Isolation of the viral RNA

Starting material for the isolation and detection of the viral genomic material was the bee homogenate dissolved in DEPC-treated water. To remove unsoluble material (like the chitin shell, legs, wings…) the homogenate was centrifuged. For the analysis we needed just 140 l of the cleared supernatant.

As a first step we had to isolate the viral RNA which is later transcribed into cDNA (complementary DNA) since only DNA can be used as template for the polymerase chain reaction. For the isolation procedure we used the QIAmp Viral RNA Mini Kit which contains a special reaction tube with a column made of a material that binds RNA but not proteins, DNA or lipids which would just interfere with our subsequent reactions.

First we added a lysis buffer (AVL), which breaks up the cells and thus releases the genomic material. The RNA was bound to the column and after some washing and purification steps it could be isolated.

Working procedure:

1. Mix 560 µl AVL buffer with 140 µl of bee homogenate

2. Incubation at room temperature for 10 minutes
This step opens the cells and releases the genomic material

3. Addition of 560 µl ethanol
The mixture with ethanol is essential for efficient binding of the RNA to the column

4. Loading of the column with 630 µl of the mixture and centrifugation at 8000 rpm (rotations per minute) for 1 minute

5. Removal of the liquid in the collection tube, loading of the column with the residual homogenate, centrifugation
The RNA is now bound on the column

6. Addition of 500 µl AW1 (wash buffer) and centrifugation for 1 minute at 8000 rpm

7. Change the collection tubes, addition of 500 µl AW2, centrifugation for 3 minutes at 14000 rpm
The subsequent use of both washing buffers ensure proper purification of the RNA

8. Removal of the old collection tube. Use of a new sterile Rnase-free collection tube. Isolation of the RNA by addition of 60 µl elution buffer (AVE) and inkubieren

9. centrifugation at room temperature for 1 minute.

The elution buffer destroys the bonds between column and RNA. The centrifugation step leads to elution of the RNA from the column. The isolated RNA is stored at -20°C.

Reverse transcription and PCR

The detection of the viral RNA was performed by using the QIAgen One Step RT-PCR Kit. First, the RNA is transcribed into cDNA (reverse transcription). In a second step, the cDNA is specifically amplified (PCR).

PCR program:

A)Reverse transcription for 30 minutes at 50°C

B)Activation of the PCR

by heating for 15 minutes at 95°C (This step leads to inactivation of the reverse transcriptase and activation of the DNA polymerase)

C)PCR

40x denaturation 30 s, 95°C
Annealing 50s, 55°C (DWV and APV);
53°C (IAPV)
Extension 1min, 72°C
1x Final extension 7min, 72°C

DNA gel and documentation:

Preparation of a 1,2% agarose gel: 1,2g of agarose is mixed with 100ml of 0,5x TBE (tris-boric acid-EDTA-buffer) and dissolved by boiling in a microwave. To stain the DNA during the run 2 µl of Gelstar (SYBR GREEN) are added.
After pouring the gel in a suitable tray and cooling for about one hour, the combs are removed, the gel placed in gel chamber, covered with 0,5x TBE buffer and the samples are loaded. The gel run was performed at 150 volts for about 45 minutes.

Preparation of the samples:
After the RT-PCR run 5 µl of loading buffer were added to each sample. This buffer contains glycerol to ensure that the samples stay in the gel slots during loading