Jun 29, 2023
TELEvir Field Protocol
- 1Statens Serum Institut;
- 2University of Copenhagen
Protocol Citation: Anna S Fomsgaard 2023. TELEvir Field Protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj694xlk5/v1
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: April 06, 2022
Last Modified: June 29, 2023
Protocol Integer ID: 60387
Funders Acknowledgement:
Horizon 2020
Grant ID: 773830
Disclaimer
This protocol was prepared for a workshop held the 23-24 June 2022 at Statens Serum Institut, Denmark. Here 25 TELEVIR partners from 10 European countries participated to train in the usage of a field-deployable point-of-incidence toolbox to identify emgerging virus threats. The protocol was used to detec SARS-CoV-2 and Human Papillomavirus a RNA and DNA virus detection test-of-concept.
Abstract
The TELE-Vir project key aim is to develop a fast point-of-incidence (poi) toolbox for identification and characterisation of emerging virus threats for humans and/or domestic and wildlife animals.
Presented here is a protocol including sample pretreatment, NA extraction and random amplification for metagenomic virus detection using the MinION device (Oxford Nanopore Technologies)
Protocol materials
DNase Set IZymo ResearchCatalog #E1010
Step 1
MagNA Pure LC Total Nucleic Acid Isolation KitRocheCatalog #03038505001
In 2 steps
REPLI-g Cell WGA & WTAQiagenCatalog #150052
Step 30
Rapid Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-RAD004
Step 37
Pretreatment
Pretreatment
15m
DNase Set IImercoCatalog #E1010
Equipment
BD Disposable Syringe with Luer-Lok Tip (5ml)
NAME
Syringe
TYPE
Becton Dickinson
BRAND
309649
SKU
Equipment
Syringe Filter pore size 0.2 μm
NAME
Filter
TYPE
Minisart
BRAND
16532
SKU
LINK
Polyethersulfone (PES), Pore Size 0.22 µm, Ethylene Oxide, Female Luer Lock, Male Luer Lock, Pack Size 50
SPECIFICATIONS
400 µL Sample
50 µL DNase I
50 µL DNase Digestion buffer
00:15:00 at room temperature
Note
If 400 µL sample is not available, downscale the reagents to fit the lower sample volume.
15m
Add 500 µL PBS (or equivalent to 1 ml sample material in total)
Poor the diluted sample into the lid of the Syringe Filter (Minsart). The filter can be placed on a sterile surface meanwhile e.g. the Syringe Filter paper lid (inner side).
Suck in air corresponding to app 1 ml in a 5 ml syringe
Extract the 1 ml sample material from the petri dish to the 5 ml syringe
Attach the 0,22 µM Minisart syringe filter to the 5 ml syringe with Luer-Lok
Filter the sample and air directly into a 4.5 ml cryotube containing 1 ml MPLB-buffer. Put lid on, mix by turning tube.
MagNA Pure LC Total Nucleic Acid Isolation KitImercoCatalog #03038505001
Inactivation
Inactivation
20m
Incubate
00:20:00 at room temperature for viral inactivation
CITATION
CITATION
20m
Extraction
Extraction
10m
Kit used for hand held NA extraction:
MagNA Pure LC Total Nucleic Acid Isolation KitImercoCatalog #03038505001
Equipment
4.5 ml cryotube
NAME
tube
TYPE
Thermo Fisher Scientific
BRAND
363452
SKU
LINK
Equipment
3.6 ml cryotube
NAME
Tube
TYPE
Thermo Fisher Scientific
BRAND
379189
SKU
LINK
Cylinder magnet
A rubber band to attach the magnet to a tube.
A 50 ml Nunc tube or similar to pour excess solutions into for disposing.
Following the protocol by:
CITATION
Per X number of samples, aliquot in cryotubes or Eppendorf tubes
960 µL Magnetic Glass Particles (MGPs) in an Eppendorf tube
4 mL Wash buffer I in a 4.5 ml cryotube
1.5 mL Wash buffer II in an Eppendorf tube
3 mL Wash buffer III in a 3.6 ml cryotube
100 µL Elution buffer in an Eppendorf tube
Prepare one sample at the time.
Approximate time 00:10:00 per sample
10m
Add 960 µl MGPs to 2 ml solution of sample and MPLB-buffer
Put lid on and turn tube in hand gently until well mixed
Attach magnet and trap beads.
Pour solution into trash tube. The MGPs should stay behind trapped on the side of the tube.
Pour Wash buffer I in the sample tube
Put a lid on, remove magnet and turn the tube in hand gently until well mixed
Attach magnet and trap beads.
Pour solution into trash tube. The MGPs should stay behind trapped on the side of the tube.
Pour Wash buffer II in the sample tube
Put a lid on, remove magnet and turn the tube in hand gently until well mixed
Attach magnet and trap beads.
Pour solution into trash tube. The MGPs should stay behind trapped on the side of the tube.
Pour Wash buffer III in the sample tube
Put a lid on, remove magnet and turn the tube in hand gently until well mixed
Attach magnet and trap beads.
