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.
| Components | Volume for 1 reaction system | Volume for N reaction system |
| PCR reaction buffer | 14μL | 14μLx N |
| PCR Enzyme mixture | 1μL | 1μLx N |
| Total volume | 15μL | 15μLx N |
(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
| Program | Temperature | Reaction duration | Number of cycles |
1 | 50°C | 2 min | 1 |
| 2 | 95°C | 2s | 1 |
| 3 | 95°C | 1s | 41 |
| 60°C | 13s/20s/35s |
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.
Channels/
Controls | Cycle threshold(Ct)value |
| FAM | VIC |
| Positive control | Ct > 40 or UNDET | Ct > 40 or UNDET |
| Negative control | Ct ≤ 35 | Ct ≤ 35 |
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.
