Specific primers for detecting mRNA expression level of bovine LPL (lipoprotein lipase) genes and fluorescent quantitative detecting kit

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

Inactive Publication Date: 2015-09-09
LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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  • Abstract
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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 mRNA expression level of bovine LPL (lipoprotein lipase) genes and fluorescent quantitative detecting kit
  • Specific primers for detecting mRNA expression level of bovine LPL (lipoprotein lipase) genes and fluorescent quantitative detecting kit

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Embodiment 1

[0032] The fluorescence quantitative PCR detection kit of LPL 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 LPL 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'-CCGCAGACAGGATTACAG-3',

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

[0040] Standard LPL gene template: the concentration is 0.8 μmol / L, and the sequence of the LPL gene template is 5'-CCGCAGACAGGATTACAGGAGGAAAAGATTTTAGAGACATTGAAAGTAAATTTGCTCTCAGGACTCCCGAAGACACAGCTGAGGACACTTGCCACCTCA...

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Abstract

The invention provides specific primers for detecting the mRNA expression level of bovine LPL (lipoprotein lipase) genes. The nucleotide sequence of the forward primer is 5'-CCGCAGACAGGATTACAG-3'; and the nucleotide sequence of the reverse primer is 5'-AGGAATGAGGTGGCAAGT-3'. The invention further provides a corresponding detecting kit. The kit fulfills the purpose of simply and quickly detecting the transcription level of the LPL genes, and can be used for monitoring the mRNA expression state of different bovine species (including dairy cow, cattle and yak), different tissues and different periods. The kit has the advantages of high sensitivity, good stability and low experimental cost on the aspect of detecting the mRNA expression level of the genes. The kit can be used for monitoring the mRNA expression level of the LPL genes in the fat deposition process of different bovine species, interpreting the fat deposition principle and identifying the correlation between the genes and the animal fat deposition and meat quality.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a specific primer and a fluorescent quantitative PCR detection kit for detecting the expression level of bovine LPL 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 a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 裴杰褚敏郭宪包鹏甲梁春年丁学智阎萍冯瑞林王宏博朱新书
Owner LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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