Pour solution into trash tube. The MGPs should stay behind trapped on the side of the tube.
Pour Elution buffer in the sample tube
Put a lid on, remove magnet and turn the gently until beads are eluted.
Transfer 10 µL Elution Buffer with MGPs to 2 x 0.2 ml PCR tube
Whole Transcriptome and Genome Amplification
Whole Transcriptome and Genome Amplification
4h
Equipment to enable portability
- A MiniPCR to run isothermal incubations
- A powerbank that can provide 20V. Use one to power the miniPCR and another to power the MinION for approximately 5 hours. Use brand of choice.
- A salad swing as a hand driven centrifuge. Use brand of choice.
- A coolbox for reagents and cooling samples. Use brand of choice.
Equipment
All-in1 Laptop Powerbank 24000
NAME
Power Bank
TYPE
Sanberg
BRAND
420-57
SKU
LINK
Any powerbank that can produce 20V is acceptable. Have 2-3 to run the MiniPCR and the MinION
SPECIFICATIONS
Equipment
miniPCR® mini8 thermal cycler
NAME
Thermal cycler
TYPE
miniPCR®
BRAND
mini8
SKU
LINK
Equipment
A Salad Spinner Centrifuge
NAME
Centrifuge
TYPE
undefined
BRAND
undefined
SKU
LINK
Kit used for WTA and WGA
REPLI-g Cell WGA & WTAImercoCatalog #150052
Quantiscript RT Enzyme Mix, Ligase Mix, and REPLI-g SensiPhi DNA Polymerase should be thawed on ice.
All other components can be thawed at room temperature (15–25°C)
The two aliquots of same sample enables uniform whole genome amplification (WGA) and whole transcriptome amplification (WTA) in parallel reactions.
Cleanup for WTA
Add 2 µL gDNA Wipeout Buffer to PCR tube with 10 µl eluted sample material
00:10:00 42 °C on MiniPCR
10m
Repair (WGA) and Reverse Transcription (WTA)
For WGA, mix following and add 10 µL Repair reagent mix to sample
| Reagents | Volume (µL) per sample | |
| RT/Polymerase Buffer | 4 | |
| gDNA Wipeout buffer | 2 | |
| H2O sc | 1 | |
| Random primer | 1 | |
| Random Hex-P primer (20µM) | 1 | |
| WGA Ready Enzym | 1 | |
| Total | 10 |
This modified protocol uses 5'-phosphorylated random hexamers (P-N6) instead of kit provided oligo-dT primers according to Rosenstierne et al (2014).
CITATION
For WTA, mix following and add 8 µL RT reagent mix to sample
| Reagents | Volume (µL) per sample | |
| RT/Polymerase Buffer | 4 | |
| H2O sc | 1 | |
| Random primer | 1 | |
| Random Hex-P primer (20µM) | 1 | |
| Quantiscript RT Enzym Mix | 1 | |
| Total | 8 |
Incubate
01:00:00 42 °C On MiniPCR
Afterwards immediatly
00:03:00 95 °C on MiniPCR
1h 3m
Ligation
Mix following and add 10 µL Ligation mix to each tube
| Reagents | Volume (µL) per sample | |
| Ligation buffer | 8 | |
| Ligase Mix | 2 | |
| Total | 10 |
Incubate
00:30:00 24 °C This is the Repli-G recommended temperature, however, the miniPCR only goes to 25C. Instead of incubation in the MiniPCR, try table-top incubation.
00:05:00 95 °C
Hold at 4 °C
35m
Amplification
Mix following and add 30 µL amplification mix to each tube
| Reagents | Volume (μL) per sample | |
| Repli-g reaction buffer | 29 | |
| Repli-g SensiPhi DNA polymerase | 1 | |
| Total | 30 |
Incubate
02:00:00 30 °C
00:05:00 65 °C
Hold at 4 °C
2h 5m
For library preparation use amplified material without MGPs.
Library Preparation
Library Preparation
Multiple rapid library preparation kits are available through Oxford Nanopore Technology.
For Workshop we use.
Rapid Sequencing KitImercoCatalog #SQK-RAD004
Equipment
MinION Mk1C
NAME
Sequencer
TYPE
Oxford Nanopore Technology
BRAND
MIN-101C
SKU
LINK
Citations
Step 33.1
Rosenstierne MW, McLoughlin KS, Olesen ML, Papa A, Gardner SN, Engler O, Plumet S, Mirazimi A, Weidmann M, Niedrig M, Fomsgaard A, Erlandsson L. The microbial detection array for detection of emerging viruses in clinical samples--a useful panmicrobial diagnostic tool.
https://doi.org/10.1371/journal.pone.0100813Step 8
Vinner L, Fomsgaard A. Inactivation of orthopoxvirus for diagnostic PCR analysis.
Step 8
Rosenstierne MW, Jensen CE, Fomsgaard A. Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples.
https://doi.org/10.3791/58001Step 9
Rosenstierne MW, Jensen CE, Fomsgaard A. Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples.
https://doi.org/10.3791/58001