Primer pair, amplification reagent, kit and detection method for detecting infectious hypodermal and hematopoietic necrosis virus (IHHNV) of prawns and application

A technique for hematopoietic tissue and necrosis virus, which is applied in the field of primer pairs for detecting infectious subcutaneous and hematopoietic tissue necrosis virus of prawns, can solve the problems of unfavorable rapid detection, large instrument dependence, long time consumption, etc., and achieves short detection time and low cost. , The effect of the simple structure of the device

Pending Publication Date: 2022-01-07
湛江海关技术中心
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AI Technical Summary

Problems solved by technology

[0007] However, the existing IHHNV detection technology mainly uses the PCR method, but this method 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, kit and detection method for detecting infectious hypodermal and hematopoietic necrosis virus (IHHNV) of prawns and application
  • Primer pair, amplification reagent, kit and detection method for detecting infectious hypodermal and hematopoietic necrosis virus (IHHNV) of prawns and application
  • Primer pair, amplification reagent, kit and detection method for detecting infectious hypodermal and hematopoietic necrosis virus (IHHNV) of prawns and application

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Experimental program
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Effect test

Embodiment 1

[0048] Raw material source:

[0049] Primers and probes were designed and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.;

[0050] RPA reaction solution and RPA lyophilized powder were purchased from TwistDx Inc, UK.

[0051] The RPA freeze-dried powder includes three enzymes: recombinase proteins uvs X and uvs Y, single-chain binding protein gp32 and Bsu DNA polymerase.

[0052] Establishment of fluorescent RPA detection kit and method for shrimp infectious subcutaneous and hematopoietic necrosis virus:

[0053] 1. Design and preparation of primers and probes

[0054] According to the comparison and analysis of the nonstructural protein 1 and nonstructural protein 2 region gene sequences of AF218266.2, EF633688.1, and AY355307.1, the conserved extension region sequence was determined, and the primer probe was designed according to the RPA primer probe design principle. Primer probes were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. Design primer pa...

Embodiment 2

[0068] Sensitivity evaluation of IHHNV fluorescent RPA detection

[0069] Use IHHNV standard substance (initial concentration of 12.5ng / ul) to perform 3-fold dilution, select samples with 9-fold, 243-fold, and 2187-fold concentration for detection, and use ddH at the same time 2 O served as a negative control. The result is as figure 1 As shown, 9 times, 243 times the concentration of the sample detection results are positive, 2187 times the concentration of the sample and ddH 2 O tested negative.

[0070] In order to test the accuracy of the detection method and the effectiveness of clinical application, a batch of clinical samples were detected by this method and the fluorescent PCR method. For the amplification results of the IHHNV fluorescent RPA detection system, see figure 2 , and the comparison chart between the detection results of clinical samples and fluorescent PCR is shown in Table 2 below.

[0071] Table 2 Comparison of IHHNV fluorescent RPA clinical sample ...

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Abstract

The invention relates to a primer pair, amplification reagent, kit and detection method for detecting an infectious hypodermal and hematopoietic necrosis virus (IHHNV) of prawns and application. According to the invention, the primer pair for detecting the IHHNV is designed, wherein the primer pair consists of primers IHHNV-F and IHHNV-R; and a fluorescent probe IHHNV-P is designed. According to the invention, amplification of a target gene of the IHHNV based on a RPA technology can be successfully achieved, the detection method is based on the RPA technology, can be used for specifically detecting the IHHNV, and has advantages of simplicity and convenience in operation, high sensitivity, mild reaction condition and short reaction time, and basic requirements of farms on field detection can be met.

Description

technical field [0001] The invention relates to the technical field of biological detection, and furthermore, relates to a pair of primers, an amplification reagent, a test kit, a detection method and an application for detecting infectious subcutaneous and hematopoietic tissue necrosis virus of prawns. Background technique [0002] Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a single-stranded DNA virus with a genome sequence size of about 4.1kb, and the size of the virus particle is only 20-22nm, which is an icosahedral spherical structure . [0003] IHHNV was first discovered in a farm in the United States in the early 1980s. It first broke out in Litopenaeus stylirosiris, and then it was also found in cultured prawns in America and Asia. IHHNV can almost all infection of shrimp. The most sensitive host of IHHNV is Penaeus redfront, which can cause high morbidity and mortality (90%), and can also cause "runt-deformity syndrome" (Runt-deformity syndr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2563/107
Inventor 张娜李家侨郑晓聪仇保丰谢艳辉刘骁杨劲李红权刘建芳李军斯泽恩郑枢尹
Owner 湛江海关技术中心
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