before reading.
| 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 |
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 ● KingFisher™ Duo Prime Magnetic Particle Processor | ThermoFisher, 5400630 (Flex) or ThermoFisher, 5400110 (Duo Prime) | |
| Bullet Blender 24 Gold | Next Advance, BB24-AU | |
| Adjustable micropipettes | Locally sourced | |
| Multi-channel micropipettes | Locally sourced | |
| Vortex | Locally sourced | |
| Tube centrifuge | Locally sourced | |
| Plate centrifuge | Locally sourced | |
| Qubit 4 Fluorometer | ThermoFisher, Q33238 | |
| Thermo Heater Mixer | Locally sourced |
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™ 4 Fluorometer, with WiFi
NAME
Fluorometer
TYPE
Invitrogen
BRAND
Q33238
SKU
LINK
Equipment
KingFisher™ Duo Prime Purification System
NAME
Purification System
TYPE
Thermo Scientific™
BRAND
5400110
SKU
LINK
| A | B | C | |
| Material | Description | Mfg / Product # | |
| ZymoBIOMICS Microbial Community Standard Material | For TNA extraction positive control | Zymo Research, D6300 | |
| Dengue virus type 1 (DENV-1) positive control | For TNA extraction positive control; BSL-2 | ATCC, VR-1856 or locally sourced | |
| Human gammaherpesvirus (EBV) positive control | For TNA extraction positive control | Naval Medical Research Center | |
| IndiMag Pathogen Kit | w/o plastics, 384 reactions | Indical Bioscience, SP947257 | |
| Buffer ATL | 200 mL, Tissue Lysis Buffer | Qiagen, 19076 | |
| Reagent DX | 1 mL, Antifoaming Reagent | Qiagen, 19088 | |
| Measuring Spoon 100 µL | RNase Free, pack of 10, reusable | Next Advance, MSP01-RNA | |
| Navy RINO RNA lysis kit | Bead lysis kits | Next Advance, NAVYR5-RNA | |
| 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 oxidase beads | 0.1 mm, 400 g | Fisher Scientific, 50-154-2950 | |
| KingFisher™ Deepwell 96 Plate | KingFisher | ThermoFisher, 95040450 | |
| 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 | |
| BRAND Self-adhesive Plate Sealing Film | Aluminum (consumable) | Fisher Scientific, 13-882-329 or equivalent | |
| MicroAmp™ Clear Adhesive Film | KingFisher | ThermoFisher, 4306311 | |
| Nonstick, RNase-Free Microfuge Tubes | 1.5 mL | ThermoFisher, AM12450 | |
| Nonstick, RNase-Free Microfuge Tubes | 2.0 mL | ThermoFisher, AM12475 | |
| Qubit assays tubes | For Qubit™ DNA/RNA measuring (consumable) | Thermo Fisher, Q32856 | |
| RNaseZap™ RNase Decontamination Solution | To remove RNase from the working area | ThermoFisher, AM9780 | |
| Qubit™ 1X dsDNA HS Assay Kit | (consumable) | ThermoFisher, Q33230 | |
| Qubit™ RNA HS Assay Kit | (consumable) | ThermoFisher, Q32852 | |
| Ethanol | 100% (molecular biology grade) | Locally sourced | |
| Isopropanol | 100% (molecular biology grade) | Locally sourced | |
| Nuclease-free Water | For negative control | Locally sourced | |
| Dry ice | To maintain cold chain during sample handling using Bullet Blender | Locally sourced | |
| Ice bucket and ice | To keep sample cold | Locally sourced | |
| Kimwipes | To dry material | Locally sourced | |
| Falcon tubes | 15 mL and 50 mL | Locally sourced | |
| Cotton swabs | 6 inches, sterile, plastic handle (consumable) | Fisher Scientific, 22-363-163 or equivalent | |
| Scalpels | Size 11 (consumable) | Fisher Scientific, 14-840-01 or equivalent | |
| Forceps | Straight and curved, fine point, Stainless steel, sterile | BioQuip, #4531, #4532 | |
| Forceps | Straight, fine point | Bioquip, #4730 | |
| Office scissors | 8-10 inches, strong enough to cut the cotton swab handle | Locally sourced | |
| Sterile 1x PBS | To clean leech sample | Locally sourced | |
| Sterile petri dishes | 100 mm diameter | Fisher Scientific, FB0875712 or equivalent |
