Swine vesicular disease virus one-step RT-LAMP

Health and Safety and Other Risks:

• Disposable gloves and a laboratory coat should be worn to avoid contact with skin and clothes when using TRIzol Reagent as it contains phenol. Tubes containing waste TRIzol Reagent must be placed into a suitable container for appropriate disposal.

• RNA extraction must be carried out in a location separate from areas where reverse transcription and PCR amplification are carried out to avoid contamination of stock reagents.

• Disposable gloves must be worn throughout the technique as RNases from the skin have an adverse affect on RNA. Precipitated extracted RNA pellets may be very small or invisible; care must be taken to avoid accidental discard. Extracted RNA should be stored at -90 to -50oC in a monitored freezer.

Health and Safety:

This protocol should be used in accordance with the current Health and Safety Policy and other relevant instructions as issued by the Institute for Animal Health. Due consideration must be given to National standards and regulations.

• Ensure any samples frozen and previously stored in TRIzol are thawed. Incubate for 5 minutes at approximately 22°C (room temperature).
• Label a fresh 2 ml skirted Sarstedt tube corresponding to each test sample to be processed; add 200 ?l of chloroform to each 2 ml tube followed by 1.0 ml of the TRIzol solution containing the sample.
• Vortex mix each tube for 10-15 seconds.
• Centrifuge the tubes for 15 minutes at 13,000 rpm at 2-8°C.
• Label a fresh 1.5 ml conical tube for each test sample and add 1.0 ?l of glycogen to each.
• Remove 500 ?l of the top phase (clear phase) from the centrifuged tubes (from stage 5.4) and add to the corresponding glycogen-containing tubes. Add 500 ?l of isopropyl alcohol to each tube.
• Vortex mix each tube for a few seconds.
• Incubate the tubes at approximately 4°C (on ice) for 10 minutes.
• Orientate the tubes in the centrifuge using the tube label so that the RNA pellet will be in an expected position. Centrifuge the tubes for 10 minutes at 13,000 rpm at 2-8°C.
• Discard the supernatant from the tubes and then add 1.0 ml of 70% ethanol to each one.
• Vortex mix the tubes for a few seconds.
• Orientate the tubes in the centrifuge as in stage 5.9 and centrifuge for 10 minutes at 13,000 rpm at 2-8°C.
• Remove as much supernatant as possible using gentle vacuum suction (or pipetting) without disturbing the RNA pellet. Air dry each tube for 2-3 minutes at approximately 22°C. Resolubilise each RNA pellet by adding 20 ?l of nuclease-free water to each tube and gentle mixing.
• Maintain the RNA samples on ice and immediately proceed with RT-PCR or store them at -90°C to -50°C until required.

Results will only become known after analysis of the subsequent reverse transcription and PCR amplification procedures.

If pipettes subsequently fail calibration checks but final results are satisfactory according to the controls, the Line Manager or Operator may still deem the results acceptable

The highly conserved 3D polymerase gene was chosen as target for the primer design. The RT-LAMP reaction is carried out in a 25 µl mixture. After production of the master mix, 23 µl portions are filled into each reaction tube and 2 µl template are added.

The volume of the master mix depends on the number of extracted samples plus the number of positive and negative controls (water). Recipe and a template to calculate final volume can be found in the adjoined protocol.

Before dividing up the master mix into portions of 23 µl per tube, the master mix must be homogenised thoroughly.

The RT-LAMP reaction is carried out in a 25 µl mixture containing 1 × Thermo buffer (New England Biolabs, Beverly, MA, USA), 0.54 × First strand buffer (Invitrogen, Carlsbad, CA, USA), 1.12 mM dNTPs (GE Healthcare, Uppsala, Sweden), 0.2 µM each of F3 and B3, 1.6 µM each of FIP and BIP, 0.8 µM each of Floop and Bloop, 0.8 µM betaine (Sigma), 4.5U cloned AMV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and 8U Bst DNA polymerase, Large Fragment (New England Biolabs).

2µl template RNA is added to each tube with 23µl master mix, except for the negative control for which water is added. After addition of template RNA, close the reaction tubes and run LAMP.

Run 5µl of the RT-LAMP product on a 1.5% agarose gel.

Digest 2µl product with BspHI (New England Biolabs) for 1 h at 37°C before analysis by gel-electrophoresis to confirm the result.

Add 0.5µl 10× Sybr-Green Gel Stain I 10,000× concentration (Molecular Probes, Leiden, the Netherlands) to 10µl RT-LAMP product to visualised the result. After a short vortex a colour change can be seen if the sample is positive.