Primer composition for detecting expression level of cynoglossus semilaevis interleukin IL-6 gene and application of primer composition

A technology of gene expression level and primer composition, applied in the field of genes, can solve problems such as undiscovered patent publications, and achieve the effects of saving manpower, high detection sensitivity, and strong specificity

Pending Publication Date: 2021-11-12
TIANJIN AGRICULTURE COLLEGE +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Through the search, no patent publications related to the patent application of the present invention have been found

Method used

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  • Primer composition for detecting expression level of cynoglossus semilaevis interleukin IL-6 gene and application of primer composition
  • Primer composition for detecting expression level of cynoglossus semilaevis interleukin IL-6 gene and application of primer composition
  • Primer composition for detecting expression level of cynoglossus semilaevis interleukin IL-6 gene and application of primer composition

Examples

Experimental program
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Effect test

Embodiment 1

[0048] This embodiment is a method for using the primer composition, which specifically includes the following steps:

[0049] Step 1: Tissue RNA Extraction

[0050] The tissue RNA was extracted with a commercial kit, and the RNA concentration, OD260 / 280 value and OD260 / 230 value of the extracted RNA were measured with an ultramicro spectrophotometer. In this example, the RNA extraction kit (TaKaRa MiniBEST Universal RNA Extraction Kit) was used to extract tissue RNA, and the specific operation was performed according to the instructions.

[0051] Step 2: cDNA reverse transcription

[0052] Take about 500ng RNA, use Takara commercial kit PrimeScript TM RT reagent Kit (Perfect Real Time) was used to synthesize cDNA, and the specific operation was carried out according to the instructions.

[0053] Step 3: Real-time quantitative PCR reaction

[0054] Real-time fluorescence quantitative amplification and data analysis were performed according to the PCR reagent (TB Green Pre...

Embodiment 2

[0063] In this example, the expression level of interleukin IL-6 gene in each tissue of tongue sole stimulated by inactivated Vibrio anguillarum was analyzed, and the specific steps were as follows:

[0064] Healthy juvenile tongue sole, 13-16 cm in length, were randomly divided into two groups, namely the experimental group and the control group. Each fish in the control group was intraperitoneally injected with 0.1 mL sterile PBS, and each fish in the experimental group was injected intraperitoneally with 0.1 mL of inactivated Vibrio anguillarum bacterial solution, the concentration of which was 10 8 CFU / mL, the liver, spleen, hindgut and kidney tissues of tongue sole were collected 4 hours after injection and stored in a -80°C refrigerator.

[0065] RNA was extracted from each tissue in the treatment group and the experimental group, and after reverse transcription, the expression level of IL-6 gene was detected by fluorescent quantitative PCR. By comparing with the contro...

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Abstract

The invention discloses a primer composition for detecting the expression level of a cynoglossus semilaevis interleukin IL-6 gene. The composition consists of a primer group 1 and a primer group 2; the primer group 1 is used for cynoglossus semilaevis IL-6 gene detection, and the primer group 2 is used for beta-actin reference gene detection; the primer group 1 is composed of primers as shown in a sequence SEQ ID NO. 1 and a sequence SEQ ID NO. 2; and the primer group 2 is composed of primers as shown in a sequence SEQ ID NO.3 and a sequence SEQ ID NO.4. According to the primer composition designed by the invention, specific amplification of the cynoglossus semilaevis IL-6 gene can be realized, amplification and melting curves of fluorescent quantitative PCR only have independent peaks, and the specificity is strong. The IL-6 gene expression level in a sample is quantified through a real-time fluorescence quantification method established by the invention, and the detection sensitivity is very high.

Description

technical field [0001] The invention belongs to the field of gene technology, in particular to a primer composition for detecting the expression level of interleukin IL-6 gene of half-smooth tongue sole and its application. Background technique [0002] Cellular interleukin (IL)-6 is secreted by many different types of cells and acts on T cells, B cells, and stem cells, thereby stimulating cell proliferation and differentiation, and regulating gene expression and cell survival. It has an important function in the body's defense and is one of the most important pro-inflammatory cytokines. When the body is infected or tissue damaged, IL-6 can activate immunity and acute phase response, and is released immediately after the host is exposed to pathogens, helping the body to clear the source of infection and restore damaged tissues. Its regulated expression is therefore associated with pathogen infection and immune stimulation. It is of great significance to study its expressio...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2563/107C12Q2545/114
Inventor 邵蓬汪笑宇汤青平贾磊王群山刘克明何晓旭
Owner TIANJIN AGRICULTURE COLLEGE
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