Specific primers for detecting bovine LF gene mRNA (messenger ribonucleic acid) expression level and fluorescent quantitative detection kit

A technology for expression level and real-time fluorescence quantification, applied in the field of genetic engineering, can solve problems such as affecting the accuracy of quantification, and achieve the effects of good stability, simple and fast quantitative detection, and low experimental cost.

Inactive Publication Date: 2015-09-30
LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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Problems solved by technology

However, since SYRB 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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  • Specific primers for detecting bovine LF gene mRNA (messenger ribonucleic acid) expression level and fluorescent quantitative detection kit
  • Specific primers for detecting bovine LF gene mRNA (messenger ribonucleic acid) expression level and fluorescent quantitative detection kit

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Embodiment 1

[0032] The fluorescence quantitative PCR detection kit of LF gene mRNA expression level of the present invention is made up of following components:

[0033] 2×SYBR GREEN MIX 5mL;

[0034] Positive control: 500 μL of standard LF gene template;

[0035] Primer mixture 500μL;

[0036] Ultrapure water 5mL.

[0037] Among them, 2×SYBR GREEN MIX consists of the following components: Taq DNA polymerase 0.1 U / μL, dNTPs substrate 0.4 mmol / L, Mg 2+5.0 mmol / L, KCl 100 mmol / L, Tris·HCl 20 mmol / L at pH 8.3, 0.02% gelatin and SYBR Green dye.

[0038] Primer mixture: upstream primer 8 μmol / L, downstream primer 8 μmol / L. Wherein, the sequence of the upstream primer is: 5'-CCCGCTACTTGACCACCTT-3',

[0039] The sequence of the downstream primer is: 5'-TCCACTGCTGGCACTTCTTC-3'.

[0040] Standard LF gene template: the concentration is 0.8 μmol / L, and the sequence of LF gene template is

[0041] 5'-CCCGCTACTTGACCACCTTGAAGAACCTCAGGGAAACTGCGGAGGAGGTGAAGGCGCGGTACACCAGGGTCGTGTGGTGTGCCGTGGGACCCGA...

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Abstract

The invention provides specific primers for detecting bovine LF gene mRNA (messenger ribonucleic acid) expression level. The nucleotide sequences of the specific primers are as follows: the forward primer sequence is 5'-CCCGCTACTTGACCACCTT-3', and the reverse primer sequence is 5'-TCCACTGCTGGCACTTCTTC-3'. The invention also provides a corresponding detection kit. The kit achieves the goal of conveniently and quickly detecting LF gene transcription level, and can monitor the mRNA expression state of the LF gene of different bovine species (including milk cow, yellow cattle and yak) and different tissues in different periods. The kit has the advantages of high sensitivity, favorable stability and low experiment cost in the aspect of detection of the LF gene mRNA expression level.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to specific primers and a fluorescent quantitative PCR detection kit for detecting the expression level of bovine LF gene mRNA. Background technique [0002] Polymerase chain reaction (Polymerase Chain Reaction, PCR) is the most commonly used technique in DNA manipulation technology in vitro. PCR technology puts template DNA, specific primers, dNTPs substrates, heat-resistant DNA polymerase, magnesium ions, etc. in the same buffer reaction system, and performs repeated thermal cycles of high-temperature denaturation, low-temperature annealing, and medium-temperature chain extension to achieve target DNA Fragments appear as 2 in the reaction solution n Double amplification (where n is the number of thermal cycles). [0003] Fluorescent quantitative PCR technology is based on ordinary PCR reaction, combined with real-time fluorescence detection technology and...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6876C12Q2600/158
Inventor 裴杰阎萍郭宪包鹏甲褚敏丁学智冯瑞林梁春年朱新书王宏博
Owner LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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