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Hematopoietic chimera real-time quantitative PCR (Polymerase Chain Reaction) detection and specimen collection method

A technology of real-time quantitative and detection methods, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., and 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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  • Hematopoietic chimera real-time quantitative PCR (Polymerase Chain Reaction) detection and specimen collection method
  • Hematopoietic chimera real-time quantitative PCR (Polymerase Chain Reaction) detection and specimen collection method
  • Hematopoietic chimera real-time quantitative PCR (Polymerase Chain Reaction) detection and specimen collection method

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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 PCR (Polymerase Chain Reaction) real-time quantitative detection method for detecting a hematopoietic chimera by using fluorescent dye SYBR green. The method comprises the following steps of: collecting a specimen; selecting genetic polymorphism markers to design a primer and a probe; detecting the hematopoietic chimera through real-time quantitative PCR; artificially synthesizing different dilute concentrations of hematopoietic chimera; and performing statistical analysis. The step of specimen collection comprises the specific procedures of clinically collecting blood samples of a donor and a receptor before transplantation and one month after transplantation; and directly extracting genome DNA by using the conventional method. In the invention, 12 specific genetic polymorphism markers capable of being used for Q-PCR to detect the hematopoietic chimera are introduced. Moreover, a melting curve is also used for verifying the specificity of the PCR product in order to overcome the defect that the fluorescent dye SYBR green brought into double-chain DNA is nonspecific. The real-time quantitative PCR method provided by the invention has the advantages of low price, long quality guarantee period and high sensitivity and is a fast, simple, convenient and reliable hematopoietic chimera detection method.

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 PCR detection and sample collection method for detecting 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 rout...

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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