A specific primer and fluorescent quantitative detection kit for detecting bovine lpl gene mrna expression level

A detection kit and fluorescence quantitative technology, applied in the field of genetic engineering, can solve the 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: 2021-09-28
LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

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

Method used

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  • A specific primer and fluorescent quantitative detection kit for detecting bovine lpl gene mrna expression level
  • A specific primer and fluorescent quantitative detection kit for detecting bovine lpl gene mrna expression level

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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’-CCGCAGACAGGATTACAGGAGGAAAAGATTTTAGAGACATTGAAAGTAAATTTGCTCTCAGGACTCCCGAAGACACAGCTGAGGACACTTGCCACCTC...

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Abstract

The invention provides a specific primer for detecting the mRNA expression level of bovine LPL gene. The nucleotide sequence is as follows: the upstream primer sequence is: 5'-CCGCAGACAGGATTACAG-3'; the downstream primer sequence is: 5'-AGGAATGAGGTGGCAAGT-3'. The invention also provides a corresponding detection kit. The invention realizes the purpose of detecting the LPL gene transcription level conveniently and quickly, and can monitor the mRNA expression state of the LPL gene in different cattle species (including cows, cattle and yaks), different tissues and different periods. The invention has the advantages of high sensitivity, good stability and low experiment cost in detecting gene mRNA expression level. The invention can monitor the expression level of LPL gene mRNA in the fat deposition process of different cattle species and be used to explain the deposition mechanism, and can identify the correlation between the gene and 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 Patents(China)
IPC IPC(8): C12N15/11C12Q1/6888C12Q1/686
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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