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Gene detection kit used for detecting cell chimerism or individual recognition

A cell and state technology, applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as difficulty in setting high-sensitivity detection schemes, complicated SNP quantitative methods, and inability to use micro-chimerism detection , to achieve the effects of easy promotion and use, improved detection sensitivity, and good versatility

Inactive Publication Date: 2010-03-17
北京市道培医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the difference between the alleles of a single nucleotide polymorphism is only one base, it is difficult to set a high-sensitivity detection scheme, and the detection sensitivity can often only reach 1%, and it still cannot be used for the detection of microchimerism
Moreover, the SNP quantification method is complex and poor in practicability

Method used

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  • Gene detection kit used for detecting cell chimerism or individual recognition
  • Gene detection kit used for detecting cell chimerism or individual recognition
  • Gene detection kit used for detecting cell chimerism or individual recognition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Design and synthesis of primers

[0051] Selection of candidate INDELs sites with strong polymorphisms in the Chinese Han population: According to the SNP database of the National Center for Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih. gov / sites / entrez?db=snp) to select INDELs polymorphism information, and initially select INDELs sites with strong polymorphism in this population in the database data.

[0052] Primer design as figure 1 , illustrates the primer design method and principle of the present invention: design and prepare corresponding primer sequences according to each INDELs site sequence selected: for each INDELs site, design a common upstream primer (F), a common Downstream primer (R), site-specific primer (+) for detecting insertion polymorphism (Insersion, I) and site-specific primer (-) for detecting deletion polymorphism (Deletsion, D). The 3' end of each site-specific primer has 4-6 bases that specifically correspond ...

Embodiment 2

[0059] Embodiment 2 The composition of kit of the present invention

[0060] The gene detection kit for detecting cell chimerism or individual identification according to the present invention, 10tests / box, stored at -20°C, consists of the following reagents:

[0061] 1) Genomic DNA extraction reagents (20 person-times), purchased from Aisijin Biotechnology (Hangzhou) Co., Ltd., item number: AP-MN-BLGDNA-50;

[0062] 2) 5ml of ultrapure water (MilliporeMILLI-Q PF PLUS pure water machine, resistivity > 18.2MΩ);

[0063] 3) 2ml of 2×PCR reaction solution, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., item number: KT201;

[0064] 4) 2ml of 2×SYBRGreen PCR reaction solution, purchased from Applied Biosystems Co., Ltd., Cat. No.: 4367659;

[0065]5) 50ul each of the various primers used in the detection (various primers are shown in Table 1, and their sequences were synthesized by IntegratedDNA Technologies in the United States, diluted with 1×TE to a concent...

Embodiment 3

[0071] Embodiment 3: This kit is used for the detection of individual identification

[0072] Genomic DNA samples of two different individuals (named A and B respectively) were randomly selected, and the polymorphism detection of the 8 INDELs loci in this kit was carried out respectively.

[0073] The PCR reaction system is 20 ul, each reaction system contains 10 ng of genomic DNA, and each INDELs site corresponds to 4 pmol of primers numbered "F, R, +" or "F, R, -" (used to detect insertion polymorphism and deletion polymorphism), 2×PCR reaction solution 10ul, supplement the reaction system with ultrapure water. The PCR reaction conditions were pre-denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 15 seconds-annealing at 58°C for 30 seconds-extension at 72°C for 45 seconds, and finally extension at 72°C for 7 minutes. The PCR product was detected by 2% agarose electrophoresis for 30 minutes, and the polymorphism of each site was judged acc...

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Abstract

The invention discloses a gene detection kit which is used for detecting cell chimerism or individual recognition, in particular relating to an oligonucleotide array sequence, a kit and a method usedfor detecting cell chimerism or individual recognition. The kit utilizes gene insertion / deletion polymorphism (INDELs) and site-specific fluorescent quantitative PCR to quantitatively detect that different individuals of human beings have chimerism or microchimerism of foreign body cells, which can be used for the detection of mother and son / daughter cell microchimerism, cell chimerism of donor / receptor after the transplantation of hematopoietic stem cells or other organs and residual diseases after the transplantation of hematopoietic stem cells of leukemia, and for the forensic individual identification, paternity test and the like. The kit in the invention has good specificity and high detection sensitivity, can accurately and quantitatively detect with convenient operation and with norequirement of special devices; and when adopting the method in the invention to carry out individual identification detection, only agaroseelectrophoresis is needed to distinguish the results.

Description

technical field [0001] The invention relates to a gene detection kit, a nucleotide sequence and a use method thereof for detecting cell chimeric state or individual identification, and belongs to the field of biotechnology. Background technique [0002] Use the unique genetic information of different biological individuals to distinguish different individuals or individual sources of cells, which can be used to detect the long-term microchimerism of mother and child cells after childbirth related to autoimmune diseases, hematopoietic stem cells or other organ transplants. Chimerism of recipient / recipient cells, minimal residual disease after leukemia hematopoietic stem cell transplantation, etc., have a wide range of applications in forensic evidence identification, paternity testing and other medical tests. The genome is a fragment containing all the genetic information of a person, which is born with and remains unchanged throughout life. This genetic information is conta...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 刘红星朱平王倩蔡鹏童春容
Owner 北京市道培医院
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