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Giant salamander iridescent virus taqman real-time fluorescent quantitative PCR kit and its application

A giant salamander iridovirus, real-time fluorescence quantitative technology, applied in fluorescence/phosphorescence, determination/inspection of microorganisms, biochemical equipment and methods, etc.

Active Publication Date: 2011-12-21
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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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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  • Giant salamander iridescent virus taqman real-time fluorescent quantitative PCR kit and its application
  • Giant salamander iridescent virus taqman real-time fluorescent quantitative PCR kit and its application
  • Giant salamander iridescent virus taqman real-time fluorescent quantitative PCR kit and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Composition and preparation of fluorescent quantitative PCR kit for giant salamander iridescent virus.

[0032] A). Reagent composition:

[0033] 10×Taq reaction buffer, 2.5mmol / L dNTPs, and 5U / μL Taq DNA polymerase were all purchased from TaKaRa Company.

[0034] B). Fluorescence quantitative reaction solution:

[0035] 10×Taq reaction buffer 2μL, 2.5mmol / L dNTPs 0.4μL, 50μmol / L forward primer and reverse primer 0.4μL each, 25μmol / L probe GSIV probe 0.4μL, 5U / μL Taq DNA polymerase 0.4μL, 14 μl of sterile double distilled water. Wherein the fluorescent probe is the nucleotide sequence shown in SEQ ID NO: 3, the 5' end of the fluorescent probe is marked with FAM, and the 3' end of the fluorescent probe is marked with ROX.

[0036] C). Self-prepared reagents: 20% PEG, proteinase K (5-10mg / ml), phenol:chloroform (volume ratio 1:1), 3mol / L sodium acetate, 70% ethanol, absolute ethanol.

Embodiment 2

[0038] Establishment of quantitative formula of fluorescent quantitative PCR kit for giant salamander iridescent virus.

[0039] A). Template preparation:

[0040] The positive plasmid containing the 1392 nucleotide fragments of the giant salamander iridescent virus MCP gene ligated into the pGEM-T vector (seeing number: Promega Corporation, Technical Manual, NO: 042: 1-30, revised edition in 1999) was transformed into Escherichia coli After DH5α proliferated, it was extracted by alkaline lysis, purified by DNA purification kit, and the concentration of positive recombinant plasmid was determined by spectrophotometer to be 300 μg / mL, and the A260 / A280 value was 1.86.

[0041] And calculate the DNA copy number of the recombinant plasmid according to the following method.

[0042] 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 D...

Embodiment 3

[0046] Detection of giant salamander iridescent virus with a fluorescent quantitative PCR kit for rapid quantitative detection of giant salamander iridescent virus.

[0047] Sample: Nucleic acid was extracted from carp epithelial tumor cells (preserved by our laboratory) inoculated with giant salamander iridescent virus.

[0048] 1 The composition and preparation of the fluorescent quantitative PCR kit for giant salamander iridescent virus,

[0049] Reagent composition:

[0050] a). The DNA extraction reagent is prepared by oneself, 20% PEG, proteinase K (5-10mg / ml), phenol:chloroform (volume ratio 1:1), 3mol / L sodium acetate, 70% ethanol, absolute ethanol.

[0051] b). Standard positive template.

[0052] c). Fluorescence quantitative reaction solution: PCR 10×buffer 2μl, forward primer and reverse primer 0.4μl (50μmol / L), fluorescent probe 0.4μl (25μmol / L), dNTPs 0.4μl (10mmol / L), Taq DNA polymerase 0.4μl (5U / μl), sterile double distilled water 14μl. The fluorescent prob...

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Abstract

The invention discloses a giant salamander iridovirus TaqMan real-time fluorescent quantitative PCR (polymerase chain reaction) kit and application thereof. The kit comprises sterile double distilled water, a 10*Taq reaction buffer, 2.5mmol / L dNTPs (deoxyribonucleotide triphosphates) and a fluorescent quantitative reaction liquid. The kit is characterized in that the fluorescent quantitative reaction liquid contains primers and a fluorescent probe, wherein the primers are a forward primer and a reverse primer, the forward primer is 5'-GCGGTTCTCACACGCAGTC-3', the reverse primer is 5'-ACGGGAGTGACGCAGGTGT-3', and the fluorescent probe sequence is 5'-AGCCGACGGAAGGGTGTGTGAC-3'; the 5' terminal marker of the fluorescent probe is FAM, and the 3' terminal marker is ROX; 5U / mu L Taq DNA (deoxyribonucleic acid) polymerase and standard positive template pMCP are a pMD19-T vector composed of 1392 nucleotide segments containing giant salamander iridovirus MCP gene; and the vector proliferates in colibacillus E.coli DH5a. The invention also discloses application of the kit in detection of giant salamander iridovirus pathogen. The kit is quantitative and accurate, and has the advantage of high detection speed (the detection only takes 1 hour, and totally takes 2-3 hours plus the extraction and preparation of the DNA); the method is easy to implement and simple to operate; and the invention can be used for simultaneously detecting high-flux samples, and the accuracy is up to 98%.

Description

technical field [0001] The invention belongs to the technical field of giant salamander disease detection, and more specifically relates to a giant salamander iridescent virus TaqMan real-time fluorescent quantitative PCR kit. The PCR kit can not only use all types of fluorescent quantitative PCR instruments currently on the market, but more importantly, can quickly Quantitative detection of giant salamander iridescent virus. At the same time, it also relates to the use of a giant salamander iridescent virus TaqMan real-time fluorescent quantitative PCR kit. technical background [0002] The giant salamander (Andrias davidiamus), commonly known as the giant salamander, is a second-class national protected animal and is included in the CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) species catalog. It is the largest existing individual amphibian and has important scientific research value. and medicinal and nutritional value. However...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 周勇曾令兵张辉孟燕范玉顶肖艺
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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