A real-time fluorescent quantitative PCR kit and application for quantitative detection of krecs gene

A technology of real-time fluorescence and kits, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of lack of quantitative KRECs, etc., and achieve accurate and reliable quantitative results, high sensitivity and specificity, and improved sensitivity. Effect

Active Publication Date: 2014-10-22
SHANGHAI ADVANCED CLINICAL LAB SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of methods and kits that can detect and quantify KRECs

Method used

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  • A real-time fluorescent quantitative PCR kit and application for quantitative detection of krecs gene
  • A real-time fluorescent quantitative PCR kit and application for quantitative detection of krecs gene
  • A real-time fluorescent quantitative PCR kit and application for quantitative detection of krecs gene

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Experimental program
Comparison scheme
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 KRECs and β-actin gene sequences (http: / / genome.ucsc.edu / cgi-bin / hgNear), use Primer3.0 to design the upstream and downstream of the nested PCR on the KRECs gene Primers, where the nested PCR upstream primers of KRECs and β-actin combine with the gene at the upstream of the real-time fluorescent quantitative PCR upstream primers and the gene, and the nested PCR downstream primers of KRECs and β-actin bind with the gene The binding position of the real-time fluorescence quantitative PCR downstream primer and the downstream of the gene binding position. 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 the primers were purified by PAGE, and the probes were purified by HPLC....

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 KRECs 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 KRECs and beta-actin genes; and the real-time fluorescence quantitative PCR system comprises forward and reverse primers and specific fluorescence probes for KRECs and beta-actin genes. The kit can be used for quickly screening the B-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 KRECs, 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 fluorescent quantitative polymerase chain reaction (Polymerase Chain Reaction, PCR) kit for quantitative detection of κ-deleting recombination excision circles (κ-deleting recombination excision circles, KRECs) 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,...

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 SHANGHAI ADVANCED CLINICAL LAB SCI
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