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TaqMan real-time fluorescence quantitative PCR kit for sturgeon iridovirus and application of kit

A technology of real-time fluorescence quantification and iridescent virus, which is applied in the determination/inspection of microorganisms, microorganisms, biochemical equipment and methods, etc.

Active Publication Date: 2015-02-04
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional PCR method takes about 7 hours to complete

Method used

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  • TaqMan real-time fluorescence quantitative PCR kit for sturgeon iridovirus and application of kit
  • TaqMan real-time fluorescence quantitative PCR kit for sturgeon iridovirus and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Composition of Fluorescent Quantitative PCR Kit for Sturgeon Iridovirus:

[0037] A). Positive template pMCP

[0038] It is a pM D19-T vector containing a 519 nucleotide fragment (shown in SEQ ID NO.4) of the MCP gene of the pheasant iridovirus.

[0039] B). Fluorescence quantitative reaction solution:

[0040] 10×Taq reaction buffer, 2.5mmol / L dNTPs, 50μmol / L forward primer and reverse primer, 25μmol / L fluorescent probe, 5U / μL Taq DNA polymerase, sterile double distilled water.

[0041] The forward primer is 5'-TCTCGCTTCCTGTCGTCG-3';

[0042] The reverse primer is 5'-AGTTGGGCGTCAGCTTGG-3';

[0043] Fluorescent probe: 5'-FAM-ACTGTGCCAATGCTCTCAAACGAAT-ROX-3'.

[0044] Self-prepared reagents: 20% PEG, proteinase K (5-10mg / ml), phenol:chloroform (volume ratio 1:1), 3mol / L sodium acetate, 70% ethanol, absolute ethanol.

[0045] Used in the following examples.

Embodiment 2

[0047] Establishment of the standard curve of the fluorescent quantitative PCR kit for the saffron iridovirus.

[0048] A). Template preparation:

[0049] Ligate the 519 nucleotide fragment (shown in SEQ ID NO.4) containing the MCP gene of Sturgeon iridovirus into pMD19-T vector, transform the positive plasmid pMCP into E. The box was purified, and the positive recombinant plasmid concentration was determined to be 450 μg / mL by a spectrophotometer, and the A260 / A280 value was 1.88.

[0050] And calculate the DNA copy number of the recombinant plasmid according to the following method.

[0051] Molecular copy number (copies / μL) = DNA mass concentration / DNA molecular weight, where: DNA mass concentration = 260nm absorbance × dilution factor × 6.02 × 10 23 ; DNA molecular weight = number of DNA bases × 324.5.

[0052] B).Standard curve preparation

[0053] The positive plasmid pMCP was serially diluted 10 times to 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1 ...

Embodiment 3

[0058] Specific detection:

[0059] The real-time fluorescent quantitative PCR specific detection was carried out using the total genomic DNA of the sturgeon iridovirus, the sturgeon herpesvirus-I, II, the spade sturgeon iridescent virus and the spleen tissue cells of the sturgeon as templates.

[0060] The results showed that the total DNA template of Sturgeon iridescent virus had a typical amplification curve and a high fluorescence signal value, and the detection result was positive. There was no specific amplification signal in the total genome DNA of the control group Acipenser herpesvirus-I and Acipenser II, Acipenser iridovirus and spleen tissue cells of Acipenser schneideri in this sample.

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Abstract

The invention discloses a TaqMan real-time fluorescence quantitative PCR kit for sturgeon iridovirus and application of the kit. The kit comprises bacteria-free double distilled water, a 10*Taq reaction buffer, dNTPs, a forward primer 5'-TCTCGCTTCCTGTCGTCG-3', a reverse primer 5'-AGTTGGGCGTCAGCTTGG-3', a fluorescent probe 5'-ACTGTGCCAATGCTCTCAAACGAAT-3', 5U / microliter Taq DNA polymerase and a standard positive template pMCP, wherein the end 5' of the fluorescent probe is marked with FAM, while the end 3' of the fluorescent probe is marked with ROX. The kit is accurate in quantification, and high in detection speed, in other words, the kit only takes 1 hour, and only 2-3 hours in total are required in combination with the preparation of DNA extraction; the method of the kit is feasible, and simple and convenient to operate; high-throughput sample detection can be carried out simultaneously; the accuracy rate reaches up to 98%.

Description

technical field [0001] The invention belongs to the technical field of sturgeon disease detection, and more specifically relates to a TaqMan real-time fluorescent quantitative PCR kit for sturgeon iridescent virus. The PCR kit can not only use all types of fluorescent quantitative PCR instruments currently on the market, but more importantly, Rapid and quantitative detection of sturgeon iridescent virus. At the same time, it also relates to the use of a sturgeon iridescent virus TaqMan real-time fluorescent quantitative PCR kit. technical background [0002] Sturgeon belongs to Osteichthyes, Actinopterygii, Chon drostei, and Acipenseriformes. It is an existing ancient biological population and is widely distributed in the northern hemisphere. There are 26 species of sturgeon in 2 families, 6 genera, and 8 species distributed in my country, mainly distributed in the Yangtze River Basin, Heilongjiang River Basin and Xinjiang. With the continuous reduction of wild sturgeon re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/70C12Q2531/113C12Q2561/101C12Q2561/113
Inventor 周勇曾令兵孙爱义解旭东范玉顶徐进马杰
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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