| Component name | Specification | Quantity | Main constituents |
PCR detection reagent
(Large package, 24 tests/kit) | MPXV PCR reaction solution A | 510 µL/tube | 1 | Specific primers and probes, Tris-HCl buffer |
| MPXV PCR reaction solution B | 105 µL/tube | 1 | Hot-start Taq antibody enzymes, Taq enzymes, dNTPs, UDG enzymes |
PCR detection reagent
(Large package, 48 tests/kit) | MPXV PCR reaction solution A | 935 µL/tube | 1 | Specific primers and probes, Tris-HCl buffer |
| MPXV PCR reaction solution B | 195 µL/tube | 1 | Hot-start Taq antibody enzymes, Taq enzymes, dNTPs, UDG enzymes |
Quality controls
(Large package, 24 tests/kit; Large package, 48 tests/kit) | MPXV negative control | 450 µL/tube | 1 | Pseudovirus containing MPXV internal standard fragments (RNase P gene) |
| MPXV positive control | 450 µL/tube | 1 | Pseudovirus containing MPXV target fragment, pseudovirus containing MPXV internal standard fragments (RNase P gene) |
Note: Components from different lot numbers of kits should not be used interchangeably.
Description of quality control: Positive control is pseudovirus containing MPXV target fragment and pseudovirus containing MPXV internal standard fragments (RNase P gene), the negative control is pseudovirus containing MPXV internal standard fragments (RNase P gene). During use, they shall be involved in nucleic acid extraction and shall be considered as substances with potential infectivity, and the operation and disposal must meet relevant regulatory requirements.
8. Applicable Instruments
ABI 7500, AGS4800, AGS8830-16.
9. Warnings and Precautions
- This product is used for in vitro detection only. Please read this Instruction for Use carefully before the test.
- To avoid any potential biological hazards in the specimens, the test specimens should be regarded as infectious substances and avoid contact with skin and mucous membranes. The specimens should be treated in the bio-safety cabinet which can prevent the outflow of aerosol. The used test tubes and pipette tips in the sample preparation area should be put into the container containing disinfectant and sterilized together with the waste before they can be discarded. The specimen operation and treatment should meet the requirements of relevant regulations.
- Disposal of products: After PCR, the products tend to cause contamination, and all the reaction tubes should be put into bio-safety garbage disposal bags or other containers by those who no longer participate in the test on that day , and make sure it is completely sealed before disposal.
- DNase contamination should be avoided during the whole experiment, and laboratory gown, disposable gloves and masks should be worn during the experiment. Complete the operation in the cleaned, disinfected and UV-strerilized bio-safety cabinet to avoid harmful substances entering the respiratory tract.
- Use autoclaved disposable centrifuge tubes and tips or buy DNase-/RNase- free centrifuge tubes and tips.
- PCR reagents should be thawed completely and centrifuge at 8,000 rpm for several seconds before use but repeated freezing and thawing should be avoided.
- Failure to control cross-contamination during specimen processing may lead to false positive results.
- The laboratory shall be managed in strict accordance with the management practice of PCR gene amplification laboratory. Professional training is necessary for the laboratory personnel. The test process should be strictly carried out in specific areas (reagent preparation area, sample preparation area, amplification and detection area). All the consumables should be sterilized for single use. Use dedicated instruments and equipment in difference stage. Cross usage is not permitted for materials used in different areas and different stages.
- After testing, treat workbench and pipette with 10% hypochlorous acid or 75% alcohol, and then irradiate with ultraviolet lamp for 20-30 minutes.
- After the nucleic acid extraction, carry out the next step of the test immediately.
- Quality control shall be carried out for each test.
10. Materials and Instruments Required but Not Provided
- Disposable gloves and mask, powderless
- Specimen collection tube
- Adjustable micropipettes and pipette tips with filters
- Timer
- Vortex mixer
- Real-time PCR amplification instrument
- Table model high speed centrifuge
- Sterile saline
- The nucleic acid extraction or purification reagent (such as YSXB No. 20170583 and YSXB No. 20170667) manufactured by Daan Gene Co., Ltd.
11. Specimen Requirements
- Applicable specimen type: Rashes, scabs, blister fluid, pustular fluid, and whole blood specimens.
- Specimen collection
- Rashes, scabs: Take the rash cover or scab, put it into a specimen collection tube, then seal the tube and send it for inspection.
- Blister fluid, pustular fluid: Use a disposable sterile syringe to draw 0.2 mL of sterile saline, and insert it into the herpes membrane, draw in and out for 2 to 3 times, and then draw the content solution, and load it into the specimen collection tube, then seal the tube and send it for inspection.
