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Atp7b Gene Large Fragment Deletion Detection Kit and Detection Method

A technology for detection kits and large fragments, which is applied in the field of ATP7B gene large fragment deletion detection kits, can solve the problems of long import time, high price, and restrictions on wide application, so as to reduce the use threshold and cost, ensure efficiency and accuracy The effect of the detection method

Active Publication Date: 2022-03-22
BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the use of MLPA technology to detect large fragment deletions of ATP7B gene has not been widely used in China. Most studies only focus on point mutations that occur in exons, ignoring possible large fragment deletion mutations; and a few using MLPA technology All the studies used foreign commercial detection kits (SALSA P098 kits, MRC-Holland), but this kit needs to be equipped with special analysis software, and the price is high due to the use of fluorescent labels, etc., and it takes a long time to import, which limits its wide application in clinical practice

Method used

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  • Atp7b Gene Large Fragment Deletion Detection Kit and Detection Method
  • Atp7b Gene Large Fragment Deletion Detection Kit and Detection Method
  • Atp7b Gene Large Fragment Deletion Detection Kit and Detection Method

Examples

Experimental program
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Effect test

Embodiment l

[0050] Probe and Primer Design

[0051] The present invention designs relevant probes for the 5'UTR promoter region and 21 exons of the ATP7B gene, and each site includes a pair of probes (left probe and right probe). The left probe sequentially (5'-3') includes an upstream universal primer binding sequence, a stuffer sequence of varying lengths, and a target-specific sequence; the right probe sequentially (5'-3') includes a target-specific sequence , stuffer sequences of varying lengths and downstream universal primer binding sequences. The probe sequences are shown in Table 1.

[0052] Table 1 ATP7B gene large fragment deletion detection probe sequence

[0053]

[0054]

[0055]

[0056]

[0057] The left and right probes are prepared by chemical synthesis, and the right probe is modified with a phosphorylation group at the 5' end during synthesis, so as to complete the connection reaction with the free hydroxyl group at the 3' end of the left probe. The synth...

Embodiment 2

[0063] Detection of ATP7B Gene Large Fragment Deletion Mutations in Healthy Human Samples

[0064] (1) Sample DNA extraction

[0065] To obtain peripheral blood DNA, use a blood genomic DNA extraction kit (such as Tiangen Biochemical Technology No. DP348 product), and follow the kit instructions to extract genomic DNA. Detect the concentration and purity of DNA, and adjust the DNA concentration to 20ng / μl.

[0066] (2) Genomic DNA denaturation

[0067] Each detection sample is divided into 4 tubes for detection, and each tube detects 6 sites. Take 5 μl of DNA sample (100 ng) from each reaction tube and incubate at 95°C for 5 minutes on a PCR machine, then rapidly cool to room temperature (25°C) to denature the genomic DNA into a single strand.

[0068] (3) Hybridization of probes to genomic DNA

[0069] Add 1.5 μl Taq high-fidelity DNA ligase (for example, product number M0647S from NEB Company) Buffer, 6 μl probe mixed solution (0.5 μl for each probe, 5 μM) and 2.5 μl DEP...

Embodiment 3

[0099] Detection of large fragment deletion mutations of ATP7B gene in patients with hepatolenticular degeneration

[0100] Steps (1)-(5) are the same as in Example 2.

[0101] (6) Detection of amplification products by capillary electrophoresis

[0102]Take the PCR product and detect it in the Qsep100 automatic nucleic acid analysis system, using a high-resolution sampling cartridge (S1 cartridge), the sampling voltage is 4KV, the sampling time is 10 seconds, the electrophoresis voltage is 6KV, and 20bp and 1000bp DNA markers are added at the same time . After the product concentration is converted into an electrical signal by the supporting software, the peak diagram is given, and the peak diagram ordinate RFU value is read, and compared with the RFU value of the healthy control in Example 2, if the RFU value is 50% of the control group or has no value, Then it can be judged that there is a heterozygous or homozygous deletion at the corresponding site of the ATP7B gene in ...

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Abstract

The invention discloses a detection kit and a detection method for large fragment deletion of ATP7B gene, and relates to the field of biological detection and identification. The kit includes probe pairs and universal primer pairs for detecting the large fragment deletion of MLPA gene for the 5'UTR and 21 exons of ATP7B respectively; A detection method for detecting large fragment deletions of MLPA gene with primer pairs. The kit and detection method provided by the invention are simple and low in cost, and can be used for rapid detection of large fragment deletion mutations of the ATP7B gene.

Description

technical field [0001] The invention relates to the field of biological detection and identification. More specifically, it relates to a detection kit and detection method for large fragment deletion of ATP7B gene. Background technique [0002] Hepatolenticular degeneration (HLD), also known as Wilson disease (WD), is an autosomal recessive disorder of copper metabolism caused by ATP7B gene mutations. ATP7B gene mutations cause massive deposition of copper ions It is an important pathogenic factor, so early diagnosis and copper removal treatment are of great significance to the prognosis of patients. At present, the detection of ATP7B gene mutation based on Sanger sequencing has become an important reference index for the molecular diagnosis of hepatolenticular degeneration. In the prior art, most of the mutations in the ATP7B gene still use the classic method of next-generation sequencing. Although this method can provide accurate detection results, it cannot detect large...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6844
Inventor 周冬虎黄坚贾思雨李潇瑾
Owner BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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