Detection method, primer pair and kit for chicken-original STING (stimulator of interferon genes) fluorogenic quantitative PCR (polymerase chain reaction)

A detection kit and fluorescence quantitative technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems that detection methods have not been reported yet

Inactive Publication Date: 2016-04-13
GUANGXI UNIV
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Problems solved by technology

Yet, the detection method that detects chicken STING with SYBRGreenI ...

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  • Detection method, primer pair and kit for chicken-original STING (stimulator of interferon genes) fluorogenic quantitative PCR (polymerase chain reaction)
  • Detection method, primer pair and kit for chicken-original STING (stimulator of interferon genes) fluorogenic quantitative PCR (polymerase chain reaction)
  • Detection method, primer pair and kit for chicken-original STING (stimulator of interferon genes) fluorogenic quantitative PCR (polymerase chain reaction)

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Embodiment 1

[0023] Embodiment 1 prepares the standard curve of STING gene

[0024] 1. Design and synthesis of primers

[0025] According to the chicken STING sequence published by NCBI, three pairs of specific primers were designed using primer5.0 and Oligo7.0, and were synthesized by Beijing Liuhe Huada Company. The primer sequences are as follows:

[0026] Primer pair one: upstream primer: S 1 -F: 5′-TGACCGAGAGCTCCAAGAAG-3′

[0027] Downstream primer: S 1 -R: 5′-CGTGGCAGAACTACTTTTCAG-3′

[0028] Primer pair two:

[0029] Upstream primer: S 2 -F: 5′-TGACCGAGAGCTCCAAGAAG-3′

[0030] Downstream primer: S 2 -R: 5′-AGCTCTTCCATGCACTCCTT-3′

[0031] Primer pair three:

[0032] Upstream primer: S 3 -F: 5′-GCTCCAAGAAGAATGTCGCTCA-3′

[0033] Downstream primer: S 3 -R: 5′-AGCTCTTCCATGCACTCCT-3′

[0034] 2. Collection of chick peripheral blood leukocytes, spleen and trachea and other tissue samples

[0035] 2 mL of fresh anticoagulated blood from healthy Sanhuang chickens was collect...

Embodiment 2

[0050] The detection of STINGmRNA transcript level in the chicken tracheal tissue of embodiment 2

[0051] The trachea is the target organ of chicken infectious bronchitis virus (IBV). In order to explore the effect of IBV infection on the transcription level of STING mRNA in the trachea of ​​chicks, the real-time fluorescent quantitative PCR detection method of the chicken-derived STING gene established above was used to detect the IBV infection. Changes in mRNA levels of STING gene in chicken tracheal tissue.

[0052] 1. 48 feathers are randomly divided into two groups of 14 days old SPF chickens, 24 pigeons in every group, and group A is the test group, with 10 6 EID 50 The IBVM41 virus of / 0.1mL carries out eye-dropping, nasal drop route to challenge virus, and every 0.2mL, B group is control group, carries out eye-dropping, nasal drop with the negative allantoic fluid of 0.2mL. At 1, 3, 5, 8, 11, 14, 21 and 28 days after the challenge, 3 feathers were randomly selected ...

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Abstract

The invention discloses a pair of specificity real-time fluorogenic quantitative PCR (polymerase chain reaction) primers, namely an upstream primer S1-F and a downstream primer S1-R. Accordingly, the inventor designs and sets up a corresponding kit and a corresponding detection method. It is shown through experiments that the detection method, the primer pair and the kit have the advantages of being high in detection speed, accurate in quantification, high in specificity and sensitivity, good in repeatability and stability and free of pollution. A technological platform is provided for fast and accurately detecting the relative expression level of chicken-original STING (stimulator of interferon genes) mRNA, and a technological base is provided for further inquiring an innate immunity mechanism of chicken-related diseases.

Description

technical field [0001] The invention belongs to the technical field of chicken-derived STING gene detection, and in particular relates to a chicken-derived STING gene fluorescence quantitative PCR detection method, a primer pair and a kit. Background technique [0002] STING (Stimulator of interferongenes) protein is a recently discovered important receptor molecule involved in the body's innate immune response. When various viruses invade the body and are recognized by pattern recognition receptors (PRRs), STING can activate NF-kB and IRF3-mediated The signal transduction pathway induces the production of type I interferon, which plays an important role in the body's antiviral innate immune response. Therefore, accurate quantification of STING in chicken tracheal tissue will help to further understand the immune status and antiviral immune mechanism of the tested chickens. [0003] Fluorescent quantitative PCR technology is a new technology often used in the quantitative d...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q2563/107C12Q2545/113
Inventor 磨美兰谢智文何怡宁曹艳杰戴锦龙李和鸣
Owner GUANGXI UNIV
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