TELEvir Field Protocol

Equipment

Value	Label
BD Disposable Syringe with Luer-Lok Tip (5ml)	NAME
Syringe	TYPE
Becton Dickinson	BRAND
309649	SKU
https://www.bd.com/en-us	LINK

Equipment

Value	Label
Syringe Filter pore size 0.2 μm	NAME
Filter	TYPE
Minisart	BRAND
16532	SKU
Polyethersulfone (PES), Pore Size 0.22 µm, Ethylene Oxide, Female Luer Lock, Male Luer Lock, Pack Size 50	SPECIFICATIONS

400µL

50µL

50µL

0h 15m 0s

Note

If 400 µL sample is not available, downscale the reagents to fit the lower sample volume.

Add 500µL (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.

Incubate

0h 20m 0s for viral inactivation

Kit used for hand held NA extraction:

Equipment

Value	Label
4.5 ml cryotube	NAME
tube	TYPE
Thermo Fisher Scientific	BRAND
363452	SKU

Equipment

Value	Label
3.6 ml cryotube	NAME
Tube	TYPE
Thermo Fisher Scientific	BRAND
379189	SKU

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

Rosenstierne MW, Jensen CE, Fomsgaard A 2018 Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples. Journal of visualized experiments : JoVE https://doi.org/10.3791/58001

Per X number of samples, aliquot in cryotubes or Eppendorf tubes

960µL Magnetic Glass Particles (MGPs) in an Eppendorf tube4mL Wash buffer I in a 4.5 ml cryotub1.5mL

Wash buffer II in an Eppendorf tub3mL

Wash buffer III in a 3.6 ml cryotu100µLe

Elution buffer in an Eppendorf tube

Prepare one sample at the time.

Approximate time 0h 10m 0s

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 10µL to a 0.2 ml PCR tube.

Transfer 10µL to 2 x 0.2 ml PCR tube

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

Value	Label
All-in1 Laptop Powerbank 24000	NAME
Power Bank	TYPE
Sanberg	BRAND
420-57	SKU
Any powerbank that can produce 20V is acceptable. Have 2-3 to run the MiniPCR and the MinION	SPECIFICATIONS

Equipment

Value	Label
miniPCR® mini8 thermal cycler	NAME
Thermal cycler	TYPE
miniPCR®	BRAND
mini8	SKU

Equipment

Value	Label
A Salad Spinner Centrifuge	NAME
Centrifuge	TYPE
undefined	BRAND
undefined	SKU

Kit used for WTA and WGA

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 2µL to PCR tube with 10 µl eluted sample material

0h 10m 0s 42°C on MiniPCR

Repair (WGA) and Reverse Transcription (WTA)

For WGA , mix following and add 10µL to sample

A	B
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).

For WTA , mix following and add 8µL to sample

A	B
RT/Polymerase Buffer	4
H2O sc	1
Random primer	1
Random Hex-P primer (20µM)	1
Quantiscript RT Enzym Mix	1
Total	8

Incubate

1h 0m 0s 42°C On MiniPCR

Afterwards immediatly

0h 3m 0s 95°C on MiniPCR

Ligation

Mix following and add 10µL to each tube

A	B
Ligation buffer	8
Ligase Mix	2
Total	10

Incubate

0h 30m 0s 24°C

0h 5m 0s 95°C

Hold at 4°C

Amplification

Mix following and add 30µL to each tube

A	B
Repli-g reaction buffer	29
Repli-g SensiPhi DNA polymerase	1
Total	30

Incubate

2h 0m 0s 30°C

0h 5m 0s 65°C

Hold at 4°C

For library preparation use amplified material without MGPs.

Multiple rapid library preparation kits are available through Oxford Nanopore Technology.

For Workshop we use.

Equipment

Value	Label
MinION Mk1C	NAME
Sequencer	TYPE
Oxford Nanopore Technology	BRAND
MIN-101C	SKU