Specification

PRODUCT PERFORMANCE INDEX
1. Sensitivity: 200 copies/mL.
2. Specificity: No cross reaction with Enterovirus (EV), Measles virus (MV), Rubella virus (RV), Varicella-zoster virus (VZV), Dengue virus (DenV), Human Parvovirus B19(HPVB19), Epstein-barr virus (EBv), human herpes virus 6(HHV-6), etc.
3. Precision: CV ≤ 5%.

PROCEDURES
1. Sample preparations
DNA extraction from clinical samples is conducted according to the corresponding requirements and procedures in the virus DNA extraction kit. The extracted
DNA can be directly used for detection. If the sample is not detected immediately after extraction, it should be stored at -70°C for standby, avoiding repeated freeze-thaw cycles.
2. Reaction system preparation
(1) System preparation: Take out the reagent from the kit, and allow it to thaw completely at room temperature. Invert the mixture and centrifuge immediately. N test reactions (N = number of samples to be tested + positive control + negative control + 1) are prepared for reaction systems, respectively, as follows.

(2) Reaction system distribution: Mix well the above reaction buffer, centrifuge, and dispense 15μL of aliquots into PCR tubes suitable for fluorescent PCR instrument. (Centrifuge the PCR tubes at 6,000rpm for 30s and transport to sample treatment zone.)
3. Sample loading (Sample treatment zone)
Add 10μL nucleic acid sample, negative control and positive control to the above PCR tubes respectively. Cap the tubes tightly and centrifuge at 6,000rpm for 10s and then transport to PCR amplification zone.
* Precaution: Adding samples in the following order is
recommended: negative control-> nucleic acid sample -> positive control.

4. PCR amplification (PCR amplification zone)
4.1 Put the caped PCR tubes into real-time PCR machine for amplification.
4.2 Fluorescence PCR cycle condition setting

Collect fluorescent signals at step 3:60℃; 35s for ABI 7500, 20s for SLAN-96S/96P, while 13s for other Real-Time PCR Systems. The total volume:25μL.
4.3 Disposal after detection
Dispose the PCR tubes in a sealed bag after reaction and treat the used tubes as medical wastes.

5. Settings for result analysis
Set the baseline at a region before the exponential amplification where the fluorescent signals of all the samples are relatively stable (no significant fluctuations in all the samples); set the starting point (cycle number) away from the signal fluctuations at the starting
phase of fluorescence collection; set the end point (cycle number)1~3 cycles before the Ct of the first sample to enter exponential amplification. 4~15 cycles are recommended.
Set the threshold right above the highest point of the negative control amplification curve (irregular noise curve).

6. Quality control criteria
Prior to evaluating the specimen results, the Positive Control and Negative Control should be interpreted using the interpretation table below, and the Positive Control and Negative Control curve must be performed correctly, otherwise the sample result is invalid.

7. Interpretation of Test Results
FAM channels for Monkeypox Virus, detection result should be interpreted as below.
a) Positive: Ct ≤ 38 and amplification curve is S-shaped.
b) Suspected: 38 < Ct ≤ 40 and amplification curve is S-shaped, a second test is needed. Consider positive if Ct ≤ 40 and amplification curve is S-shaped for the second test. Considered negative if Ct > 40 or null Ct and Ct ≤ 35 in VIC channel for the second test.
c) Negative: Ct > 40 or null Ct and Ct ≤ 35 in VIC channel.
d) Re-test: Ct > 40 or null Ct and Ct > 35 in VIC channel.