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

A real-time fluorescence quantitative and real-time fluorescence technology, which is applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of shortage, achieve convenient storage, reduce false positive results, and facilitate long-term preservation Effect

Active Publication Date: 2013-06-26
无锡联合利康临床检验所有限公司
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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 for the combined quantitative detection of TRECs and KRECs

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: the preparation of kit

[0048]1. Design and synthesis of primers and probes

[0049] Query TRECs, KRECs and β-actin gene sequences according to UCSC Human Gene Sorter on UCSC website (http: / / genome.ucsc.edu / cgi-bin / hgNear), use Primer3.0 on TRECs, KRECs and β-actin genes Design the upstream and downstream primers of nested PCR and the upstream and downstream primers and probes of fluorescent quantitative PCR, in which the nested PCR upstream primers of TRECs, KRECs and β-actin bind to their genes at the same time as their real-time fluorescent quantitative PCR upstream primers bind to the gene Upstream of the position, the binding position of the downstream primers of TRECs, KRECs and β-actin to their gene is downstream of the binding position of their real-time fluorescent quantitative PCR downstream primers to the gene. The selected primers have good specificity for gene binding and high PCR amplification efficiency. Both primers and probes were entru...

Embodiment 2

[0072] Embodiment 2: the use of kit

[0073] 1. Extraction of DNA from Dried Blood Filter Paper

[0074] The operation steps are as follows:

[0075] 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;

[0076] B. After standing for 15 minutes, centrifuge at 3700rpm for 5 minutes to absorb the solution as much as possible;

[0077] C. Repeat steps A-B, wherein the standing time is 10 minutes;

[0078] D. Add sterile milli-Q water, centrifuge at 3700rpm for 30 seconds, and absorb the milli-Q water as much as possible;

[0079] E. Add 30μl Generation DNA Elution Soln II, centrifuge at 3700rpm for 1 minute, and place in a 99℃ water bath for 25 minutes;

[0080] F. After cooling to room temperature, centrifuge at 3700rpm for 30 seconds, store at 4°...

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Abstract

The invention relates to a real-time fluorescence quantitative PCR (polymerase chain reaction) kit for quantitatively detecting TRECs and KRECs genes 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, KRECs and beta-actin genes; and the real-time fluorescence quantitative PCR system comprises forward and reverse primers and specific fluorescence probes for TRECs, KRECs and beta-actin genes. The kit can be used for quickly screening the T-cell level and 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 combined quantitative detection of TRECs and 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 method for quantitatively detecting free T cell receptor excision circles (T-cell receptor excision circles, TRECs) and κ-deleting recombination excision circles (κ-deleting recombination excision circles, KRECs) genes Fluorescent quantitative polymerase chain reaction (Polymerase Chain Reaction, PCR) kit and its application. 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. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
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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