- Whole blood: Collect 5 mL of anticoagulant whole blood from the vein of the suspected patients or animals suspected to carry the virus, then seal the tube and send it for inspection.
- Specimen storage
Specimens collected can be used immediately for testing, or stored at -20±5°C for 6 months or stored at -70°C for long-term storage. Repeated freeze-thaw of specimens should be avoided.
12. Test Methods
12.1. Specimen processing and nucleic acid extraction (Specimen preparation area)
The nucleic acid extraction or purification reagent (such as YSXB No. 20170583 and YSXB No. 20170667) manufactured by Daan Gene Co., Ltd. can be used. Take 200 μL of specimens for nucleic acid extraction.
The MPXV negative control and MPXV positive control in this kit are all involved in nucleic acid extraction process.
12.2. PCR amplification (Reagent preparation area)
Take MPXV PCR reaction solution A and MPXV PCR reaction solution B from the kit, thaw them at room temperature and vortex to mix well, then centrifuge at 8,000 rpm shortly for later use.
Take N PCR reaction tubes (N = number of specimens to be tested + MPXV negative control + MPXV positive control). The preparation of MPXV reaction mixture for single test is as follows:
| MPXV PCR reaction solution A | MPXV PCR reaction solution B | Total |
| 17 µL | 3 µL | 20 µL |
12.3. Specimen loading (Specimen preparation area)
Add 10 μL of each extracted nucleic acids of the test specimen, MPXV negative control, MPXV positive control to the reaction tubes mentioned above. Tighten the tube cap, centrifuge the tubes for 15 s and transfer them to the amplification detection area.
12.4. PCR Amplification (Amplification and detection area)
12.4.1. Put the reaction tube into the sample slot.
12.4.2. ABI Prism 7500 instrument setup
12.4.2.1. Open the “Setup” window, set negative control (NTC), positive control and unknown specimens (Unknown) according to the corresponding order of specimens, and set the specimen name in the column of “Sample Name”. The detection mode of the probe is set as: Reporter 1: FAM, Quencher 1: NONE, Reporter 2: VIC, Quencher 2: NONE, Passive Reference: NONE.
12.4.2.2. Open the instrument window and set the cycle conditions as follows:
| Stage | Reps | Target (°C) | Running Time | Data Collection |
| 1 | 1 | 50 | 00：02：00 | |
| 2 | 1 | 95 | 00：05：00 | |
| 3 | 45 | 95 | 00：00：05 | |
| 60 | 00：00：10 | √ |
After setting, save the file and run the program.
12.4.2.3. AGS4800 instrument setup
12.4.2.3.1. First turn on the computer, then turn on the PCR instrument host power, and finally start the AGS4800 software, select the corresponding layer (upper, middle, lower).
12.4.2.3.2. Click New, edit the experiment name and file path in the experiment name, and click OK to open a blank file after saving.
12.4.2.3.3. Program Parameters: On the Program Parameters page. The amplification parameters are as follows:
| Stage | Reps | Target (°C) | Running Time | Data Collection |
| 1 | 1 | 50 | 00：02：00 | |
| 2 | 1 | 95 | 00：05：00 | |
| 3 | 45 | 95 | 00：00：05 | |
| 60 | 00：00：10 | √ |
12.4.3.4. Sample Parameters: Click on the “Sample Parameters” page, select the current program dye settings (channel 1 FAM, channel 2 VIC) in the upper menu shortcut key, then select sample wells in the 48-well table below, select FAM, VIC at the dye type picture on the right side, and select the corresponding sample type in the button below: negative control, unknown sample, etc.
12.4.3.5 Initiating amplification: After confirming that the 48-well plate is properly placed in the host, click the Start button to start the PCR cycle.
12.4.3.6 Monitoring Process: Switching the program running page can display the actual situation of the PCR curve gradually increasing as the cycle increases in real time. If the label is selected FAM or VIC, only the changes of the corresponding probes are displayed.
12.4.3.7 Save the result: After the program ends, it is automatically saved to the file path.
12.4.4 AGS8830-16 instrument setup
12.4.4.1 Turn on the AGS8830-16 instrument.
12.4.4.2 Click “Custom” to set up the program.
12.4.4.3 Program Parameters: On the Program Parameters page. The amplification parameters are as follows:
| Stage | Reps | Target (°C) | Running Time | Data Collection |
| 1 | 1 | 50 | 00：02：00 | |
| 2 | 1 | 95 | 00：05：00 | |
| 3 | 45 | 95 | 00：00：05 | |
| 60 | 00：00：10 | √ |
12.4.4.4 Dye Parameters: Click the Dye Parameters page, select the current program dye settings (Channel 1 FAM, Channel 2 VIC), then select the sample wells, and select in the Sample Type section: Negative Control, Positive Control, Unknown Sample, etc.
