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Infectious hypodermal and hematopoietic necrosis virus(IHHNV) Nucleic Acid Detection Kits
(PCR-Fluorescent Probe Method)
Intended use:
This kit is suitable for the specific detection of IHHNV in water samples, shrimp seedlings, bait, etc., and is used for the etiological diagnosis of suspected infected persons.
Principle:
This kit is chosen infectious subcutaneous and hematopoietic organ necrosis virus (IHHNV) specific fragment to design primers probe probe, the probe with primer amplification area in the middle of a specific binding DNA template, in the process of PCR extension, the circumscribed Taq polymerase enzymes from the 5 'end of fluorescent groups on the probe will be cut down, make it free in the reaction system, Thus, the 3 '-end quenched group was separated and fluorescence was emitted, which was detected by fluorescence PCR instrument to realize the detection of infectious subcutaneous and hematopoietic necrosis virus (IHHNV) in a completely closed reaction system.
Main components:
| Component | Specification |
| PCR reaction liquid | 24 tubes |
| Positive control | 1 tube |
| Negative control | 1 tube |
Sample requirements:
1. Sample type
Seedlings: parent, shrimp eggs, seedlings, coarse standard products
Pond: bottom mud, excrement, water body
Bait: live bait, chilled bait and non-high-temperature treated bait samples
2. Transport
Sample collection and transportation shall be carried out in accordance with NY/T 541. Samples shall be transported to the laboratory as soon as possible under refrigerated conditions to avoid repeated freeze-thaw.
3. Sample preservation
It can be stored for 24 hours at 2℃~8℃, and can be stored for a long time at -20℃.
Using Micgene 242/244/244IVD and ABI QuantStudio5:
procedures are set as below:
| Steps |
Cycle number |
Temperature | Time |
| 1 | 1 | 95℃ | 3min |
| 2 | 45 | 95℃ | 10s |
| 60℃ |
30sCollect the fluorescent |
FAM was selected for detection channel.
The results of interpretation
| Results | Ct Value |
| Positive | Ct≤45 |
| Negative | No Ct value or Ct value≥45 |
| Gray area | 40＜Ct≤45 |
Note: The result is judged to be grey area, which needs to be rechecked. If the recheck result is Ct value < 45 is considered as positive, and no Ct value or Ct value ≥45 is considered as negative.
Result analysis:
Automatic analysis using instrumentation
Quality control:
Negative control: No obvious amplification curve of FAM detection channel;
Positive control: FAM channel showed obvious amplification curve, Ct value ≤30;
The above conditions are met simultaneously in the same experiment; otherwise, the experiment is invalid and re-testing is required.