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Fluorescence-quantitative PCR detection kit for gene transcription level of agasicles hygrophila actin

A technology of A. chinensis and a detection kit, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., and can solve problems affecting the accuracy of quantification and achieve highly specific effects

Inactive Publication Date: 2015-10-07
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since SYBR Green binds to all double-stranded DNA, false positives caused by primer-dimers, single-stranded secondary structures, and erroneous amplification products will affect the accuracy of quantification

Method used

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  • Fluorescence-quantitative PCR detection kit for gene transcription level of agasicles hygrophila actin
  • Fluorescence-quantitative PCR detection kit for gene transcription level of agasicles hygrophila actin
  • Fluorescence-quantitative PCR detection kit for gene transcription level of agasicles hygrophila actin

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Effect test

example 1

[0032] Prepare the actin fluorescent quantitative PCR reaction solution in proportion, and the 25 μL reaction system is as follows:

[0033] Table 2

[0034]

[0035] Negative control and positive control should be set in the same quantitative reaction.

[0036] Take SYBR ? Premix Ex Taq TM 1250 μL, primer mixture 100 μL, ultrapure water 1000 μL, mix thoroughly, and prepare

[0037] 2350 μL of FQ-PCR master mix.

[0038] Thoroughly mix the above liquids, divide the premix into 100 small tubes according to 23.5 μL / tube, and add them to the special PCR reaction tubes for fluorescence quantification.

[0039] Prepare 5 tubes of standard actin gene template with serial dilutions of 10E+8 / ml, 10E+7 / ml, 10E+6 / ml, 10E+5 / ml, 10E+4 / ml, 10 μL in each tube.

[0040] FQ-PCR amplification: Add 1.5 μL of the cDNA to be tested (replaced with the above concentrations of standards here), water or standard actin gene template into each reaction tube containing 23.5 μL of...

example 2

[0044] Prepare the actin fluorescent quantitative PCR reaction solution in proportion, and the 25 μL reaction system is as follows:

[0045] table 3

[0046]

[0047] Negative control and positive control should be set in the same quantitative reaction.

[0048] Take SYBR ? Premix Ex Taq TM 1250 μL, 100 μL of primer mixture, and 1000 μL of ultrapure water were thoroughly mixed to prepare 2350 μL of FQ-PCR master mix.

[0049] Thoroughly mix the above liquids, divide the premix into 100 small tubes according to 23.5 μL / tube, and add them to the special PCR reaction tubes for fluorescence quantification.

[0050] Prepare 5 tubes of standard actin gene template with serial dilutions of 10E+8 / ml, 10E+7 / ml, 10E+6 / ml, 10E+5 / ml, 10E+4 / ml, 10 μL in each tube.

[0051] FQ-PCR amplification: Add 1.5 μL of standard actin gene template (negative control tube plus ultrapure water) to each reaction tube containing 23.5 μL of PCR reaction master mix, vortex and mix, ce...

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Abstract

The invention relates to a kit for gene transcription level of agasicles hygrophila actin ( actin ), and belongs to the technical field of fluorescence-quantitative DNA expansion in vitro in the molecular biological detecting techniques. The invention firstly protects a detecting primer, and the sequence of the detecting primer is showed in SEQ. ID NO. 1 and SEQ. ID NO; secondly the fluorescence-quantitative PCR detection kit capable of detecting the gene transcription level of the agasicles hygrophila actin quantitatively is provided, the operation of the kit is simple, convenient and quick, and the kit is composed of SYBR Premix Ex Taq, primer mixture, a standard actin gene template and ultrapure water. The invention further protects the application, detecting the expresssion quantity of the agasicles hygrophila actin, of the kit. According to the kit for the gene transcription level of the agasicles hygrophila actin, the transcription level of the actin gene can be measured accurately, the high specificity is achieved, the expansion curve shows that the fluorescence signal value of the actin gene is in accord with a standard S-shaped curve, and a melting curve shows that the fluorescent quantitation has high detecting specificity.

Description

technical field [0001] The invention relates to a kit for detecting actin (actin) gene transcription level of A. chinensis. The invention belongs to the technical field of fluorescence quantitative DNA in vitro amplification in molecular biology detection technology. Background technique [0002] Polymerase Chain Reaction (Polymerase Chain Reaction, PCR), that is, DNA in vitro amplification technology, is a biological high-tech pioneered by Mullis in the United States in 1985. With the emergence of high-temperature-resistant Taq enzyme, the improvement of amplification automation, PCR has been popularized rapidly in recent years, and it only needs picogram-level templates to quickly and specifically replicate any desired gene or DNA fragment in large quantities. [0003] Fluorescent quantitative PCR is a nucleic acid quantitative technology. This technology introduces specific fluorescent marker substances into the PCR reaction system, and monitors the amplification of DNA i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/158
Inventor 郑丽祯傅建炜史梦竹李建宇游泳林涛
Owner INST OF PLANT PROTECTION FAAS
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