12.4.4.5 Initiating Amplification: After confirming that the PCR reaction tube is properly placed in the host, click the Start button to start the PCR cycle.
12.4.4.6 Monitoring Process: Switching the program running page can display the actual situation of the PCR curve gradually increasing with the increase of the cycle in real time. If the marker is selected FAM or VIC, only the changes of the corresponding probes are displayed.
12.4.4.7 Save result: After the program ends, it is automatically saved to the file path.
12.5. Result analysis (Please refer to the instructions for each instrument, taking the ABI7500 instrument as an example)
Save the test data file after the reaction finishes. Adjust the start value, end value of Baseline, and the Threshold value according to the analyzed image (the user can adjust according to the actual situation, the Start value can be set to 3 to 15, and the End value can be set to 5 to 20, and adjust the Threshold value at the Log graphic window so that the threshold line is located in the exponential phase of the amplification curve). Click Analysis to automatically obtain the analysis results, and check the results in the Report interface.
12.6 Quality control
Negative control: No amplification curve shows in FAM detection channel or Ct value > 40; while an amplification curve shows in VIC detection channel and Ct value ≤ 35.
Positive control: An amplification curve shows in FAM detection channel and Ct value ≤ 32; while an amplification curve shows in VIC detection channel and Ct value ≤ 35.
The above requirements must be met in the same test at the same time, otherwise, this test is invalid and needs to be repeated.
13. Positive Judgment Value
The positive judgment value of this reagent is 40, and the positive judgment value of the internal standard is 36.
14. Interpretation of Test Results
14.1 Each test needs to detect MPXV negative control, MPXV positive control, and the determination of the test results can be carried out only when the quality control results meet the quality control requirements.
14.2 In the case of quality control is normal, the result is interpreted as follows:
Negative results judgment: If the test result of the test specimen shows no amplification curve in the FAM channel, while an amplification curve shows in VIC detection channel and Ct value ≤ 36, it can be determined that the specimen is negative for Monkeypox virus.
| | FAM channel | VIC channel | Results judgment |
| Negative results judgment | Ct value > 40 or no Ct value | Ct value ≤ 36 | Negative |
Positive results judgment: If the test result of the test specimen shows amplification curve in the FAM channel and Ct value ≤ 40, while an amplification curve or not shows in VIC detection channel, it can be determined that the specimen is positive for Monkeypox virus.
| | FAM channel | VIC channel | Results judgment |
| Positive results judgment | Ct value ≤ 40 | Has or no Ct value | Positive |
14.3 When the test result of the FAM detection channel is positive, the VIC channel (for internal standard) may be tested negative due to the competition of the system.
14.4 When the internal standard result is negative, if the test results of FAM detection channel of the tested tube is also negative, it is indicated that the system is inhibited or the operation is wrong, and the test is invalid, the specimen should be retested.
14.5 The recommended report format is as following:
The negative result report format: no MPXV DNA was detected in the sample, and the concentration was lower than the sensitivity of the kit.
The positive result report format: MPXV DNA was detected in the sample.
15. Limitations of Test Method
15.1 Specimen test results are related to the specimen collection, processing, transportation and preservation, improper sample collection, transportation, storage and processing, improper test operation or test environment may lead to incorrect test results.
15.2 Failure to control cross contamination during specimen processing may lead to false positive results.
15.3 Mutations of the virus in epidemic may lead to false negative results.
15.4 The test results are for clinical reference only. If confirmation of diagnosis is needed, please correlate clinical symptoms and combine with other test methods.
16. Performance Characteristics
16.1 The LOD of the kit to detect the Monkeypox virus is 200 copies/mL.
16.2 Compliance rate of positive and negative reference: the compliance rate of enterprise positive reference is 100%; the compliance rate of enterprise negative reference is 100%.
16.3 The coefficient of variation of the Ct value of the enterprise precision references R1 and R2 is ≤5.0%, and the test result of R3 is negative.
16.4 There is no cross-reaction to other pathogens with the same site of infection or similar symptoms of infection.
17. References
17.1 Arita l, Jezek Z, Khodakevich L, et al. Human monkeypox: a newly emerged orthopoxvirus zoonisis in the tropical rain forests of Africa[J].AmJ Trop Med Hyg,1985,34(4): 781-789.
17.2 Guan Xixi,Tian Houwen. Research progress in monkeypox virus detection[J].Chinese Journal of Experimental and Clinical Virology,2017,31(3):273-276.
18. Reference Standards
EN ISO 18113-2:2011 In vitro diagnostic medical devices - Information supplied by the manufacturer (labelling) - Part 2: In vitro diagnostic reagents for professional use.
