Specific primer for detecting bovine IGF-1 genes, fluorescent quantitative PCR detection kit of bovine GHR genes, and detection method and application of kit

A technology of IGF-1 and kit, applied in the field of genetic engineering, can solve the problems affecting the accuracy of quantification, and achieve the effects of good stability, low experimental cost and high sensitivity

Inactive Publication Date: 2015-01-28
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

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  • Specific primer for detecting bovine IGF-1 genes, fluorescent quantitative PCR detection kit of bovine GHR genes, and detection method and application of kit

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Embodiment Construction

[0027] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0028] The fluorescence quantitative PCR detection kit of IGF-1 gene of the present invention is made up of following components:

[0029] 2×SYBR GREEN MIX 5mL;

[0030] Primer mixture 500μL;

[0031] Ultrapure water 5mL;

[0032] Positive control: 500 μL of standard IGF-1 gene template.

[0033] 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, 100 mmol / L KCl, 20 mmol / L Tris.HCl pH8.3, 0.02% gelatin and 20 ng / mL SYBR Green dye.

[0034] Primer mixture: upstream primer 8 μmol / L, downstream primer 8 μmol / L, ...

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Abstract

The invention relates to a specific primer for detecting bovine IGF-1 genes, a fluorescent quantitative PCR detection kit of the bovine GHR genes, and a detection method and an application of the kit. The fluorescent quantitative PCR detection kit of the bovine IGF-1 genes comprises a PCR reaction solution, a TaqDNA polymerase mixture, a SYBRGreen dye and primers. The sequences of the primers are as follows: the sequence of an upstream primer is 5'-AGCAGTCTTCCAACCCAA-3 '; and the sequence of a downstream primer is 5'-AGATGCGAGGAGGATGTG-3'. The invention also provides a detection method and an application of the above kit and the specific primer for detecting the bovine IGF-1 genes. The object of conveniently and rapidly detecting an mRNA expression level of the genes is achieved. The detection kit has the advantages of high sensitivity, good stability and low experimental cost in detecting the mRNA expression level of the genes. The method provided by the invention can effectively amplify IGF-1 genes derived from three bovine species including dairy cow, cattle and yak, and can detect the mRNA expression quantity of the IGF-1 genes.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a specific primer for detecting bovine IGF-1 gene and a fluorescent quantitative PCR detection kit for bovine IGF-1 gene. 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 flexible PCR reaction, combined with real-time fluorescence detection technology and co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q2563/107C12Q2545/113
Inventor 裴杰郭宪包鹏甲梁春年褚敏朱新书丁学智冯瑞林阎萍
Owner LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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