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Gene chip kit for aquatic animal DNA (deoxyribonucleic acid) virus detection, and preparation method and application thereof

A DNA virus and aquatic animal technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of difficulty, large differences in the structure of DNA virus and RNA virus, etc., to achieve enhanced sensitivity and detection range Expansion and reduction of testing costs

Active Publication Date: 2014-04-30
SHENZHEN AUDAQUE DATA TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are mainly 13 kinds of viral infectious diseases infecting fish that are seriously concerned by countries all over the world, among which there are 6 kinds of double-stranded DNA viruses. At present, rapid and simultaneous detection of all viruses is an urgent need for aquatic animal disease detection. Due to the virus structure This is a very challenging subject. The structure of DNA virus and RNA virus is quite different. It is also difficult to detect RNA virus and DNA virus at the same time.

Method used

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  • Gene chip kit for aquatic animal DNA (deoxyribonucleic acid) virus detection, and preparation method and application thereof
  • Gene chip kit for aquatic animal DNA (deoxyribonucleic acid) virus detection, and preparation method and application thereof
  • Gene chip kit for aquatic animal DNA (deoxyribonucleic acid) virus detection, and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: the preparation of gene chip

[0034] (1) Selection of target sequences for molecular beacon probes

[0035] Target gene sequence of channel catfish virus DNA polymerase (NCBI accession number GenBank No.: NC_001493.1), epidemic hematopoietic organ necrosis virus DNA polymerase target gene sequence (NCBI sequence accession number GenBank No.: FJ433873.1) , DNA polymerase target gene sequence of goldfish hematopoietic organ necrosis virus (NCBI accession number GenBank No.: JQ815364.1), koi herpesvirus DNA polymerase target gene sequence (NCBI sequence accession number GenBank No.: AY939862.1) Compared with the homology of the red sea bream iridescent virus DNA polymerase target gene sequence (NCBI sequence retrieval number GenBank No.: AB007366.1), the selection range of hybridization probes for each virus was determined, and Primer5.0 software was used to find all Possible probe sequences were screened, and the specificity of the probes was analyzed usin...

Embodiment 2

[0046] Embodiment 2: Design and preparation of PCR primers

[0047] iv. Comparative analysis of channel catfish virus, epidemic hematopoietic organ necrosis virus, goldfish hematopoietic organ necrosis virus, koi herpes virus and red sea bream iridescent virus DNA polymerase target gene sequence gene, designed a DNA virus specific for the above five The PCR demerging primer of DNA polymerase homology region, comprises the nucleotide sequence described in SEQ ID NO.1-2:

[0048] Forward primer: 5'-GAYTTYGCNWSNYTNTAYCC-3' (SEQ ID NO.1)

[0049] Reverse primer: 5'-TCNGTRTCNCCRTA-3' (SEQ ID NO.2)

[0050] The 5' of the primer nucleotide SEQ ID NO.1-2 is fluorescently labeled to obtain a PCR degenerate primer:

[0051] Han-forward primer: 5'-Cy5-GAYTTYGCNWSNYTNTAYCC-3',

[0052] Han-reverse primer: 5'-Cy5-TCNGTRTCNCCRTA-3'.

[0053] Using this primer for PCR can amplify trace DNA fragments in the sample. The scope of PCR amplification covers the DNA polymerase genes of all doub...

Embodiment 3

[0054] Embodiment 3: Aquatic animal DNA virus detection

[0055] (1) Sample preparation

[0056] Take a sample of suspected diseased fish to be tested, take a small amount of tissue samples from the liver, spleen, kidney and other organs of the fish, use a commercial kit (Qiagen51304QIAamp DNA Mini Kit) to extract DNA, and use the extracted sample DNA as a template to do Touchdown PCR , to obtain purified DNA samples.

[0057] The Touchdown PCR reaction system is shown in Table 1:

[0058] The reaction system composition of the PCR reaction of table 1, 50 μ L reaction system

[0059]

[0060] Amplify according to the following conditions:

[0061] 95℃, 4min;

[0062] 94°C, 1min, 63°C, 1min, 72°C, 1min, 2 cycles;

[0063] 94°C, 1min, 62°C, 1min, 72°C, 1min, 2 cycles;

[0064] 94°C, 1min, 61°C, 1min, 72°C, 1min, 2 cycles;

[0065] 94°C, 1min, 60°C, 1min, 72°C, 1min, 2 cycles;

[0066] 94°C, 1min, 59°C, 1min, 72°C, 1min, 2 cycles;

[0067] 94°C, 1min, 58°C, 1min, 72°C...

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PUM

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Abstract

The invention relates to a gene chip for detecting aquatic animal DNA (deoxyribonucleic acid) viruses, which comprises a solid-phase carrier and an aquatic animal DNA virus molecule beacon probe fixed to the solid-phase carrier, wherein the molecule beacon probe comprises nucleotide sequences disclosed as SEQ ID NO.3-7, the 5' end of the nucleotide sequences disclosed as SEQ ID NO.3-7 is connected with FAM fluorescein, and the 3' end is connected with a Dabcyl quenching group. The invention also relates to a kit for detecting aquatic animal DNA viruses, and a preparation method and application thereof. The gene chip and kit for detecting aquatic animal DNA viruses can implement one-step quick detection on all infected aquatic animal DNA viruses, have higher sensitivity, and reduce the detection cost for individual samples.

Description

technical field [0001] The invention belongs to the gene chip kit in the field of biotechnology, in particular to a gene chip kit for aquatic animal DNA virus detection and its preparation method and application. Background technique [0002] my country is a big breeding country, a big importer and exporter of aquatic animals and their products, and a big consumer. It is also a country with high and frequent outbreaks of epidemics and diseases. With the sharp reduction of catchable resources, aquaculture has become an important measure that our country has to adopt to meet the increasing protein demand of the people. However, the loss of disease seriously restricts the healthy and sustainable development of the aquaculture industry. Breeding models, changes in the ecological environment, and the spread of pathogens among multiple hosts all affect the prevalence of aquatic animal diseases. Diseases caused by viruses have caused great harm to modern aquatic product farming. Du...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6837C12Q1/70C12Q2531/113C12Q2537/143C12Q2565/501
Inventor 兰文升刘荭王津津史秀杰郑晓聪贾鹏于力何俊强黄佳鸣
Owner SHENZHEN AUDAQUE DATA TECH
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