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A primer, kit and detection method for detecting epidemic hematopoietic organ necrosis virus using pyrosequencing technology

A technology of hematopoietic organ necrosis and pyrosequencing, applied in the field of molecular biology, can solve problems such as false positives and inability to achieve rapid confirmation, and achieve the effects of small sample volume, accurate short DNA sequence analysis, and short time-consuming

Active Publication Date: 2019-04-05
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, the standard method for EHNV detection is mainly to separate and culture the virus from cells, and then identify it by means of histopathology, immunofluorescence, ELISA, electron microscopy and PCR. Antigen capture ELISA can effectively identify the virus, but the sensitivity is only 60%. %; Although the PCR method is sensitive, there is still the possibility of false positives; other methods cannot achieve the purpose of rapid confirmation at the molecular level

Method used

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  • A primer, kit and detection method for detecting epidemic hematopoietic organ necrosis virus using pyrosequencing technology
  • A primer, kit and detection method for detecting epidemic hematopoietic organ necrosis virus using pyrosequencing technology
  • A primer, kit and detection method for detecting epidemic hematopoietic organ necrosis virus using pyrosequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 detects the design of the primers of epidemic hematopoietic organ necrosis virus

[0027] By comparing and analyzing the gene sequences of the epidemic hematopoietic organ necrosis virus published in the NCBI gene bank (GenBank) of the National Center for Bioinformatics in the United States, it was determined that the specific sequence of the EHNV ORF65 gene was 174bp, and specific PCR primers and primers were designed according to the specific sequence. Pyrosequencing primers, screen out a set of optimal primers for subsequent PCR and sequencing.

[0028] The epidemic hematopoietic organ necrosis virus specific primer designed according to the specific region sequence of the epidemic hematopoietic organ necrosis virus ORF65 gene of the present invention can amplify a specific single band for the epidemic hematopoietic organ necrosis virus DNA, and the PCR amplification fragment length It is 137bp. Then design pyrosequencing primers according to the specif...

Embodiment 2

[0033] Embodiment 2 kit composition

[0034] The kit includes the primer set designed in Example 1 for detecting the epidemic hematopoietic organ necrosis virus, that is, the amplification primer pair and the sequencing primer. The kit also includes the materials and reagents needed to complete the PCR reaction and pyrosequencing, such as DNA extraction reagents (DNeasy Blood & Tissue Kit, QIAGEN Company), PCR reaction reagents (PyroMark PCR Kit, QIAGEN Company), single-stranded template preparation reagents (Strand Mycoavidin-coated magnetic beads, GE Company), pyrosequencing reagents (PyroMark Gold Q96SQA Reagents, denaturing buffer, washing buffer, QIAGEN Company), etc., the above materials and reagents can be mixed and packaged, or individually packaged, Preferably, it is packaged individually.

Embodiment 3

[0035] The establishment of embodiment 3 detection method

[0036] 1. DNA extraction of epidemic hematopoietic organ necrosis virus

[0037] Using EHNV (the EHNV strain donated by the Danish Veterinary Laboratory of OIE Fish Disease Reference Laboratory) as raw material, DNeasyBlood&Tissue Kit (QIAGEN company, product number 69506) was used to extract DNA. The kit operation steps are as follows:

[0038] (1) Pipette 20 μl proteinase K to the bottom of a 1.5ml centrifuge tube. (2) Add 200 μl of the virus liquid to be extracted into the centrifuge tube. (3) Add 200 μl Buffer AL to the sample, and vortex for 15 seconds to mix. (4) Incubate at 56°C for 10 minutes. (5) Centrifuge quickly to remove the liquid droplets remaining in the lid of the 1.5ml centrifuge tube. (6) Add 200 μl (equal volume of the sample) of ethanol (96-100%), and vortex for 15 seconds to mix. After shaking, quickly centrifuge to remove the liquid droplets remaining in the cap of the 1.5ml centrifuge tube...

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Abstract

The invention discloses primers for detecting the epidemic haematopoietic necrosis virus with a pyrosequencing technology, a kit and a detection method, and particularly relates to a group of primers for detecting the epidemic haematopoietic necrosis virus with the pyrosequencing technology. The primers comprise an amplification primer pair shown in sequence tables SEQ ID No.2 and SEQ ID No.3 as well as a sequencing primer shown in the sequence table SEQ ID No.4. The invention further discloses the detection kit containing those primers. The pyrosequencing technology is used for detecting the epidemic haematopoietic necrosis virus and has the advantages of high flux, rapidness and accuracy, short DNA sequence synthesis can be performed accurately, the sequencing process takes a short time, and construction of the standard operation process is facilitated.

Description

technical field [0001] The invention relates to a primer, a kit and a detection method for detecting epidemic hematopoietic organ necrosis virus by using pyrosequencing technology, belonging to the field of molecular biology. Background technique [0002] Epizootie hematopoietie necrosis (EHN) is a systemic disease of fish, which mainly infects redfin perch and rainbow trout, leading to necrosis of liver, spleen and hematopoietic tissues including kidney, and even death. A fish infectious disease caused by a class of viruses represented by Epizootiehematopoietie necrosis virus (EHNV). EHNV can cause high case fatality rates in redfin bass, cause significant economic losses to fisheries, and cause dramatic declines in wild populations. Rainbow trout have low infection rates and high mortality rates, making it difficult to detect the virus in apparently healthy fish . The International Office of Epizootics (OIE) has included EHNV in the must-report list, and many countries a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/6869C12Q1/701C12Q2600/158C12Q2565/301C12Q2563/107
Inventor 王娜张旻李霆王彩霞景宏丽吴绍强张利峰江育林
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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