Primer pair, amplification reagent, amplification kit and detection method for detecting Enterocytozoon hepatopenaei of penaeus vannamei and application

A technology of Enterocystoma hepatica and Penaeus albicans, which is applied in the field of primer pairs for detection of Enterococcus hepatica of Penaeus vannamei, can solve the problems of unfavorable rapid detection, long time consumption, and large instrument dependence, and achieve low cost, simple equipment structure, and instrument The effect of simple structure

Pending Publication Date: 2021-08-20
湛江海关技术中心
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Problems solved by technology

However, the existing EHP detection technology mainly uses the fluorescent PCR method, which is too dependent on the instrument and takes a long time, which is not conducive to rapid on-site detection.

Method used

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  • Primer pair, amplification reagent, amplification kit and detection method for detecting Enterocytozoon hepatopenaei of penaeus vannamei and application
  • Primer pair, amplification reagent, amplification kit and detection method for detecting Enterocytozoon hepatopenaei of penaeus vannamei and application
  • Primer pair, amplification reagent, amplification kit and detection method for detecting Enterocytozoon hepatopenaei of penaeus vannamei and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Establishment of real-time fluorescent RPA detection kit and method for Shrimp Enterococcus hepatica:

[0050] Source of raw materials:

[0051] Primers and probes were designed by themselves and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.;

[0052] RPA reaction solution and RPA freeze-dried powder were purchased from TwistDx Inc, UK;

[0053] The RPA freeze-dried powder includes three enzymes: recombinase proteins uvs X and uvs Y, single-chain binding protein gp32 and Bsu DNA polymerase.

[0054] 1. Design and preparation of primers and probe sequences

[0055] According to MT539781.1, MN308365.1, MN453588.1, MN453587.1, MH260592.1, MH260591.1, MH260590.1,, MH260589.1, MH259891.1, MH259890.1, MH259889.1, MH259 The 18s rRNA gene homologous sequences of MH259887.1, MH259886.1, MF134829.1, MF134828.1, MF134827.1, MF134822.1, MF134817.1, MF134816.1, MF134814.1 and MF134811.1 were compared and analyzed, Determine the conserved sequence of the amplified reg...

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Abstract

The invention relates to a primer pair, amplification reagent, amplification kit and detection method for detecting Enterocytozoon hepatopenaei of penaeus vannamei and application. The primer pair for detecting the Enterocytozoon hepatopenaei is designed and is composed of primers EHP-F and EHP-R; and a fluorescent probe EHP-P is designed. Amplification of a target gene of the Enterocytozoon hepatopenaei based on a RPA technology can be successfully achieved; the detection method disclosed by the invention is based on the RPA technology and can be used for specifically detecting the Enterocytozoon hepatopenaei; and the method is simple and convenient in operation, high in sensitivity, mild in reaction conditions and short in reaction time and can meet the primary needs of a farm on site detection.

Description

technical field [0001] The invention relates to the technical field of biological detection, and furthermore, relates to a primer pair, an amplification reagent, an amplification kit, a detection method and an application for detecting Enterocystoma hepatica of Penaeus vannamei. Background technique [0002] With the continuous expansion of the scale of Penaeus vannamei aquaculture, the occurrence of prawn disease has become one of the bottlenecks in the development of the shrimp aquaculture industry. Emerging diseases such as acute hepatopancreatic necrosis / early shrimp death syndrome (AHPND / EMS) and enterosporidiosis (EHP) of shrimp have increased the risk and burden of farming enterprises, making the success rate of shrimp farming in my country decline year by year, seriously affecting It has promoted the development of shrimp import and export trade and shrimp farming industry. [0003] Enterocytozoon hepatopenaei is a new pathogen newly discovered in recent years that p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/6844C12Q1/04C12N15/11C12R1/90
CPCC12Q1/6844C12Q1/6893C12Q2521/507C12Q2522/101C12Q2521/101C12Q2563/107
Inventor 张娜谢艳辉李家侨李红权杨劲仇保丰郑晓聪斯泽恩郑枢尹刘骁李军刘建芳
Owner 湛江海关技术中心
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