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[Result Analysis]
Following the completion of reactions and detection, the instrument will automatically save the results.
1. Setting analysis condition: According to the image obtained from the PCR reaction, adjust the start value, end value of baseline and threshold. The user can adjust these values according to their situation. The start value can be set at 3-15; end value can be set at 5-20.
2. Adjust the threshold to just above the curve of MPV N Ctrl.
3. Click “Analyze” icon to update the analysis.
4. Enter “Report” window and record unknown sample values (Ct):
Ct value | MPV (FAM) | Internal control (VIC) |
Definition of “+” in each channel | Ct≤38.5 with typical amplification curve | Ct≤36 with typical amplification curve |
Definition of “-” in each channel | Ct＞38.5 or no typical amplification curve | Ct＞36 or no typical amplification curve |
Interpretation of quality control
Positive and negative controls should be examined prior to interpretation of clinical sample results.
Positive and Negative Controls | MPV (FAM) | Internal Control (VIC) | Results | Actions |
MPV N Ctrl | - | - | Valid | Continue to result interpretation |
MPV P Ctrl | + | + | ||
If controls do not meet these criteria, the samples on the plate are invalid and retest is required. | ||||
Interpretation of clinical sample results
Assessment of clinical sample test results should be performed after the positive and negative controls have been determined to be valid. If the controls are not valid, the patient results cannot be interpreted.
MPV (FAM) | Internal Control (VIC) | Results |
+ | +/- | Monkeypox virus Positive |
- | + | Monkeypox virus Negative |
- | - | Invalid, retest1 |
1 If the retest still invalid, resampling. | ||
[Limitations of the Procedure]
1. The test results of this kit are only for clinical reference. The clinical diagnosis and treatment of patients should be considered in combination with clinical observations, patient history, and epidemiological information.
2. The test results may be affected by the quality of the samples collected as well as their handling, transportation and storage. Deficiencies in these factors may lead to false negative results.
3. False positive results may occur if cross-contamination is not controlled during sample processing.
4. Amplification of internal control may fail if virus concentration in the specimen is high.
5. Viral nucleic acid extraction reagents from other companies need to be tested on several samples to verify their suitability
[Performance Index]
1. The analytical sensitivity (LoD) of the detection kit is 200 copies /mL.
2. No Cross reactivity with: human genome, Camelpox virus, Variola virus, Ectromelia virus, Vaccinia virus, Cowpox virus, Mouse hemorrhagic fever virus, Meningitis virus, Capri pox virus, Fowl pox virus, Mouse pox virus, Pigeon pox virus, Herpes simplex virus type 1&2, Varicella-zoster virus.
3. Precision: The intra-batch/ batch precision, intra-day/ day precision and precision variation coefficients between different operators are not greater than 5%.
[Precautions]
1. Read instructions carefully prior to testing. The test must be performed according to the instructions provided.
2. Laboratory management shall be strictly in accordance with management standards of a nucleic acid test laboratory.
3. Conduct quality control for every experiment.
4. Fully thaw PCR reagents prior to use but avoid repeated freeze-thaw cycles.
5. Pipettes used with this test must be calibrated regularly.
6. The entire process should be divided into three separate areas within the laboratory: The first area for reagent preparation, the second area for specimen processing and reaction system preparation and the third area for amplification, Fluorescence detection and result analysis. The instruments, equipment, and personal protective equipment (PPE) in each area should be used independently to prevent cross contamination.
7. Operators of the test should always avoid potential contamination of RNase and DNase. Test operators should not directly touch the reaction tube by hand. Operators must use disposable gloves and testing materials without Fluorescent properties.
8. Follow biohazard safety standard precautions. Biological safety cabinets should be used when handling specimen to ensure the safety of operators and prevent pollution. Harmful and/or toxic specimens and reagents should be properly stored and maintained by designated personnel. Waste should be properly disposed in special containers. Instruments and equipment such as operating tables, pipettes, centrifuges, and amplification instruments should be frequently wiped and disinfected with 10% sodium hypochlorite and/or 75% ethanol. Laboratory rooms and ultra-clean workbenches should be regularly treated with UV lamps and after each experiment.
9. The reagents in the centrifuge tube should be fully thawed and mixed before use. Centrifuge for a few seconds to concentrate the liquid at the bottom of the centrifuge tube. When preparing the reaction system, it should be noted that the mixing of all liquids should be carried out on the vortex mixer as much as possible. Once the reaction system is completed, centrifuge for a few seconds at a low speed.
10. Do not mix reagents from different batches.
[References]
[1] Kulesh D A , Baker R O , Loveless B M , et al. Smallpox and pan-Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms[J]. Journal of Clinical Microbiology, 2004, 42(2):601-609.
[2] Jezek Z , Marennikova S S , Mutumbo M , et al. Human monkeypox: a study of 2,510 contacts of 214 patients.[J]. Journal of Infectious Diseases, 1986(4):551-555.
[3] Neubauer H , Reischl U , Ropp S , et al. Specific detection of monkeypox virus by polymerase chain reaction[J]. Journal of Virological Methods, 1998, 74(2):201.
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