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Method for synthesizing chimera in hemopoiesis chimera real-time quantitative polymerase chain reaction (PCR) detection

A real-time quantitative and synthetic method, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of high price and short shelf life

Inactive Publication Date: 2012-04-04
江苏迈健生物科技发展股份有限公司
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Problems solved by technology

The real-time quantitative PCR method of this TaqMan technology requires the application of a specific TaqMan probe, which has the disadvantages of high price and short shelf life

Method used

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  • Method for synthesizing chimera in hemopoiesis chimera real-time quantitative polymerase chain reaction (PCR) detection
  • Method for synthesizing chimera in hemopoiesis chimera real-time quantitative polymerase chain reaction (PCR) detection
  • Method for synthesizing chimera in hemopoiesis chimera real-time quantitative polymerase chain reaction (PCR) detection

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Embodiment Construction

[0021] Such as figure 1 As shown, according to the purpose of the present invention, a kind of novel method that utilizes fluorescent dye SYBR green to detect hematopoietic chimerism that one embodiment of the present invention establishes, comprises the following steps:

[0022] S1. Collection of specimens: Blood samples from donors and recipients before and one month after transplantation were collected clinically, and genomic DNA (genomic DNA) was directly extracted by conventional methods, and the DNA concentration was measured with a Nanodrop spectrophotometer, and then stored at 4°C for later use .

[0023] S2. Select polymorphic genetic markers and design primers and probes: nucleotide sequences of polymorphic genetic markers human allele short insertion / deletion polymorphisms (human biallelic short insertion / deletion polymorphisms) from Marshfiled Clinic (http: / / research.marshfieldclinic.org / genetics / Genetic Research). Among them, the selection criteria are: includin...

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Abstract

The invention provides a method for artificially synthesizing chimera in hemopoiesis chimera real-time quantitative polymerase chain reaction (PCR) detection by utilizing fluorescent dye SYBR green for detection. The PCR detection method comprises the following steps that: specimens are collected; polymorphism genetic marker design primers and probes are selected; the real-time quantitative PCR hemopoiesis chimera detection is carried out; 12 hemopoiesis chimeras with different diluting concentrations are artificially synthesized; and statistics analysis is carried out, wherein the method for artificially synthesizing the chimera comprises the following step that donator deoxyribonucleic acid (DNA) is used for diluting receptor DNA or the receptor DNA is used for diluting the donator DNA for artificially synthesizing 12 chimeras with different diluting concentrations. The invention introduces 12 specific polymorphism genetic markers capable of being used for Q-PCR (real-time fluorescence quantitative) detection of hemopoiesis chimera. In addition, in order to overcome the defect that the double-chain DNA inclusion of the fluorescent dye SYBR green is non-specific, and simultaneously, a fusion curve is used for verifying the specificity of the PCR products. The real-time quantitative PCR method has the advantages that the required cost is low, the quality guarantee period is long, in addition, the sensitivity is high, and the method belongs to the fast, simple, convenient and reliable hemopoiesis chimera detection method.

Description

technical field [0001] The invention relates to a real-time quantitative PCR detection method for hematopoietic chimeras, in particular to a method for synthesizing chimeras in the real-time quantitative PCR detection of hematopoietic chimeras using fluorescent dye SYBR green. Background technique [0002] With the use of allogeneic hematopoietic stem cells to treat various malignant and non-malignant blood diseases, the overall survival time and disease-free survival time of many patients are prolonged. The main causes of treatment failure are disease recurrence, graft rejection, and graft-versus-host disease. Therefore, quantitative analysis of donor chimerism after allogeneic stem cell transplantation in patients is an important tool for diagnosis of graft survival, early detection and treatment of disease recurrence, and graft rejection. The monitoring of chimerism after allogeneic hematopoietic stem cell transplantation has become a routine inspection method, especiall...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 虞强
Owner 江苏迈健生物科技发展股份有限公司
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