Enterocytozoon Hepatopenaei-18s Nucleic Acid Amplification Multiplex Real Time PCR

Enterocytozoon hepatopenaei-18s (EHP-18S)Nucleic Acid Detection Kits

(PCR-Fluorescent Probe Method)
 
 
Intended use:
 
This kit is suitable for the quarantine of Enterocytozoon hepatopenaei -18S (EHP-18S) before the purchase of larvae in the shrimps 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:
 
In this kit, a primers probe was designed by using a specific fragment of Enterocytozoon hepatopenaei -18S (EHP-18S). The probe could specifically bind to a DNA template in the middle of the primer amplification region. During the PCR extension process, the exonuclease activity of Taq enzyme cut the 5 '-terminal fluorophore from the probe and made it free in the reaction system. Thus, the 3 '-end quenched group was separated, and the fluorescence was emitted, which was detected by fluorescence PCR instrument, and the detection of eHP-18S nucleic acid was realized in the completely closed reaction system.
 
Main components:
 

 
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:

FAM was selected for detection channel.
 
The results of interpretation

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.