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[Procedures]
Specimen Preparation
1. Nucleic acid extraction should be performed using an automatic nucleic acid extractor and Viral Nucleic Acid Extraction Reagents according to their package insert.
2. It is recommended to use the Viral Nucleic Acid Extraction Kit provided by Anitoa Systems, LLC，200-300μL of sample were lysed, combined, washed twice, and finally eluted to obtain 60-100μL of DNA solution for subsequent detection.
3. The extracted DNA should be immediately tested or stored at -20℃.
NOTE:
For REF: AMPV01, the MPV N Ctrl and MPV P Ctrl should be treated simultaneously.
For REF: AMPV01D, the MPV N Ctrl and MPV P Ctrl do not need to be extracted. The MPV P Ctrl Lyophillization need to be redissolved before use. Add 200μL MPV N Ctrl to MPV P Ctrl tube and vortex 20-30s to mix thoroughly. Then stored at - 25℃ to -15℃ after re-dissolution.
PCR Reaction Preparation
For REF: AMPV01
1. Take out all components from the kit and place them at room temperature. Vortex 20s and transient centrifuge at 2000- 6000rpm.
2. Prepare the MPV Master mix as shown in the table below, based on the total number of samples (including patient specimen(s), MPV P Ctrl and MPV N Ctrl). Vortex 20-30s to mix thoroughly and transient centrifuge at 2000-6000rpm.
MPV Master mix | Component name | Volume pre test | Total of N specimens (e.g.) |
MPV B-S | 10 μL | 10×(N+3) μL | |
MPV P-P | 5 μL | 5×(N+3) μL |
3. Aliquot 15µL “MPV Master mix” into each clean PCR tube or well.
4. Pipet 10µL sample (including extracted patient specimen(s), MPV P Ctrl and MPV N Ctrl) and add to the PCR tube or well containing PCR master mix, respectively. Cover PCR tubes or wells, mix and transient centrifuge.
5. Put it into the PCR instrument.
For REF: AMPV01D
1. Take out several 8-tubes-strips that containing MPV Mix from the kit based on the total number of samples (including patient specimen(s), MPV P Ctrl and MPV N Ctrl). PCR tube(s) can be cut down from the strip.
2. Open the “MPV Mix” PCR tube caps, then add 23µL sample (including extracted DNA from patient specimen(s), MPV P Ctrl and MPV N Ctrl) to the PCR tube.
3. Cover the tubes with the new Caps of 8-strip Tube provided, mix and transient centrifuge at 2000-6000 rpm, then put them into the PCR instrument.
PCR Assay
1. Create a new experiment.
2. Select the FAM channel for Monkeypox virus (MPV) . Select the VIC channel for internal control.
3. Select “none” for Passive Reference (For ABI Real-Time PCR systems).
4. Set cycle conditions. Enter the reaction volume (25μL), and edit the PCR program as follows:
Steps | Temperature | Time | Cycle |
1 | 50˚C | 2 minutes | 1 |
2 | 95˚C | 2 minutes | 1 |
3 | 95˚C | 5 seconds | 40 |
4* | 58˚C | 30 seconds | |
5 | 37˚C | 1 second | 1 |
*: Data Collection at Step 4 (58˚C, 30S)
5. Save the file. Run the program
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