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Primer and reagent kit for detecting CYP21 gene mutation and application of primer and reagent kit

A technology of CYP21A2 and kit, which is applied in specific-purpose bioreactor/fermenter, bioreactor/fermenter combination, biomass post-treatment, etc., which can solve the problems of low accuracy of results, cumbersome operation, and unknown cause of disease. Clear and other issues to achieve the effect of reducing pollution and comprehensive and accurate detection

Inactive Publication Date: 2020-07-31
CAPITALBIO GENOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method needs to use multiple pairs of primers for multiple rounds of PCR amplification. There are a large number of primer dimers / polymers in the system, and tiny pollution may be infinitely amplified. The operation is cumbersome and the accuracy of the results is low.
At the same time, the existing kits cannot detect 30kb deletions and recombination / fusion mutations of CYP21 true and false genes, so that about 30% of the pathogenic causes of 21-OHD cannot be clarified

Method used

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  • Primer and reagent kit for detecting CYP21 gene mutation and application of primer and reagent kit
  • Primer and reagent kit for detecting CYP21 gene mutation and application of primer and reagent kit
  • Primer and reagent kit for detecting CYP21 gene mutation and application of primer and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Primer Design and PCR Amplification

[0049] According to the reference sequence of the CYP21A2 gene in the UCSC database, and consult relevant literature and pathogenic databases, design the full-length specific primers of the CYP21A2 gene; design the corresponding genotype according to the complex genotype structure of the 30kb large fragment deletion and recombination / fusion of the CYP21 gene Amplification primers, the details of the primer sequences are shown in Table 1, and the primers were synthesized by Thermo Fisher Corporation of the United States.

[0050] Table 1 Sequence information of CYP21A2 gene amplification primers

[0051]

[0052]

[0053] Use the three pairs of primers shown in Table 1 to prepare the reaction system shown in Table 2, mix well and perform PCR amplification: pre-denaturation at 94°C for 3 minutes; denaturation at 98°C for 15 seconds, annealing at 67°C for 30 seconds, and extension at 72°C for 5 minutes , a total of 20 ...

Embodiment 2

[0056] Example 2 CYP21A2 point mutation detection

[0057] (1) Recover the amplified product of PCR amplification using CYP21A2_L / R, use 0.8 times the volume of magnetic beads to purify, and remove redundant PCR primers and dimers according to the standard steps of Beckman's purification of magnetic beads;

[0058] (2) Carry out end-repair of the purified CYP21A2 gene sequence, using the Qiagen kit, the system is shown in Table 3, and the conditions are shown in Table 4;

[0059] Table 3 enzyme end repair system

[0060] Reagent Volume (μL) PCR reaction product 17.5 Custom 10×DNA shearing buffer 2.5 Custom 5×DNA shearing enzyme master mix reagent 5

[0061] Table 4 Reaction conditions for enzyme-cut end repair

[0062] temperature Reaction time 4℃ 1min 32℃ 18min 65℃ 30min 4℃ ∞

[0063] (3) Prepare the adapter ligation reaction solution according to Table 5, complete the ligation of the Torrent sequenci...

Embodiment 3

[0071] Example 3 CYP21A2 30kb deletion and recombination / fusion mutation detection

[0072] Take 10 μL of the PCR products amplified by the three pairs of primers, load them on 1% agarose gel, use DS10000Marker as DNA molecular weight standard, and electrophoresis at 150V for 30min; after completion, stain the agarose gel with SYBR Green I, and use gel The gel imaging system took pictures and followed the standard steps of the instrument.

[0073] The result is as figure 1 As shown, among them, lane 1 is the amplification product of primer set 1, which is a 5.6kb target fragment, indicating that the detection result is normal; swimming lane 2 is the amplification product of primer set 3, which is a 3.4kb target fragment, indicating that the detection result is recombinant / Fusion; Lane 3 is the amplified product of primer set 2, which is an 8.5kb target fragment, indicating that the detection result is a 30kb deletion.

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Abstract

The invention provides primers and reagent kits for detecting CYP21 gene mutation and an application of the primer and reagent kit. The primers comprise a specific primer for an overall length sequence of a CYP21A2 gene, a specific primer with 30kb deletion and mutation of a CYP21 authenticity gene and a specific primer of recombination / fusion mutation of the CYP21 authenticity gene. The constructed reagent kits and primers are small in quantity, detection of various mutation types of the CYP21 gene can be completed once, the process is simple, the results are high in precision, and the primers and the reagent kits have broad application prospects in the field of diagnosis of congenital adrenal hyperplasia.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and relates to primers, kits and applications for detecting CYP21 gene mutations. Background technique [0002] 21-hydroxylase deficiency (21-OHD) is an autosomal recessive genetic disease and is the most common type of congenital adrenal hyperplasia (CAH), accounting for about 90% to 95% of CAH. According to the severity of clinical phenotype, 21-OHD can be divided into classic type and non-classic type. 1 / 500~1 / 1000, and up to 2% in hyperandrogen women. At present, the main diagnosis of CAH is based on the patient's clinical manifestations, hormone levels, ACTH stimulation test results and genetic testing results. However, the symptoms of patients with non-classic 21-OHD are often not obvious, which is a major difficulty in clinical diagnosis. There is a good correlation between the patient's genotype and the clinical manifestations of CAH. The guidelines for the diagnosis and treatme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11C12M1/34
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2525/191C12Q2531/113C12Q2565/125
Inventor 黄铨飞黄晓燕王杨刘情景丽芳
Owner CAPITALBIO GENOMICS
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