Real-time fluorescent quantitative PCR (polymerase chain reaction) kit for one-step quantitative detection of KRECs gene and its application

A real-time fluorescence quantitative and real-time fluorescence technology, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problem of high probability of missing critical positive samples, and reduce the occurrence of false negative results. High specificity and stable sample properties

Inactive Publication Date: 2016-01-27
SHANGHAI ADVANCED CLINICAL LAB SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] In view of the above problems, especially the problem that the two-step method may miss critical positive samples, the purpose of the present invention is to provide a method that can reduce amplification errors and sample contamination, has higher sensitivity and specificity, and is easy and fast to operate. One-step KRECs gene detection kit to fill this gap in China

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  • Real-time fluorescent quantitative PCR (polymerase chain reaction) kit for one-step quantitative detection of KRECs gene and its application
  • Real-time fluorescent quantitative PCR (polymerase chain reaction) kit for one-step quantitative detection of KRECs gene and its application
  • Real-time fluorescent quantitative PCR (polymerase chain reaction) kit for one-step quantitative detection of KRECs gene and its application

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Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1: the preparation of kit

[0041] 1. Design and synthesis of primers and probes

[0042] According to the UCSCHumanGeneSorter query KRECs and β-actin gene sequences on the UCSC website (http: / / genome.ucsc.edu / cgi-bin / hgNear), use Primer3.0 to design fluorescent quantitative PCR upstream and downstream primers on KRECs and β-actin genes and probes. The selected primers have good specificity for gene binding and high PCR amplification efficiency. Both primers and probes were commissioned to LifeTechnologies to synthesize, where primers were purified by PAGE, and probes were purified by HPLC. The 5' end of the KRECs probe is a VIC fluorescent group, and the 3' end is a TAMRA fluorescent group; the 5' end of the β-actin probe is a VIC fluorescent group, and the 3' end is a TAMRA fluorescent group. Primer sequences are listed in Table 1.

[0043] Table 1 Insertion gene, specific primer and probe sequence

[0044]

[0045] 2. Construction of KRECs and β-ac...

Embodiment 2

[0060] Embodiment 2: the use of kit

[0061] 1. Extraction of DNA from dried blood filter paper of the sample to be tested

[0062] The operation steps are as follows:

[0063] A. Put three pieces of dried blood filter paper of the sample to be tested with a diameter of 3 mm obtained by punching holes into a sterilized 1.5 ml centrifuge tube, and add 180 μl of cell lysate 1.

[0064] B. Incubate at 85°C for 10 minutes. Gently flick the droplet from the cap into the tube.

[0065] C. Add 20 μl proteinase K stock solution, pipette slowly with a pipette tip, and incubate at 56°C for 1 hour. Gently flick the droplet from the cap into the tube.

[0066] D. Add 200 μl of cell lysate 2, slowly pipette with a pipette tip to mix thoroughly, and incubate at 70°C for 10 minutes. Gently flick the droplet from the cap into the tube.

[0067] E. Add 200 μl of absolute ethanol, and slowly pipette with the pipette tip to mix thoroughly. Gently flick the droplet from the cap into the tu...

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Abstract

The invention relates to a real-time fluorescent quantitative PCR (polymerase chain reaction) kit for one-step quantitative detection of KRECs gene and its application. The kit comprises a real-time fluorescent quantitative PCR reaction system based on real-time fluorescent PCR technique. The fluorescent PCR reaction system comprises forward and reverse primers specific to KRECs and beta-actin genes, and a specific fluorescent probe. The kit allows quick screening of neonatal immune system B-cell level, has high sensitivity and stability and provides excellent reproducibility, and this method is applicable to the quantitative detection of KRECs and to the functional screening of the neonatal immune system and is worthy of practical clinical application.

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 one-step quantitative detection of κ-deleting recombination excision circles (κ-deleting recombination excision circles, KRECs) genes and an application thereof. Background technique [0002] Primary immunodeficiency disease (Primary Immunodeficiency Disease, PID) is a kind of immunodeficiency disease caused by immune dysfunction caused by genetic defect of immune system or congenital hypoplasia, and more than 200 diseases have been found. PID often occurs in infants and young children, and repeated infections can occur, which can be life-threatening in severe cases. Some of these may be treated effectively, so prompt diagnosis remains important. In 2008, the US CDC carried out newborn screening for severe combined immunodeficiency (Severe Combined Immunodeficienc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2531/113C12Q2537/16C12Q2545/101C12Q2561/101C12Q2561/113
Inventor 王晓川王牧李芳序刘丹如
Owner SHANGHAI ADVANCED CLINICAL LAB SCI
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