ZymoBIOMICS Microbial Community StandardZymo ResearchCatalog #D6300
IndiMag Pathogen Kit w/o plastics (384 reactions)INDICAL BIOSCIENCECatalog #SP947257
Buffer AL, Lysis bufferQiagenCatalog #19076
Navy RINO RNA Lysis Kit 250 pack (1.5 mL)Next AdvanceCatalog #NAVYR5-RNA
Reagent DXQiagenCatalog #19088
Measuring Spoon 100 uL RNase Free pack of 10Next AdvanceCatalog #MSP01-RNA
Sterile Microcentrifuge Tube 1.5 mL (RINO®) 500/caseNext AdvanceCatalog #TUBE1R5-S
Bertin Corp 0.1mm Zirconium oxide beads (450g) (qty 500)Fisher ScientificCatalog #50-154-2950
KingFisher™ Plastics for 96 deep-well formatThermo Fisher ScientificCatalog #95040450
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
BRAND™ Self-adhesive Plate Sealing FilmFisher ScientificCatalog #13-882-329
MicroAmp Clear Adhesive FilmApplied Biosystems (ThermoFisher Scientific)Catalog #4306311
Nonstick, RNase-free Microfuge Tubes, 1.5 mLThermo FisherCatalog #AM12450
Nonstick, RNase-free Microfuge Tubes, 2.0 mLThermo FisherCatalog #AM12475
Qubit assay tubesThermo Fisher ScientificCatalog #Q32856
RNaseZap™ RNase Decontamination SolutionThermo Fisher ScientificCatalog #AM9780
Qubit 1X dsDNA High Sensitivity Assay KitThermo Fisher ScientificCatalog #Q33230
Qubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852
Safety warnings
RISKS 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, please, try to avoid contact with skin while preparing workbench for nucleic acid extraction.
Before start
Note
To prevent contamination samples nucleic acid extraction and amplification (PCR) should be performed in separate rooms.
- 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 Temperature 56 °C until the precipitates have fully dissolved. Prepare buffer ATL-DX: add Amount 100 µL Reagent DX to Amount 15 mL Buffer ATL. If smaller amounts are needed, transfer Amount 1.5 mL of Buffer ATL into a sterile 2 mL vial and add Amount 10 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 00:03:00 (before first use) or 00:01:00 (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended.
- When processing viable leeches, freeze at -20 °C for 01:00:00 or in dry ice for 00:20:00 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.
1. LEECH IDENTIFICATION
1. LEECH IDENTIFICATION
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 °C for up to 2 weeks and at -80 °C for up to 1 month. Long term storage at 4 °C is not recommended.
2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
2. BIG LEECH BLOOD MEAL COLLECTION AND INNER ORGAN
Note
If leeches are about a pinky fingernail size, homogenize leech tissues as section 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 Start" section.
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, add 50 µL bloodmeal to a tube prepared in step 3.
Note
For those leeches without blood meal, only prepare the cotton swab. See Appendix 1 for the photos of blood meal and cotton swab collected from leeches.
The cotton swabs and blood meal in lysis buffer can be stored at -20 °C for a few days before processing.
3. SMALL LEECH TISSUE HOMOGENIZATION
3. SMALL LEECH TISSUE HOMOGENIZATION
Clean forceps with 70% ethanol and Kimwipes before use and between samples.
Label navy RINO RNA lysis tubes on the cap, and add 60 µL cold, sterile 1x PBS into each navy 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 3 mm x 3 mm in the Petri dish.
Add one piece of 3 mm x 3 mm leech tissue into a navy 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 00:03:00 . Press Start.
3m
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 00:03:00 .
3m
Centrifuge the suspension at 100 x g, 00:01:00 to pellet debris.
