TITLE:  A protocol for the differentiation of the common monkeypox virus (all clades) and its mutation (1b clade) by real-time PCR
AUTHORS: Sudhir Bhatia, Gudrun Baersch

[DESCRIPTION]
The monkeypox virus was first isolated in Copenhagen in 1958. It causes similar lesions to smallpox and should be distinguished from other pathogens such as Mycobacterium leprae, Varicella zoster virus and cowpox, as these viruses can also cause similar types of lesions. Smallpox was eradicated by vaccination in the early 1980s, so the new generation is not vaccinated; therefore, there is a risk that there is no protection against smallpox and related viruses. In Africa, this virus breaks out due to close contact between people. Such a viral infection can be contained by better hygienic conditions and the use of disinfectants, but in many African countries the hygienic conditions are so poor and poverty is probably another reason why the monkeypox virus cannot be controlled. The virus is spreading in other countries, such as the USA, where more than 30,000 positive cases have been reported with many deaths. This virus is also mutating and to control the spread of this virus, people need to be tested as the 1b clone is spreading in many countries. To control the spread of the virus, it is therefore necessary to differentiate the strains. Therefore, we present a protocol to distinguish between the monkeypox virus (all clades) and the 1b clade.

This test can be used on different isolated samples from different lesions like skin, plasma, serum, oral swabs, genital lesion samples. 



[BEFORE_START]
-Consult the manual before start.
-Check the equipments needed before use. 
-The use of internal control is highly recommended. This should be used as extra real time assay.
-First Probe: namely Carboxy-fluorescein (reporter, FAM) and 6-Carboxy tetramethyl rhodamine (quencher, TAM). The results will be shown as Ct -Values. Up to 40 Ct -Values should be taken positive. Ct -Values between 40-45 should be taken as marginal positive (doubtful). It indicates the presence of all clades
 (Tube A). 
2. Second probe: namely Carboxy-fluorescein (reporter; FAM) and 6-Carboxy tetramethyl rhodamine (quencher, TAM). The results will be shown as Ct -Values. Up to 40 Ct -Values should be taken positive. Ct -Values between 40-45 should be taken as marginal positive (doubtful). It indicates the absence of clade Ib and the presence of all clades (Tube C).
 
 
 

[STEPS]
1. Thaw one tube each: A, B, C, D1 and D2. If the kit is not in use, store them at -20°C. Keep tubes away from sunlight.
2. Mark your microtubes with a sample number, positive and negative Control. 
All samples must be carried out for tube A and in parallel for tube C.
3. Thaw tube A. Add 8µl of Tube A to each tube. Otherwise use a 96 microwell plates.
5. Add 10µl of B to each microtube. Avoid touching the wall of the microtubes.

TIP: User can calculate the total requirement of chemicals needed. User can mix 8µl of A (C) + 10µl of B together in one tube for one reaction, but to have 10 reactions, there will be total volume of 180µl (80µl A (C) + 100µl B). From this, 18µl can be distributed into each tube. This step saves time and hardware.
6.     Add 2µl of your DNA template (DNA isolated from samples) with a pipette tip with filter into each microtube according to your labelling, except positive- and negative Control (Do not touch the wall). Use a new pipette tip for each sample.
 
 


  
7. Use new pipette tip with filter. Add 2µl of Tube D1 to the positive Control. (Do not touch the wall).
8. Use a new pipette tip. Add 2µl of Tube D2 to the negative Control. (Do not touch the wall).
9. Check if everything is added correctly, as the volume of each microtube must be almost the same.
4. Thaw tube C. Add 8µl of Tube C to each tube. Otherwise use a 96 microwell plates.
10. Now enter reporter (FAM) and quencher dye (TAM) to set up your software and run the following program:
  
         15 seconds at 95°C               
         60 seconds at 60°C    -- x 45 cycles
11. Before the start of the PCR program, check whether the plate or tubes are properly sealed. The wells of the plate or microtubes must be in contact with metal block (important!). There should be no air or lose contact with metal block of thermocycler. In case of 96-well plate, it should be sealed with adhesive cover. Now run your PCR.
12. After step 11 is finished take out the microtubes.
13. STEP B
Click the Result-Tab and the Amplification-Tab: Place the Threshold line above the background, then select: “Analyse”. Calculate the threshold cycle (Ct) for each well. There should be no signal in the negative control. Successful positive control and positive samples must give a curve in the software graphics. 


15. 
 

14.  
 
Interpretation of results: A positive result in Tube A means that all clades are present in the sample. A positive result in Tube C indicates the presence of all clades and the absence of clade Ib. This means that if a clade Ib mutation is present, the result in Tube A is positive and in Tube C negative. Negative samples must be negative in both tests.