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Method for detecting dose of ionizing radiation on human peripheral blood lymphocytes

A technology of lymphocytes and ionizing radiation, which is applied in the field of radiation biological dose estimation, can solve the problems of complex technology, which cannot meet the requirements of high-throughput detection, rapid quantitative detection of biological radiation dose, etc., and achieve good repeatability and stability, and save time.

Inactive Publication Date: 2014-05-21
NAT INST FOR RADIOLOGICAL PROTECTION & NUCLEAR SAFETY CHINESE CENT FOR DISEASE CONTROL & PREVENTION
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Problems solved by technology

However, these technologies are relatively complicated and require good professional knowledge and training before they can be carried out smoothly, and cannot meet the needs of high-throughput detection and rapid quantitative detection of biological radiation doses when a large number of people are irradiated

Method used

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  • Method for detecting dose of ionizing radiation on human peripheral blood lymphocytes
  • Method for detecting dose of ionizing radiation on human peripheral blood lymphocytes
  • Method for detecting dose of ionizing radiation on human peripheral blood lymphocytes

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Experimental program
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Effect test

Embodiment 1

[0019] Embodiment 1: TaqMan-MGB probe and primer pair design

[0020] 1) Design upstream and downstream primers and probe sequences according to the expression sequence of the lymphocyte pig3 gene.

[0021] The inventors used gene chip technology to screen the differential expression of 45,200 genes in the three dose groups of 0.5, 3 and 8Gy. Among them, the dose change trend of the positively regulated gene of lymphocyte pig3 is obvious, and it is of great significance to explore the expression change of pig3 gene in peripheral blood. The important practical value is closer to the application.

[0022] Lymphocyte pig3 gene expression sequence SEQ ID NO: 1 is:

[0023] ATTTCTCTGAAGCAACGCTGAAATTCACCAAAGGTGCTGGAGTTAATCTTATTCTAGACTGCATAGGCGGATCCTACTGG.

[0024] The upstream primer SEQ ID NO: 2 is: 5'-TCTCTGAAGCAACGCTGAAATTC-3'.

[0025] The downstream primer SEQ ID NO: 3 is: 5'-GTAGGATCCGCCTATGCAGTCTA-3'.

[0026] The probe nucleotide sequence of SEQ ID NO: 4 is: AAAGGTGCTGGA...

Embodiment 2

[0027] Embodiment 2: Construction of standard substance recombinant pUC57 plasmid

[0028] 1), routine method separates single lymphocyte from human peripheral blood;

[0029] 2), conventional methods extract the RNA of a single lymphocyte, and reverse transcribe it into cDNA;

[0030] 3), specific primer PCR amplifies the lymphocyte pig3 gene;

[0031] 4), after the PCR product is purified, according to figure 1 Schematic diagram of the construction of the standard material recombinant pUC57 plasmid, the PCR product was connected to the pUC57 plasmid (provided by Shanghai Sangong Co., Ltd.), and the recombinant pUC57 plasmid was constructed. figure 2 It is the agar gel electrophoresis picture of the recombinant pUC57 plasmid of the standard substance.

[0032] 5) The recombinant pUC57 plasmid was sequenced and verified, and the result was that the lymphocyte pig3 gene expression sequence SEQ ID NO: 1 was correctly inserted into the pUC57 plasmid.

Embodiment 3

[0033] Embodiment 3: the preparation of standard curve

[0034] 1) 10-fold serial dilution of the standard substance recombinant pUC57 plasmid. Utilize the NanoDrop 2000 ultraviolet spectrophotometer to measure the concentration of the recombinant pUC57 plasmid of the standard substance, and dilute the standard substance with double distilled water to 10 8 copies / μl. Add 9 μl of double-distilled water to five 0.5ml centrifuge tubes, draw 1 μl of 10 8 Copy / μl standard substance, add 9μl double-distilled water and mix thoroughly, and then carry out gradient dilution successively, and dilute the standard substance to 10 7 、10 6 、10 5 、10 4 and 10 3 Copies / μl, ready for use when sampling on 96-well plates.

[0035] 2) Fluorescent quantitative PCR was carried out using the 10-fold diluted standard substance recombinant pUC57 plasmid as a template.

[0036] The real-time fluorescence quantitative PCR instrument is the 7500fast Real Time PCR measuring instrument of ABI Compan...

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Abstract

The present invention relates to a method for detecting a dose of ionizing radiation on human peripheral blood lymphocytes. The method mainly comprises: designing primers and a Taqman-MGB probe according to a lymphocyte pig3 gene expression sequence, and constructing a recombinant vector containing the lymphocyte pig3 gene expression sequence as a standard substance; adopting the 10-fold serial diluted standard substance to carry out real-time fluorescence PCR, and drawing an absolute quantification standard curve; carrying out quantitative determination on the expression levels of the pig3 gene of lymphocytes with different culture times after ionizing radiations with different doses to obtain a dose-effect fitting curve of the radiation dose and the pig3 gene expression level; and carrying out quantitative determination on the expression level of the pig3 gene of lymphocytes with the radiation dose to be detected, and calculating the dose of the ionizing radiation on the lymphocytes requiring detection. According to the present invention, the dose of ionizing radiation on human peripheral blood lymphocytes can be rapidly and quantitatively detected so as to meet requirements of simpleness, rapid quantitation and high throughput, and the advantages can be provided when the large-scale radiation accident occurs.

Description

technical field [0001] The invention belongs to the field of radiation biological dose estimation, and in particular relates to a detection method for the dose of ionizing radiation received by human peripheral blood lymphocytes. Background technique [0002] The damage of ionizing radiation to the body is through the direct and indirect effects of the primary effect, causing changes in the structure and properties of biomolecules, and the damage at the molecular level further causes damage at the cell level, organ level, and overall level, resulting in corresponding biochemical metabolic disorders. And thus produce a series of manifestations or cause death and produce some long-term effects. [0003] In nuclear wars and nuclear or radiological accidents, the sorting and first aid of a large number of exposed people is the key to rescuing curable people (below 10Gy), and successful rescue requires rapid and accurate estimation of the exposure dose. There are many methods fo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/02G01N21/64
CPCC12Q1/6851C12Q2531/113C12Q2561/101C12Q2545/114
Inventor 刘青杰张庆召张德钦高玲封江彬
Owner NAT INST FOR RADIOLOGICAL PROTECTION & NUCLEAR SAFETY CHINESE CENT FOR DISEASE CONTROL & PREVENTION
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