Multiple fluorescence PCR (polymerase chain reaction) kit capable of detecting five DNA (deoxyribonucleic acid) viruses of aquatic animal simultaneously

A DNA virus and multiple fluorescence technology, applied in 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: 2014-05-07
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 5 kinds of double-stranded DNA virus diseases. Rapid and simultaneous detection of all viruses is an urgent need for the current testing of farmed fish and aquatic products at ports. , due to the difference in virus structure, this is a very challenging topic. The structure of DNA virus and RNA virus is quite different, and it is difficult to detect these two types of viruses at the same time

Method used

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  • Multiple fluorescence PCR (polymerase chain reaction) kit capable of detecting five DNA (deoxyribonucleic acid) viruses of aquatic animal simultaneously
  • Multiple fluorescence PCR (polymerase chain reaction) kit capable of detecting five DNA (deoxyribonucleic acid) viruses of aquatic animal simultaneously
  • Multiple fluorescence PCR (polymerase chain reaction) kit capable of detecting five DNA (deoxyribonucleic acid) viruses of aquatic animal simultaneously

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Design and preparation of PCR primers

[0019] 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 target gene sequence of red sea bream iridescent virus DNA polymerase (NCBI sequence retrieval number GenBank No.: AB007366.1), PCR demerger primers were designed for the homologous region of DNA polymerase of the above five DNA viruses, including SEQ Nucleotide sequences described in ID NO.1-2:

[0020] Han-forward primer: 5'-GAYTTYGCNWSNYTNTAYCC-3' (SEQ ID NO.1)

[0021] Han-reverse primer: 5'-TCNGTRTCNCCRT...

Embodiment 2

[0023] Example 2: Synthesis of Specific Primers and Molecular Beacon Probes

[0024] (1) Design of specific primers

[0025] Five pairs of specific primers were synthesized according to the DNA polymerase target gene sequences of channel catfish virus, epidemic hematopoietic organ necrosis virus, goldfish hematopoietic organ necrosis virus, koi herpes virus and red sea bream iridovirus respectively.

[0026] (2) Selection of target sequences for molecular beacon probes

[0027] The homology of DNA polymerase target gene sequences of channel catfish virus, epidemic hematopoietic organ necrosis virus, goldfish hematopoietic necrosis virus, koi herpes virus and red sea bream iridescent virus was compared to determine the specificity of hybridization probes for each virus. Select the range, use Primer5.0 software to find all possible probe sequences, screen qualified probe sequences, and use BLAST software to analyze the specificity of the probes, and obtain 5 target sequences of...

Embodiment 3

[0037] Embodiment 3: Aquatic animal DNA virus detection

[0038] (1) Sample preparation

[0039] To sample the suspected diseased fish, take a small amount of tissue samples from the liver, spleen, kidney and other organs of the fish, extract DNA with a commercial kit, use the extracted sample DNA as a template, and perform Touchdown PCR to obtain a purified DNA sample.

[0040] The Touchdown PCR reaction system is shown in Table 3:

[0041] The reaction system composition of the PCR reaction of table 3, 50 μ L reaction system

[0042]

[0043] Amplify according to the following conditions:

[0044] 95℃, 4min;

[0045] 94°C, 1min, 63°C, 1min, 72°C, 1min, 2 cycles;

[0046] 94°C, 1min, 62°C, 1min, 72°C, 1min, 2 cycles;

[0047] 94°C, 1min, 61°C, 1min, 72°C, 1min, 2 cycles;

[0048] 94°C, 1min, 60°C, 1min, 72°C, 1min, 2 cycles;

[0049] 94°C, 1min, 59°C, 1min, 72°C, 1min, 2 cycles;

[0050] 94°C, 1min, 58°C, 1min, 72°C, 1min, 2 cycles;

[0051] 94°C, 1min, 57°C, 1min, ...

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Abstract

The invention relates to a multiple fluorescence PCR (polymerase chain reaction) kit capable of detecting five DNA (deoxyribonucleic acid) viruses of an aquatic animal simultaneously. The kit comprises a buffer solution, and also comprises five pairs of fluorescence PCR primers and five molecular beacon probes and a PCR primer, wherein the five pairs of fluorescence PCR primers and the five molecular beacon probes are respectively corresponding to channel catfish virus, epizootic haematopoietic necrosis virus, goldfish haematopoietic necrosis virus, fancy carp herpes virus and red porgy iridescent virus, the fluorescence PCR primers and the molecular beacon probes comprise nucleotide sequences shown in SEQ ID NO.3-17, and the PCR primer comprises nucleotide sequences shown in SEQ ID NO.1-2. The multiple fluorescence PCR kit capable of detecting five DNA viruses of the aquatic animal simultaneously can rapidly detect all the DNA viruses infecting the aquatic animals once, the sensitivity is increased, and the cost for detecting single sample is reduced.

Description

technical field [0001] The invention belongs to the fluorescent PCR kit in the field of biotechnology, in particular to a multiple fluorescent PCR kit for simultaneously detecting five kinds of DNA viruses in aquatic animals. 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 produc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2537/143C12Q2563/107
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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