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Kit used for screening CYP21A2 gene of Chinese population

A technology of CYP21A2 and reagent kit, which is applied in the direction of microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of instability and inability to apply clinically, and achieve the effect of saving detection time and improving diagnostic efficiency

Active Publication Date: 2013-12-04
SUZHOU MUNICIPAL HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The MLPA technology itself is relatively mature, and is widely used in the rapid screening of aneuploidy, the detection of microdeletions in chromosomes, and the detection of single-gene genetic diseases such as DMD. There are also kits developed for the detection of adrenal hyperplasia, but it is not yet available. Stable, not yet clinically applicable

Method used

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  • Kit used for screening CYP21A2 gene of Chinese population
  • Kit used for screening CYP21A2 gene of Chinese population
  • Kit used for screening CYP21A2 gene of Chinese population

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1, detection of gene mutation hotspots in CYP21A2 patients

[0050] Sample selection: The samples came from the adrenal hyperplasia detection DNA library of the Reproductive and Genetic Center of Suzhou Municipal Hospital. All patients in the library signed the informed consent form before collection, and agreed to cooperate with follow-up diagnosis, treatment and follow-up.

[0051] In step 1, a nested PCR amplification reaction is performed for two segments of the CYP21A2 gene.

[0052] Primary amplification: PCR reaction system 10 μL, containing 10 ng of genomic DNA extracted in step 1, dNTP 2 mM, 10×Buffer (containing Mg 2+ ) 1.0 μL, MgCl 2 25mM, FastStart Taq enzyme 0.15U, the final concentration of each amplification primer pair is 0.1μM. The PCR primary amplification reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 1 min, annealing at 57°C for 1 min, extension at 72°C for 1 min, 30 cycles, and full extension at 72°C...

Embodiment 2

[0066] Example 2, in vitro detection of newborn CYP21A2 gene mutation hotspot screening

[0067] 1. Suitable for newborn heel blood test genetic screening kit (96 copies) contains the following components:

[0068] 1) PCR reaction reagent group I, including 2mM dNTP150μL, 10×PCR buffer 100μl, 25mM MgCl 2 Solution 100 μL, deionized water 200 μL, primary amplification primer mixture I 400 μL, secondary amplification primer mixture II 400 μl, 5 U / μL FastStart Taq enzyme 15 μL;

[0069] 2) Purification reagent set, including 300 μL of 1U / μL SAP enzyme, 40 μL of 5U / μL ExonI enzyme, and 260 μL of deionized water;

[0070] 3) PCR reaction reagent group II, 5×seq Buffer 120 μL, primer mixture III 100 μL, SNaPShot Mix 100 μL, deionized water 180 μL;

[0071] 4) Homozygous and heterozygous mutation positive control DNA samples are 10 μL each, and negative control sample DNA samples are 10 μL;

[0072] 5) Instruction manual.

[0073] The samples came from high-risk dried blood sample...

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Abstract

The invention discloses a one-off qualitative screening kit for seven mutation hot spots about the 21-hydroxylase gene (CYP21A2) of the Chinese population. The kit takes the seven common mutation sites on the CYP21A2 of the Chinese population as detection objects, an amplification primer and an extension primer are respectively designed, the single base group primer extension genotype (SNaPshot) technology is adopted to perform multiple nest type PCR amplification and single base group marker extension on all the target segments, and the genotypes of the seven sites are obtained in one reaction tube through capillary electrophoresis analysis. The screening method and the screening kit are convenient to use, easy to operate, low in cost, high in flux, direct and reliable in detection result and suitable for screening the CYP21A2 gene of the Chinese population on a large scale.

Description

technical field [0001] The invention relates to the field of gene mutation detection in the technical field of genetic engineering, in particular to a kit for screening common mutation hotspots of 21-hydroxylase gene CYP21A2 in Chinese population. Background technique [0002] The incidence of congenital adrenal hyperplasia (CAH) in newborns is 1 / 16000-20000. Sexual genetic disease. According to the different synthetase defects, it is divided into 17α-hydroxylase deficiency, 21-hydroxylase deficiency, 11β-hydroxylase deficiency, 3β-hydroxysteroid dehydrogenase deficiency and their electron donor P450 oxidation There are five types of reductase deficiency, among which 21-hydroxylase deficiency is the most common type in congenital adrenal hyperplasia, accounting for about 90%-95%. [0003] 21-hydroxylase deficiency is an autosomal recessive genetic disease, and its causative gene is 21-hydroxylase gene (CYP21A2). The gene is located in the human leukocyte antigen (Human Le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陈瑛刘英华
Owner SUZHOU MUNICIPAL HOSPITAL
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