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Method for detection of Italian bee immunogene defensin-1 expression by fluorescence RT-PCR technology

A kind of Italian honeybee, RT-PCR technology, applied in the direction of biochemical equipment and methods, microbial determination/examination, etc., can solve diseases and parasites, bee colony collapse syndrome, bee immune system damage, etc. problems, to shorten the experiment time, avoid subjectivity, and improve sensitivity

Inactive Publication Date: 2017-12-15
SERICULTURAL RES INST ANHUI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, due to the destruction of the ecological environment due to the excessive use of pesticides in agricultural and forestry production, the widespread use of antibiotics in the process of bee disease control, environmental pollution, interference from human activities, and destruction of vegetation, the immune system of bees has been destroyed, making In the process of bee breeding, bees are frequently attacked by diseases and parasites, and even bees in Europe and the United States have colony collapse syndrome, which has caused huge economic losses to beekeepers

Method used

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  • Method for detection of Italian bee immunogene defensin-1 expression by fluorescence RT-PCR technology
  • Method for detection of Italian bee immunogene defensin-1 expression by fluorescence RT-PCR technology
  • Method for detection of Italian bee immunogene defensin-1 expression by fluorescence RT-PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Obtaining the sequence of the Italian honeybee defensin-1 gene

[0039]Open the Beebase (http: / / hymenopteragenome.org / beebase / ) database, enter the keyword defensin-1 in the search bar, and find two results of defensin-1-1 and defensin-1-2. Click on defensin-1-1 to get its gene number GB41428 in the Beebase database. Click to enter the gene description page, select the link of the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database, enter NCBI, get the gene number LOC406143, and the mRNA number of Ref-seq is NM-001011616.2, Protein The number is NP_001011616.2, and the CDS and amino acid sequence of defensin-1 are obtained.

Embodiment 2

[0041] Extraction of Total RNA from Apis mellifera

[0042] Two groups (three parallel samples in each group) of healthy Italian bees were selected to conduct experiments on the effect of antibiotics on the expression of defensin-1. The three bee colonies in the experimental group were fed for one week according to the instructions for the use of benomyl. The three bee colonies in the experimental group were fed with the same amount of sugar water for one week. After treatment, appropriate amount of Italian worker bees were selected and stored at -80°C.

[0043] (1) Take 10 Italian worker bees and place them in a sterile mortar, pour liquid nitrogen into them and quickly grind them thoroughly with a grinding rod. Set for 5min.

[0044] (2) Add 200 μL of chloroform into the centrifuge tube, shake vigorously for 5-10 minutes, and let stand for 5 minutes.

[0045] (3) Centrifuge at 12,000 rpm for 10 min at 4°C, transfer the upper colorless aqueous phase into a new sterile cen...

Embodiment 3

[0050] cDNA synthesis

[0051] The total RNA of Apis mellifera described in Example 2 was used as a template. Take a sterilized 0.2mL centrifuge tube and add the following reaction system (20μL system):

[0052] (1) Add the following reagents to the nuclease-free PCR tube in ice bath:

[0053]

[0054]

[0055] (2) Gently mix and centrifuge for 3-5s. After incubating the reaction mixture at 65°C for 5 minutes, ice-bath for 2 minutes, and then centrifuge for 3-5s.

[0056] (3) Place the test tube in an ice bath, and then add the following reagents:

[0057] 5×RT Buffer 4.0μL

[0058] Thermo Scientific RiboLock RNase Inhibitor (20U) 0.5μL

[0059] RevertAid Premium Reverse Transcriptase (200U) 1.0μL

[0060] (4) Gently mix and centrifuge for 3-5s

[0061] (5) Carry out the reverse transcription reaction on the PCR instrument according to the following conditions

[0062] ①Incubate at 25°C for 10 minutes

[0063] ②cDNA synthesis at 50°C for 30min

[0064] ③ Stop th...

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Abstract

Particulally relating to the field of biotechnology, the invention discloses a method for detection of Italian bee immunogene defensin-1 expression by fluorescence RT-PCR technology. Establishment of the Italian bee immunogene defensin-1 real-time fluorescence RT-PCR detection method comprises the steps of: (1) gene detection and acquisition with primers; (2) extraction of total RNA; (3) cDNA synthesis; (4) real-time PCR; and (5) calculation of the Italian bee defensin-1 relative expression level before and after benomyl treatment. The invention lays the foundation for study of Italian bee defensin-1 expression regulation mechanism and immunological functions thereof, the disease-resistant molecular mechanism of Italian bees, scientific utilization of antibiotics to ensure safety production of bee products and disease control of bees, study of spatial and temporal expression condition of Italian bee immunogene defensin-1 and the influence of antibiotic benomyl feeding on the Italian bee immunogene defensin-1 expression level, and provides a technical platform for defensin-1 gene identification on the mRNA level and relative quantitative analysis.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to specific primers for detecting the expression of the Italian honeybee immune gene defensin-1, and a method for detecting the expression of the Italian honeybee immune gene defensin-1 by fluorescent RT-PCR technology. Background technique: [0002] Defensin is the largest subfamily of the antimicrobial peptide family. It is isolated from mammals, insects, and plants and acts as an effector molecule of innate immunity. It has a broad-spectrum antibacterial effect and is effective against G + The killing effect is greater than that of G - It also acts on fungi and some eukaryotic cells. It is the first step for the body to effectively resist pathogenic infection, and it plays a role in regulating the body's immune system. [0003] Bee defensin-1 is a protein produced by bees that has strong resistance to pathogenic bacteria. This protein is a component of the innate immune system of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q1/686C12Q2600/124C12Q2600/158C12Q2561/113C12Q2563/107C12Q2545/114C12Q2521/107
Inventor 代君君范涛舒蕊刘健章玉萍张丽丽陈明
Owner SERICULTURAL RES INST ANHUI ACADEMY OF AGRI SCI
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