Primers and probe for detecting amount of gene expression of interleukin 17-5 of pacific oyster by digital PCR

A technology for interleukin and gene expression, which is applied in the fields of molecular biology and biochemistry, and can solve the problem of detecting the time-series changes of the expression of interleukin 17-5 genes in the oyster long oyster, and the health status of the long oyster cultured without digital PCR detection. Long oyster interleukin 17-5 gene expression and other issues, to achieve high accuracy and specificity

Pending Publication Date: 2019-11-19
DALIAN OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far, there is no relevant report on the primers and probes for digital PCR detection of interleukin-17-5 gene expression in long oyster, so that there is no digital PCR-based detection of long oyster long oyster interleukin-17-5 gene expression The time-series changes of the quantity and use it as an index to evaluate the health status of cultured oysters

Method used

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  • Primers and probe for detecting amount of gene expression of interleukin 17-5 of pacific oyster by digital PCR
  • Primers and probe for detecting amount of gene expression of interleukin 17-5 of pacific oyster by digital PCR
  • Primers and probe for detecting amount of gene expression of interleukin 17-5 of pacific oyster by digital PCR

Examples

Experimental program
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experiment example 1

[0023] Experimental example 1: In long oyster blood lymphocytes C g Detection of the Absolute Expression of IL17-5 Gene

[0024] 1. Sample Collection

[0025] After prying open the long oyster shell with an oyster knife, puncture the blood sinus with a 10 mL syringe and extract hemolymph; filter through a 500-mesh sieve and add to a 1.5 mL EP tube, 800 g Centrifuge for 10 min to collect blood lymphocytes.

[0026] 2. Total RNA extraction and cDNA library construction

[0027] Add 1 mL Trizol reagent to each EP tube containing blood lymphocytes. Add 200 μL of pre-cooled chloroform (stored at -20°C) to every 1 mL of sample, shake vigorously for 3 min, 4°C, 12,000 g Centrifuge for 15 min. Carefully draw 400 μL of the upper transparent liquid, add the same volume of pre-cooled isopropanol (stored at -20°C), mix well, and place in a -80°C ultra-low temperature refrigerator to settle naturally. After 10 min, take out the sample, 4°C, 12,000 g Centrifuge for 15 min. Discard t...

experiment example 2

[0034] Experimental Example 2: Detection by Digital PCR C g Absolute expression level of IL17-5 gene

[0035] Using the digital PCR primers and probes of the embodiment of the present invention, the experiment was carried out according to the following reaction system (14.5 μL):

[0036] Reagent volume QuantStudio™ 3D Digital PCR Master Mix v2 7.3 μL Probe (final concentration 200 nM) 0.7 μL Forward primer (final concentration 900 nM) 0.7 μL Reverse primer (final concentration 900 nM) 0.7 μL cDNA template 1.4 μL DEPC water 3.7 μL

[0037] With the help of ProFlex™ 2x Flat PCR System (or Dual Flat Block GeneAmp™ PCR System9700) system, follow the following reaction conditions: 96°C for 10 min (denaturation), 60°C for 2 min (annealing and extension), 98°C for 30s, 39 cycles; 60°C for 2 min, 10°C ∞, detect the C g Absolute copy number of IL17-5 gene.

[0038]QuantStudio™ 3D Digital PCR Instrument system was used to read...

experiment example 3

[0040] Experimental Example 3: In blood lymphocytes of long oyster cultured in different periods C g Absolute expression of IL17-5 gene

[0041] Application of digital PCR primers and probe pairs of the present invention in Oyster hemocytes from May to August 2019 C g The mRNA expression level of IL17-5 was detected, and the results were as follows: image 3 shown. Among them, in May oyster blood lymphocytes C g The absolute amount of IL17-5 was 265.79 copies / μl, and in the oyster blood lymphocytes in July C g The absolute amount of IL17-5 was 261.60 copies / microliter, which was similar to the level in May. In August, oyster blood lymphocytes C g The absolute amount of IL17-5 was 133.95 copies / microliter, which was significantly lower than that in May and July ( p C g The absolute expression level of IL17-5 gene indirectly reflects the immune defense ability of cultured long oyster. The relevant research results have potential application value in early warning and fore...

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Abstract

The invention discloses primers and probe for detecting amount of gene expression of interleukin 17-5 of a pacific oyster by PCR. According to primer and probe nucleotide sequences, a forward primer is 5'-tccctggtccaccatcatg-3', a reverse primer is 5'-ggaacaccactgaggacttgg-3', and the probe is 5'-cgcgtcagctacc-3'. A fluorescent group added to the probe is 6-carboxyfluorescein. The accuracy and specificity of the primers and the probe are high, so the digital PCR technology is used for detecting the sequential variation of the interleukin 17-5 of the pacific oyster, and the result is used as anindex to evaluate the health status of the farmed pacific oyster. An established evaluation system has potential application value in early warning and prediction of diseases of farmed shellfishes, acontrol group or an internal control gene is not required, and the application limitation of a traditional fluorescence real-time quantitative PCR detection method is broken through.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and biochemistry, and in particular relates to a primer and a probe for digital PCR detection of interleukin 17-5 gene expression in long oyster. Background technique [0002] Interleukin 17 (Interleukin17, IL17) plays an important role in the body's defense system. IL17 can act indirectly on neutrophils by inducing downstream cytokines (such as chemokines or IL8) to promote their proliferation and migration; it can also cooperate with other cytokines (IL1, IL6 and TNF, etc.) neutrophils clear pathogens; in addition, IL17 can also promote the expression of a variety of downstream effector molecules involved in pathogen clearance, for example, IL17 in the intestine can enhance the synthesis of seal protein Claudin and tight junction protein Zona occludens-1, so that the epithelial The connection between cells is tighter, which promotes the isolation of the body from intestinal contents ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/158C12Q2600/124
Inventor 王玲玲刘兆群张玉坤宋林生宗亚男
Owner DALIAN OCEAN UNIV
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