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Enterocytozoon hepatopenaei Nucleic Acid Detection Kits
(PCR-Fluorescent Probe Method)
Intended use:
This kit is applied for quarantine inspection of dectecting Enterocytozoon hepatopenaei(EHP) virus before buying
larvae in shrimp desalt plant to ensure that larvae do not carry
the disease pathogen. Aquaculture enterprises have conducted EHP
specific detection on the bottom mud of aquaculture ponds with slow
growth of prawn to exclude the hidden danger of liver and
intestinal cytiworm parasitization.
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≤40 |
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.
Details please check from IFU!