A primer, kit and detection method for detecting channel catfish virus using pyrosequencing technology

A technology of pyrosequencing and channel catfish, applied in the field of molecular biology, can solve the problems of false positives, unable to achieve rapid confirmation at the molecular level, low sensitivity of serological methods, etc. Easy-to-use effects

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

The sensitivity of serological methods is low; 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 channel catfish virus using pyrosequencing technology
  • A primer, kit and detection method for detecting channel catfish virus using pyrosequencing technology
  • A primer, kit and detection method for detecting channel catfish virus using pyrosequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 detects the design of the primer of channel catfish virus

[0029] Through the comparative analysis of the published channel catfish virus gene sequences in the NCBI gene bank (GenBank) of the National Center for Bioinformatics in the United States, specific PCR primer pairs and pyrosequencing primers were designed according to the specific sequence of the CCV PK gene, and a set of optimal PCR primers was screened out. Use the best primers for subsequent PCR reactions and sequencing.

[0030] The channel catfish virus-specific primer designed according to the specific region sequence of the channel catfish virus PK gene can amplify a specific single band for the channel catfish virus DNA, and the length of the PCR amplification fragment is 144 bp. Then, pyrosequencing primers were designed according to the specific nucleotide sequence contained in the amplified region (shown by the specific target sequence SEQ ID No. 1) to determine the channel catfish viru...

Embodiment 2

[0035] Embodiment 2 kit composition

[0036] The kit includes the primer set designed in Example 1 for detecting channel catfish virus, that is, a pair of amplification primers and a 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 (PyroMarkGold Q96 SQA Reagents, denaturing buffer, washing buffer, QIAGEN Company), etc., the above materials and reagents can be mixed and packaged, or packaged separately, Preferably, it is packaged individually.

Embodiment 3

[0037] The establishment of embodiment 3 detection method

[0038] 1. Extraction of channel catfish virus DNA

[0039] Using CCV (preserved in our laboratory) as raw material, DNeasy Blood&Tissue Kit (QIAGEN, Cat. No. 69506) was used to extract DNA. The kit operation steps are as follows:

[0040] (1) Pipette 20 μl proteinase K to the bottom of a 1.5ml centrifuge tube. (2) Add 200 μl of the virus solution of the genome to be extracted (the virus titer is 10 7 TCID 50 / 0.1ml) into a 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. (7) Transfer the mixture obtained in step 6 to a ...

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Abstract

The invention discloses a primer and kit for detecting channel catfish viruses through the pyrosequencing technology and a detecting method, and particularly relates to the primer for detecting channel catfish viruses through the pyrosequencing technology. The primer includes an amplification primer pair shown in SEQ ID No.2 and SEQ ID No.3 in the sequence list and a sequencing primer shown in SEQ ID No.4 in the sequence list. The invention further discloses the detecting kit with the primer. When channel catfish viruses are detected through the pyrosequencing technology, the advantages of high flux, rapidness and accuracy are achieved, short DNA sequence analysis can be accurately conducted, the time consumed by the sequencing process is short, and the standard operation procedure can be conveniently established.

Description

technical field [0001] The invention relates to a primer, a kit and a detection method for detecting channel catfish virus by using pyrosequencing technology, belonging to the field of molecular biology. Background technique [0002] Channel catfish virus (CCV) is an enveloped double-stranded DNA virus, also known as Ictalurid herpesvirus 1. The latest virus classification system reported by the International Committee on Taxonomy of Viruses classified it as Classified as Herpesviridae, Rhinovirus genus. The channel catfish virus disease (CCVD) caused by this virus is an acute and fatal infectious disease, which can cause serious losses to the channel catfish breeding industry. Class II infectious diseases in the Infectious Diseases and Parasitic Diseases List. Channel catfish and other catfish are susceptible to CCV. In natural water, the virus only infects catfish juveniles and fry, and the mortality rate of hatchlings reaches 100%. The disease is mainly prevalent in No...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q1/701C12Q2565/301C12Q2563/107C12Q2531/113
Inventor 王娜吴绍强李霆张旻景宏丽王彩霞张利峰江育林
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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