1m
Without disturbing the tubes, carefully transfer the top 320 µL supernatant into the 1.5 mL tubes of "Before Start" section step 3.
Add 80 µL ATL-DTX buffer into the tube.
4. SAMPLE LYSIS
4. SAMPLE LYSIS
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 DENV-1 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.
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.
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 00:05:00 . Press Start.
5m
Let the samples settle for 00:01:00 and then repeat step 31.
1m
After beating, centrifuge the tubes with lysate for 12000 x g, 4°C, 00:05:00
Note
STOPPING POINT: lysed samples can be stored at 4 °C Overnight .
5m
5. INSTRUMENT SET UP (KingFisher Flex only, if using KingFisher Duo Prime, go to step 9
5. INSTRUMENT SET UP (KingFisher Flex only, if using KingFisher Duo Prime, go to step 9
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.
6. SET UP THE PROCESSING PLATES
6. SET UP THE PROCESSING PLATES
Set up the Wash, Elution, and Tip Comb 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 | 990 µL | |
7. EXTRACTION
7. EXTRACTION
Add 20 µL of Proteinase K into wells (based on number of samples) of a new deep-well plate.
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.
Add 135 µL Buffer VXL, 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 (can be mixed on Hula mixer for 2 min). Add 700 µL 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.
8. QUANTIFICATION AND STORAGE
8. 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.
Proceed with sample testing following the REDI-NET SOP L-4 Leech 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 L-3 Leech Storage.
9. INSTRUMENT SET UP (KingFisher Duo Prime only, if using KingFisher Flex, go to section 5)
9. INSTRUMENT SET UP (KingFisher Duo Prime only, if using KingFisher Flex, go to section 5)
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.
10. SET UP THE SAMPLE PLATE AND ELUTION STRIP
10. 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 | |
| Tip Comb | 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 | |
| Row ID | Plate Row | Reagent | Volume per well | |
| Elution | A | Nuclease-free water | 75 µL |
11. EXTRACTION
11. EXTRACTION
Add 20 µL of Proteinase K into wells (based on number of samples) of a sample row.
Transfer 270 µL supernatant without any particle carryover to the wells of the sample row 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 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.
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.
12. QUANTIFICATION AND STORAGE
12. QUANTIFICATION AND STORAGE
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 1 µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer.
Note
Kits needed: Qubit 1X dsDNA HS Assay Kit and Qubit RNA HS Assay Kit (see Appendix 4).
Proceed with sample testing following the REDI-NET SOP L-4 Leech 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 L-3 Leech Storage.
Protocol references
REFERENCES
1. Andrade Justi, S., Soghigian, J., Pecor, D. B., Caicedo-Quiroga, L., Rutvisuttinunt, W., Li, T., ... & Linton, Y. M. (2021). From e-voucher to genomic data: Preservingarchive specimens as demonstrated with medically important mosquitoes (Diptera:Culicidae) and kissing bugs (Hemiptera: Reduviidae). Plos one, 16(2), e0247068.
2. Hall RJ, Wang J, Todd AK, Bissielo AB, Yen S, Strydom H, Moore NE, Ren X, Huang QS, Carter PE, Peacey M. Evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery. J Virol Methods. 2014 Jan;195:194-204. doi: 10.1016/j.jviromet.2013.08.035. Epub 2013 Sep 13. PMID: 24036074; PMCID: PMC7113663.
3. Temmam S, Monteil-Bouchard S, Robert C, Pascalis H, Michelle C, Jardot P, Charrel R, Raoult D, Desnues C. Host-Associated Metagenomics: A Guide to Generating Infectious RNA Viromes. PLoS One. 2015 Oct 2;10(10):e0139810. doi: 10.1371/journal.pone.0139810. PMID: 26431175; PMCID: PMC4592258.
4. User Guide: MagMAX Microbiome Ultra Nucleic Acid Isolation kit, Applied Biosystems, Pub. No. MAN0018070 Rev. C.0.
5. User Guide: Indical IndiMag Pathogen Kit user’s manual.