Method for real time quantitative PCR detection of hematopoietic chimeras and designing of genetic marker primers of hematopoietic chimeras

A technology for real-time quantification and primer design, which can be used in biochemical equipment and methods, microbial determination/inspection, DNA preparation, etc., and can solve problems such as short shelf life and high price.

Inactive Publication Date: 2012-05-09
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 real time quantitative PCR detection of hematopoietic chimeras and designing of genetic marker primers of hematopoietic chimeras
  • Method for real time quantitative PCR detection of hematopoietic chimeras and designing of genetic marker primers of hematopoietic chimeras
  • Method for real time quantitative PCR detection of hematopoietic chimeras and designing of genetic marker primers of hematopoietic chimeras

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Embodiment Construction

[0021] like 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: including a...

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Abstract

The invention brings forward a method for PCR real time quantitative detection of hematopoietic chimeras and designing of genetic marker primers of the hematopoietic chimeras by using the fluorescent dye SYBR green. The PCR detection method comprises the following steps: collecting a specimen; selecting designed primers of polymorphic genetic markers and probes; carrying out real time quantitative PCR detection on a hematopoietic chimera; artificially synthesizing 12 hematopoietic chimeras of different diluted concentration; carrying out statistical analysis; wherein, selection criterion for the designed primers of the genetic markers is that the genetic markers contain at least more than two continuous allelic polymorphism with different bases and show high heterozygosity in a general population. The invention introduces 12 specific polymorphic genetic markers applicable to real time fluorescence quantitative detection of hematopoietic chimeras; moreover, a melting curve is used to verify specificity of PCR products so as to compensate for non-specificity of inclusion of double-chain DNA in the fluorescent dye SYBR green. The real time quantitative PCR detection method provided in the invention has the advantages of low cost, a long quality guarantee period and good sensitivity and is a fast, simple and reliable detection method for hematopoietic chimeras.

Description

technical field [0001] The invention relates to a real-time quantitative PCR (Polymerase Chain Reaction, polymerase chain reaction) detection method for hematopoietic chimeras, in particular to a real-time quantitative PCR detection and genetic marker design primers for detecting hematopoietic chimeras using fluorescent dye SYBR green Methods. 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 c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/10G01N21/64
Inventor 虞强
Owner 江苏迈健生物科技发展股份有限公司
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