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Special primer for detecting SNP site of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation and application

A hematopoietic stem cell and invasive technology, applied in the biological field, can solve the problems of aspergillosis and other problems, and achieve the effect of good accuracy, high specificity and cost saving

Active Publication Date: 2021-02-23
PEOPLES HOSPITAL PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the use of MALDI-TOF-MS to detect the risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation

Method used

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  • Special primer for detecting SNP site of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation and application
  • Special primer for detecting SNP site of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation and application
  • Special primer for detecting SNP site of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] This example is used to illustrate the special primers provided by the present invention for detecting the risk SNP sites of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation.

[0074] Design specific PCR primers and UEP primers for rs4257674, rs3921, and rs1800629 loci, and use matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technology to identify risk genes for invasive aspergillosis after allogeneic hematopoietic stem cell transplantation detection.

[0075] Specific PCR primers include:

[0076] Sequence 1, rs1800629 PCR primer 1:

[0077] 5'-ACGTTGGATGGATTTGTGTGTAGGACCCTG-3';

[0078] Sequence 2, rs1800629 PCR primer 2:

[0079] 5'-ACGTTGGATGGGTCCCCAAAAGAAATGGAG-3';

[0080] Sequence 3, namely rs4257674 PCR primer 1:

[0081] 5'-ACGTTGGATGGTATCATGTGCTTTCTACTG-3';

[0082] Sequence 4, namely rs4257674 PCR primer 2:

[0083] 5'-ACGTTGGATGACTGGATGATGAATCGTGGC-3';

[0084] Sequence 5, nam...

Embodiment 2

[0096] This example is used to illustrate the method provided by the present invention for detecting the risk SNP sites of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation.

[0097] 1. Extraction of template DNA

[0098] Genomic DNA was extracted from 800 cases of isolated peripheral blood, and the concentration was uniformly diluted to 20-30ng / μL.

[0099] 2. PCR reaction

[0100] Prepare the reaction system in a 5μL PCR 96-well plate:

[0101] Template DNA 1μL, Amplification Primer Mix 1μL, 10×PCR Buffer (containing 15mM Mg 2+ )0.5μL, MgCl2 (25mM) 0.4μL (MgCl in the reaction system 2 The final concentration of dNTP (25mM) is 2mM), dNTP (25mM) 0.1μL (the final concentration of dNTP in the reaction system is 0.5mM), Hotstar Taq (5U / μL) 0.2μL (the final concentration of Hotstar Taq in the reaction system is 0.2U / μL ), filled to 5 μL with MBG water. Seal the 384-well plate with Parafilm.

[0102] The amplification primer Mix consists of prime...

Embodiment 3

[0131] This example is used to illustrate the method of detecting the risk SNP sites of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation and the application of special primers.

[0132] 1. Extraction of template DNA

[0133] Genomic DNA was extracted from the peripheral blood of 8 subjects, and the concentration was uniformly diluted to 25ng / μL. The 8 subjects were all bone marrow donors for allogeneic hematopoietic stem cell transplantation.

[0134] 2. PCR reaction: see step 2 of Example 2.

[0135] 3. Alkaline phosphatase treatment (SAP digestion reaction): see step 3 of Example 2.

[0136] 4. Single base extension reaction: refer to step 4 of Example 2.

[0137] 5. Resin purification: refer to Step 5 of Example 2.

[0138] 6. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) detection: see step 6 of Example 1.

[0139] After testing, in the 8 tested samples in this embodiment, the detection rate ...

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Abstract

The invention belongs to the technical field of biology, and provides a special primer for detecting SNP sites of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation.The special primer comprises a first primer group and a second primer group; the first primer group is a specific PCR primer consisting of three pairs of primer groups; and the second primer group isa single base extension primer consisting of three primers. The invention also provides a preparation of the SNP sites for detecting the invasive aspergillosis risk after allogeneic hematopoietic stemcell transplantation, and an SNP sites information method for detecting the invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation. By adopting the special primer for detecting the SNP sites of the invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation provided, the correlation between the gene polymorphism of the three SNP sites relatedto the attack risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation and the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation can be efficiently and simultaneously completed through one-time detection, so that the detection time is greatly shortened, and the cost is saved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a special primer and application of a SNP site for detecting the risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation. Background technique [0002] Invasive aspergillosis (IA) is a common and important complication after allogeneic hematopoietic stem cell transplantation (HSCT). %, it can even be as high as 20% after incompatibility / haplotype hematopoietic stem cell transplantation; the prognosis is poor: the mortality rate of patients is as high as 30-90%. The long-term survival rates were 24.4% and 71.2% respectively; 3. The treatment time is long and the treatment cost is high. [0003] In view of the above characteristics, the prevention of IA is very important in the clinical practice of HSCT. The current prevention strategy in clinical practice is still relatively rough, and it is not possible to carry out stratified prevention based on the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/113C12Q2521/525C12Q2533/101C12Q2565/627
Inventor 孙于谦
Owner PEOPLES HOSPITAL PEKING UNIV
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