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Real-time quantitative polymerase chain reaction (PCR) detection method for hematopoietic chimeras

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 short shelf life and high price.

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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  • Real-time quantitative polymerase chain reaction (PCR) detection method for hematopoietic chimeras
  • Real-time quantitative polymerase chain reaction (PCR) detection method for hematopoietic chimeras
  • Real-time quantitative polymerase chain reaction (PCR) detection method for hematopoietic chimeras

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Embodiment Construction

[0021] Such as figure 1 As shown, according to the purpose of the present invention, a new method for detecting hematopoietic chimera using the fluorescent dye SYBR green established by an embodiment of the present invention includes the following steps:

[0022] S1. Collect specimens: clinically collect blood samples from donors and recipients before and one month after transplantation, and directly extract genomic DNA (genomic DNA) from them by conventional methods, measure the DNA concentration with Nanodrop spectrophotometer, and store them at 4°C for use .

[0023] S2. Select polymorphism genetic markers to design primers and probes (probe): the nucleotide sequence of polymorphism genetic markers, human biallelic 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 at least two consecutive base-di...

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Abstract

The invention provides a real-time quantitative polymerase chain reaction (PCR) detection method for detecting hematopoietic chimeras by utilizing a fluorescent dye SYBR green. The method comprises the following steps of: collecting samples; designing primers and probes by selecting polymorphic genetic markers; performing real-time quantitative PCR detection on the hematopoietic chimeras; synthesizing 12 hematopoietic chimeras with different diluted concentrations; and counting and analyzing. In the real-time quantitative PCR detection method, the twelve special polymorphic genetic markers for detecting the hematopoietic chimeras by fluorescent quantitative PCR are related, and in order to overcome the defect that double-stranded deoxyribonucleic acid (DNA) introduced into the fluorescent dye SYBR green is non-special, the specificity of a PCR product is verified by running a melting curve. In the real-time quantitative method, the cost is low, a quality guarantee period is long and the sensitivity is high, so the method is a method for detecting the hematopoietic chimeras simply, conveniently and reliably.

Description

Technical field [0001] The present 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 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, many patients have prolonged the overall survival time and disease-free survival time. The main causes of treatment failure are disease recurrence, graft rejection, and graft-versus-host disease. Therefore, quantitative analysis of donor chimera after allogeneic stem cell transplantation in patients is an important tool for diagnosing 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, es...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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