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on using the reagents set of the test system for Ebola virus RNA detection by reverse transcription and polymerase chain reaction

“Ebola”

(A test-system for detection the virus Ebola by reverse transcription-polymerase chain reaction)

 

The current instruction describes a procedure of using the test system reagent set for detection of Ebola virus by reverse transcription and polymerase chain reaction.

The complete test system kit includes four sets of reagents:

– lysis solution – 1 flask, 25 ml;

– wash solution № 1 – 1 flask, 40 ml;

– wash solution № 2 – 1 flask, 30 ml;

– wash solution № 3 – 1 flask, 20 ml;

– adsorbent – 1 tube, 1.25 ml;

– DEPC-Н₂О – 4 tubes, 0.5 ml each.

–  reverse transcription mix (RT-mix) – 55 tubes, 10 mg each;

–  RT-mix solvent (RT-solvent)  – 1 tube, 0,3 ml.

– PCR mix № 1 – 1 bag, 55 tubes with 5 μl each, under wax layer;

– PCR mix № 2 – 1 tube, 0,6 ml;

– positive control sample (PCS), cDNA of Ebola virus - 1 tube, 0,1 ml;

– negative control sample (NCS), deionized water - 1 tube, 0.1 mg;

– PCR oil – 1 flask, 3 ml.

– TBE buffer – 2 flasks, 10 g each;

– agarose – 2 flasks, 2 g each;

– Ethidium bromide – 1 tube, 0.2 ml.

The specific components of the test system are: an equimolar primers mix being part of PCR mix № 1 (the primer mix is a water solution of chemically synthesized oligonucleotides), a positive control sample (PCS), cDNA of Ebola virus – a recombinant plasmid рGlow/Ebola_214, containing a DNA copy of Ebola virus genome.

The kit is intended for use with 55 samples, including control samples.

 

For sample preparation (RNA isolation) set # 1 is used.

Only liquid samples are used in the analysis. The sample with organ structure should be homogenized in a mortar prior to the analysis to get a 10% suspension in normal saline. When taking blood samples for the PCR analysis heparin should not be used. To prevent coagulation, 1/10 part (by volume) of 0.5 M EDTA solution is added to the blood. The serum collected above the erythrocyte clot is used for analysis. If is it not possible to separate serum for the analysis a hemolysed blood can be used.

Prior to preparing the samples, a necessary number of 0.5 ml Eppendorf tubes is taken and labeled. 100 μl of liquid sample containing the material to be analyzed is put into every tube. Then set # 1 is used in the following order:

- Add 450 μl of lysis solution into each tube;

- Vortex the adsorbent for 10 sec and add 20 μl (one drop) into each tube;

- Vortex the contents of each tube for 14 sec;

- Keep the tubes with samples at room temperature for 10 minutes, periodically mixing at Vortex;

- Spin the tubes in a microcentrifuge for 30 sec at 10000 rpm and carefully remove the supernatant, not touching the pellet;

- Add 350 μl of Wash solution # 1, vortex for 15 seconds, pellet by spinning for 5 sec at 10000 prm, remove the supernatant;

- Repeat the washing procedure one more time using the wash solution # 1;

- Remove the remaining of Wash solution # 1 by spinning for 15 seconds with consequent discarding the supernatant;

- Add 500 μl of Wash solution # 2 into each tube;

- Spin the tubes with samples for 15 sec at 10000 rpm, remove supernatant;

- Add 350 μl of Wash solution # 3 and vortex for 15 seconds, then spin for 15 sec at 10000 rpm and remove the supernatant;

- Place open tubes in a thermostat at 55 to 60^oC for 5 minutes to dry out the adsorbent;

- Add 35 μl of DEPC-water into each tube, vortex for 15 seconds and place closed tubes in a thermostat at 55 to 60^oC for 5 minutes;

- Spin the tubes in a microcentrifuge for 1 minute at 10000 rpm; and carefully, not touching the adsorbent, transfer the supernatants into afresh Eppendorf tubes and use them as RNA samples under investigation for the reverse transcription reaction.

Storing of the samples is not recommended.

