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High-sensitivity target gene for enterocytozoon hepatopenaei disease, primer pair, kit and detection method

A microsporidiosis, high-sensitivity technology, applied in the direction of microbial-based methods, biochemical equipment and methods, microbial determination/inspection, etc.

Active Publication Date: 2019-03-19
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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Problems solved by technology

Similarly, the 157 bp ribosomal gene fragment amplified by this method is also highly homologous to other species ( image 3 , Figure 4 )

Method used

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  • High-sensitivity target gene for enterocytozoon hepatopenaei disease, primer pair, kit and detection method
  • High-sensitivity target gene for enterocytozoon hepatopenaei disease, primer pair, kit and detection method
  • High-sensitivity target gene for enterocytozoon hepatopenaei disease, primer pair, kit and detection method

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[0048] A highly sensitive detection method for prawn hepatoenteric microsporidiosis, said method comprising the steps of:

[0049] 1. Extract the total DNA from the prawns to be inspected;

[0050] (1) Take 50-100 mg of shrimp tissue (with intestinal tissue) in a 2 mL centrifuge tube;

[0051] (2) Add 200 uL GA and 1 magnetic bead, and grind for 30 s;

[0052] (3) Add 20 uL proteinase K (100 mg / mL) and incubate at 56°C for 3 h;

[0053] (4) Add 200 uL GB and incubate at 70°C for 10 min;

[0054] (5) Add 200 uL absolute ethanol, mix well and add to the spin column;

[0055] (6) After washing once with GD and twice with WP, let it dry for 5-10 minutes;

[0056] (7) Add 100 uL TE, centrifuge to get shrimp DNA, store at -20°C for later use.

[0057] 2. Perform PCR amplification using the DNA obtained in step 1 as a template;

[0058] The primers used consisted of a forward primer and a reverse primer. The forward primer was: 5-CAAGAACAACCCACGAAGCAAG-3', and the reverse prime...

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Abstract

The invention belongs to the technical fields of agriculture and animal quarantine and particularly relates to a high-sensitivity target gene for enterocytozoon hepatopenaei disease, a primer pair, akit and a detection method. The sequence of the high-sensitivity target gene is represented by SEQ ID NO:1. The invention further provides the primer pair for detecting the target gene represented bySEQ ID NO:1. The invention further provides a high-sensitivity detection method for the enterocytozoon hepatopenaei disease. The detection method comprises the following steps: (1) extracting total DNA from a to-be-detected prawn; (2) carrying out PCR amplification through the primer pair by taking DNA as a template; and (3) detecting a PCR product obtained in the step (2) by virtue of 1.5% agarose gel electrophoresis, and if a target strip is generated, determining that the prawn is infected by enterocytozoon hepatopenaei. Primers are designed based on a sporoderm protein gene (SWP) of EHP, have strong specificity, have a definite corresponding relation with pathogen EHP and are suitable for being taken as detection primers for detecting enterocytozoon hepatopenaei by virtue of a PCR method.

Description

technical field [0001] The invention belongs to the technical field of agriculture and animal quarantine, and in particular relates to a highly sensitive target gene, a primer pair, a kit and a detection method for prawn hepatoenteric microsporidiosis. Background technique [0002] Microsporidia (microsporidia) are single-celled eukaryotic endoparasites that widely parasitize shrimp, crustaceans and other animals. Shrimp hepatopenaei (Enterocytozoon hepatopenaei, EHP) is a kind of microsporidia, which belongs to Microsporidiaceae and the genus Enterocystis. EHP has been detected successively in Litopenaeus vannamei, White shrimp and Chinese shrimp farming areas in Asian countries. Studies have shown that EHP has a wide range of infection routes. It can be transmitted horizontally through polluted aquaculture water and diseased shrimp, and can also be transmitted vertically through fertilized eggs and shrimp seedlings. EHP-infected prawns have no obvious symptoms at the ini...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/686C12N15/11C12R1/90
CPCC12Q1/686C12Q1/6893C12Q2565/125
Inventor 沈卫锋牛宝龙翁宏飚杨颖
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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