Method of use:
1. Sample DNA extraction
It is recommended to use the virus nucleic acid co-extraction kit of Shijiazhuang Shengbo
Biotechnology Co., Ltd. for sample DNA extraction. Please operate according to the reagent
instructions.
2. Sample adding
After the natural thawing of qPCR reaction solution, take 17μ l of it into the PCR tube, add 3
μ l of nucleic acid extracted in step 1, cover the tube cover, mix and centrifugate.
3. PCR amplification
3.1 Place the reaction tube in the fluorescent quantitative PCR reaction tank;
3.2 Set the sample name and channel information.
4. Result analysis
4.1 Result analysis condition setting
Set baseline and threshold: generally, analyze the results directly according to the machine.
4.2 Result judgment
See the results of H5 subtype in the FAM channel, the H5 subtype in the HEX channel (or
VIC, JOE),and the H9 subtype in the Cy5 channel.
Positive: CT value of detection channel ≤ 37.0, and the curve has obvious exponential growth
curve;
Suspicious: if the CT value of detection channel is among 37.0 and 40(37.0< CT value≤40)
,
it is recommended to repeat the detection. If the test result is also the same , and the curve has
an obvious growth curve, it is determined as positive, otherwise it is negative;
Negative: there is no CT value.
5. Limitations of detection methods
Step
Number of cycles
Temperature
time
Collecting signal
1
1 cycle
90 ºC
30 sec
No
2
1 cycle
60 ºC
5 min
No
3
95 ºC
1 min
No
4
40 cycle
95 ºC
10 sec
No
5
60 ºC
31 sec
YesSample test results are related to sample collection, processing, transportation and storage
quality;
If cross contamination is not well controlled during sample extraction, false positive results
will appear;
Leakage of positive control and amplification products will lead to false positive results;
Different extraction methods have different extraction efficiency, which will lead to false
negative results;
Reagent transportation, improper storage or inaccurate reagent preparation lead to the decline
of reagent detection efficiency, resulting in false negative or inaccurate quantitative detection
results;
The test results are for reference only. If you need to be diagnosed, please combine with
clinical symptoms and other test methods.
6. Quality control standard
Negative quality control material: no obvious amplification curve or CT value display;
Positive quality control material: the amplification curve has obvious index amplification
S-shape, and CT value ≤ 32;
The above conditions should be met at the same time, otherwise the experiment will be
considered invalid.
7. Product performance index
The coincidence rate of positive and negative References: 5 positive references were 100%;
10 negative references were 100%.
The minimum detection limit was 5 × 102 copies/ml.
Precision: the coefficient of variation (CV) of CT value of precision detection within and
between batches is ≤ 3%.
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