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RB1 mutant gene, primers, detection method, kit and application

A mutation gene and detection method technology, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problem of no clear mutation hotspots, achieve stable gene mutation sites, easy detection, The effect of improving sensitivity and specificity

Pending Publication Date: 2019-11-22
SHENZHEN CITY BAOAN DISTRICT MATERNAL & CHILD HEALTH HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although RB1 gene detection is of great significance for the early diagnosis and genetic counseling of RB, there are still a considerable number of unknown pathogenic gene loci due to the different types of RB1 gene mutations, wide distribution, and no clear mutation hotspots.

Method used

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  • RB1 mutant gene, primers, detection method, kit and application
  • RB1 mutant gene, primers, detection method, kit and application
  • RB1 mutant gene, primers, detection method, kit and application

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Genomic DNA from the peripheral blood of a child (deceased) who was clinically diagnosed as retinoblastoma was extracted and amplified by PCR, followed by Sanger sequencing to obtain relevant results. The sequencing process followed the standard operation of Sanger sequencing , artificially design Sanger sequencing primers, the specific steps are:

[0057] 1. Genomic DNA extraction. Genomic DNA was extracted from peripheral blood leukocytes using general methods or kits.

[0058] 2. Remove protein, precipitate and wash DNA.

[0059] 3. Measure the concentration and purity of DNA: usually 20-50ng / μL DNA can be obtained, and the purity (ratio of ultraviolet 260OD to 280OD) is 1.6-2.0.

[0060] 4. The PCR reaction system is shown in Table 1 (unit: μl).

[0061] Table 1

[0062] wxya 2 o

4.7 10×PCR Buffer 1 MgCl 2

0.6 dNTP 0.8 upstream primer 0.4 downstream primer 0.4 Taq enzyme 0.1 dna 2

[0063] Wherein...

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Abstract

The invention discloses a RB1 mutant gene, primers, a detection method, a kit and application. Compared with traditional biomarkers, the gene mutation site discovered by the invention is stable, minimally invasive and easy to detect, which will significantly improve the sensitivity and specificity of disease diagnosis. The mutation at c.116 of exon 1 of RB1 gene discovered by the invention can beused for early screening of carriers of retinoblastoma gene mutation, and further provides genetic consultation and eugenic guidance. At the same time, it can also be used to develop diagnostic kits to provide molecular diagnosis basis for patients with retinocytoma. The invention lays an important foundation for the research on the pathogenesis of retinoblastoma, and also provides a possible drugtarget for the treatment of patients with retinoblastoma.

Description

technical field [0001] The present invention relates to the fields of molecular biology and biotechnology, in particular to an RB1 mutant gene, primers, detection method, kit and application. Background technique [0002] Retinoblastoma (RB), originating from retinal embryonic nuclear layer cells, is the most common intraocular malignant tumor in infants with an incidence rate of 1:14000–20000. Its clinical manifestations include leukocoria, sensory strabismus, red and painful eyes, corneal opacity, and aseptic orbital cellulitis. Since 90% of children with RB develop before the age of 3, it is not easy to detect in the early stage. Although advances in comprehensive adjuvant treatments such as surgery, radiotherapy, and chemotherapy can improve the prognosis of patients, the mortality and incidence of secondary tumors in RB are still high worldwide, especially in developing countries. Statistics show that the survival rate of children with RB in China is only 63%, which i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156C12Q2600/118
Inventor 李发科许雪青王丰符胜煜谢小红雷佳凡李欢闫睿
Owner SHENZHEN CITY BAOAN DISTRICT MATERNAL & CHILD HEALTH HOSPITAL