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Real-time fluorescence quantitative PCR (polymerase chain reaction) kit for quantitatively detecting TRECs gene and application thereof

A technology of real-time fluorescence and reagent kits, which is applied in the measurement/testing of microorganisms, biochemical equipment and methods, etc., and can solve the problems that children miss the best time for diagnosis and treatment, it is difficult to judge the critical value, and newborns are missed. , to achieve accurate and reliable quantitative results, simple and fast detection methods, and convenient storage

Active Publication Date: 2014-08-06
无锡联合利康临床检验所有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Currently existing TRECs kits include two parts: DNA extraction and real-time quantitative PCR. Since TRECs are circular genes excised during the developmental rearrangement of the human genome, the content of TRECs is extremely low compared to genomic DNA, requiring extremely high DNA purity. , and the one-step real-time quantitative PCR is difficult to determine the critical value, resulting in false negatives, missed detection in the process of screening newborns, and children miss the best opportunity for diagnosis and treatment

Method used

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  • Real-time fluorescence quantitative PCR (polymerase chain reaction) kit for quantitatively detecting TRECs gene and application thereof
  • Real-time fluorescence quantitative PCR (polymerase chain reaction) kit for quantitatively detecting TRECs gene and application thereof
  • Real-time fluorescence quantitative PCR (polymerase chain reaction) kit for quantitatively detecting TRECs gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1: the preparation of kit

[0040] 1. Design and synthesis of primers and probes

[0041] According to the UCSC Human Gene Sorter on the UCSC website to query the gene sequences of TRECs and β-actin (http: / / genome.ucsc.edu / cgi-bin / hgNear), use Primer3.0 to design the upstream and downstream of nested PCR on the gene of TRECs Primers and fluorescent quantitative PCR upstream and downstream primers and probes, wherein the nested PCR upstream primer of TRECs and the gene binding position is upstream of the real-time fluorescent quantitative PCR upstream primer and the gene binding position, and the nested PCR downstream primer of TRECs and The binding position of the gene is downstream of the binding position of the real-time fluorescent quantitative PCR downstream primer and the gene. The selected primers have good specificity for gene binding and high PCR amplification efficiency. Both primers and probes were entrusted to Life Technologies to synthesize, and...

Embodiment 2

[0061] Embodiment 2: the use of kit

[0062] 1. Extraction of DNA from Dried Blood Filter Paper

[0063] The operation steps are as follows:

[0064] A. Obtain a dry blood filter paper piece with a diameter of 3 mm with a hole puncher, put it into a sterilized 1.5ml centrifuge tube, add 90 μl of Generation DNA purif.Soln I, and centrifuge at 3700 rpm for 30 seconds, so that the filter paper piece is immersed in the solvent;

[0065] B. After standing for 15 minutes, centrifuge at 3700rpm for 5 minutes to absorb the solution as much as possible;

[0066]C. Repeat steps A-B, wherein the standing time is 10 minutes;

[0067] D. Add sterile milli-Q water, centrifuge at 3700rpm for 30 seconds, and absorb the milli-Q water as much as possible;

[0068] E. Add 30μl Generation DNA Elution Soln II, centrifuge at 3700rpm for 1 minute, and place in a 99℃ water bath for 25 minutes;

[0069] F. After cooling to room temperature, centrifuge at 3700rpm for 30 seconds, store at 4°C...

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Abstract

The invention relates to a real-time fluorescence quantitative PCR (polymerase chain reaction) kit for quantitatively detecting TRECs gene and application thereof. The kit comprises a PCR system based on nested PCR technology and a real-time fluorescence quantitative PCR system based on real-time fluorescence PCR technology, wherein the nested PCR system comprises forward and reverse primers for TRECs and beta-actin genes; and the real-time fluorescence quantitative PCR system comprises forward and reverse primers and specific fluorescence probes for TRECs and beta-actin genes. The kit can be used for quickly screening the T-cell level of a neonatal immune system, and has the advantages of high sensitivity, high stability and excellent reproducibility. The method is suitable for quantitative detection of TRECs, can be used for screening functions of the neonatal immune system, and has practical clinical application value.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid diagnosis, and relates to a real-time fluorescence quantitative polymerase chain reaction (Polymerase Chain Reaction, PCR) kit for quantitative detection of free T cell receptor excision circles (T-cell receptor excision circles, TRECs) genes and its use. Background technique [0002] Primary Immunodeficiency Disease (PID) is an immunodeficiency disease caused by immune dysfunction caused by genetic defects of the immune system or congenital hypoplasia. , 1 / 5000 people in Japan and Sweden, 1 / 8000 people in Hong Kong, my country. There is a lack of comprehensive statistical data in China. If the incidence rate is 1 / 10,000, there are 2,500 new PID cases among the 25 million newborns born in my country every year, and the cumulative number of patients in childhood reaches 30,000-60,000. Primary immunodeficiency disease, related to genetics, often occurs in infants and young children, and recurre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 王晓川刘丹如王牧房聪
Owner 无锡联合利康临床检验所有限公司
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