 

For running a reverse transcription reaction, use the № 2 set:

-         Take out and label a necessary number of tubes with the RT mix, according to the number of tubes with the RNA samples;

- Add 5 μl of RT solvent into each tube;

- Add 5 μl of an isolated RNA sample into each tube;

- Vortex the contents of the tubes until the reagents are fully dissolved;

- Keep the tubes in the thermostat at 37^oC for 40 minute.

- The resulting cDNA samples can be stored at -18 to -22^oC for 1 month.

 

For running a PCR use set № 3:

- Take out and label a necessary number of tubes with the PCR mix № 1, including tubes for positive control (PCS) and negative control;

- Add 10 μl of PCR mix № 2 into each tube;

- Add 10 μl of the cDNA samples from the previous step. In the negative control tube, add 10 μl of NCS; in the positive control sample tube, add 10 μl f the PCS;

- Add 20 μl (one drop) of the PCR oil into each tube;

- Perform the amplification reaction according to the temperature and time schedule (for amplificators with the active regulation regime) from Table 1.

Table 1.

Amplification temperature and time program

Temperature and time program	Number of cycles
94 ºС – 3 min.	1 cycle
94 ºС – 10 sec 56 ºС – 15 sec 72 ºС – 15 sec	42 cycles
72 ºС – 3 min.	1 cycle
10 ºС – keeping

 

For electrophoresis detection of PCR products, mix № 4 is used.

4.1 Preparation of the electrophoresis running buffer.

Put TBE buffer solution in a 1000 ml measuring flask, adjust the volume with deionized water to the required point, add 80 μl of Ethidium bromide, and mix until the concentrated buffer (enough to make one plate of agarose gel) is completely dissolved.

4.2 Agarose gel preparation

Put agarose powder into a 250 ml thermo-stable conical flask, add 100 ml of working TBE buffer solution, and put the flask onto the electric stove. Bring to boil, and, after the agarose is completely melted, take the flask off the stove. The agarose should be fully transparent and not containing any  non-melted particles.

4.3 Preparing the gel box for running an electrophoresis.

4.3.1 Put the gel tray onto a height-regulated preparation platform. Set the platform in a horizontal position using level. Put a gel mold into the tray, insert a comb (two or three combs may be inserted, depending on the number of samples).

4.3.2 Cool the melted agarose down approximately to 50 ºС. Add 5 μl of ethidium bromide into the agarose flask, and carefully mix the flask contents by slow, even rotation.

4.3.3 Pour the cooling down agarose onto the tray. The set agarose layer should be approximately 4 mm thick. After the agarose gel is set (in approximately 30 minutes) carefully remove the combs, trying to not damage the wells. The tray with gel is then transferred to the gel box.

4.3.4 Pour the working buffer solution in the gel box so that the buffer would completely cover the gel, and the buffer layer above the gel would be approximately 2 to 5 mm thick.

4.4 Take out 10 μl of the amplification product (of blue color) from under the PCR oil, and add to the respective well on the gel.

4.5 Put the lid onto the gel box, turn on the current, and set the power supply at 120 V (approximately 10 V/sm).

4.6 Depending on the gel box model, run the electrophoresis for 40 to 60 minutes, then turn the current off, and transfer the gel into the UV-chamber in a special room for viewing. The gel is viewed using the UV irradiation with wave length from 300 to 310 nm.

 

1. The RT-PCR results are considered not authentic and are not to be registered in case:

    - There is a fluorescent band in the negative control sample lane;

    - There is no fluorescent band in the positive control sample lane.

In such cases, a second analysis of the samples should be done using a fresh set of reagents.

 

2. The RT-PCR results are considered authentic and are to be registered in case:

    - There is no fluorescent band in the negative control sample lane;

    - There is a fluorescent band in the positive control sample lane.

3. The result of detecting the Ebola virus in the sample is considered positive if there is a fluorescent band in the sample lane at the level of the positive control sample band (214 bp).

Shelf life. The test system shelf life is 12 months.

Storage. The test system should be stored according to СП 3.3.2.1248-03 in a dry, dark place at 2 to 8 °С during its whole shelf life.

Shipping. The shipping of the test system should be done according to the СП 3.3.2.1248-03 at 2 to 8 °С. A short-time, no longer that one day, shipping at 9 to 20 °С is permitted. Freezing the product is not permitted. For a long distance shipping air transport should be used.