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Detection kit and system for detecting fragile X chromosome syndrome

An X chromosome and kit technology, applied in the biological and medical fields, can solve the problems of inability to determine the number of CGG repeats, difficult and effective PCR amplification, etc., and achieve the effects of high accuracy, low DNA amount, and optimized amplification system

Inactive Publication Date: 2018-09-14
北京信诺佰世医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the CGG repeat sequence of the FMR1 gene is high in GC, and some of the repeat numbers are more than 200. PCR is difficult to effectively amplify, and it cannot be solved by Sanger sequencing; and the common agarose electrophoresis identification technology cannot determine the number of CGG repeats.

Method used

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  • Detection kit and system for detecting fragile X chromosome syndrome
  • Detection kit and system for detecting fragile X chromosome syndrome
  • Detection kit and system for detecting fragile X chromosome syndrome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Extraction of Venous Peripheral Blood DNA

[0028] Follow the DNA extraction steps below to extract the DNA of the four samples of ABCD:

[0029] 1. Dispense 400 μL of erythrocyte lysate (QIAGEN, Cat: 158904) into a 1.5 mL centrifuge tube, add 200 μL of venous peripheral blood from four patients, and mix well;

[0030] 2. Incubate at 56°C for 10 minutes, during which time mix well;

[0031] 3. Centrifuge at 13,000 g for 1 min, discard the supernatant, add 200 μL of cell lysate (QIAGEN, Cat: 158906), and mix well;

[0032] 4. Add 10 μL proteinase K (QIAGEN, Cat: 158918) and mix well;

[0033] 5. Centrifuge at 13000g for 1min, transfer the supernatant to a clean 1.5mL centrifuge tube, add 400μL isopropanol, mix well, and you can see DNA precipitate after mixing;

[0034] 6. Centrifuge at 13,000 g for 1 min, discard the supernatant, and add 400 μL of 70% ethanol to rinse the precipitate;

[0035] 7. Centrifuge at 13000g for 1min, discard the supernatant, and...

Embodiment 2

[0038] Embodiment 2: PCR amplification

[0039] The primers shown in the following table 1 and the amplification system shown in table 2 are used to amplify the DNA sample prepared in Example 1:

[0040] Table 1: Primer sequences

[0041]

[0042]

[0043] Note: FAM indicates that the 5' end of the primer sequence is modified by FAM type, FAM: 6-carboxyfluorescein, the signal color is blue; NO indicates that the sequence is not modified.

[0044] PCR amplification system: Each sample is amplified in two tubes, labeled as Q system and TP system, and the amplification systems are as follows:

[0045] Table 2: Amplification System

[0046]

[0047]

[0048] The PCR amplification procedure is as follows:

[0049] Table 3: PCR Amplification Procedure

[0050]

[0051] After the reaction is completed, the amplified product is recovered for future use. The product of the obtained Q scheme was subjected to ordinary agarose gel electrophoresis, and the results were ...

Embodiment 3

[0053] Example 3: Analyzing the four amplified products according to the fragment analysis process

[0054] 1. ABI fragment analysis

[0055] 1) Mix the Q system and TP system products in a volume ratio of 2:1;

[0056] 2) Add 1 μl of mixed PCR to 9 μl formamide containing 0.2 μl Liz 1200 internal standard (ABI, Cat: 4379950, as a reference for judging sample size);

[0057]3) Denature at 95°C for 4-10 minutes, put it into an ABI 3130XL sequencer for detection, and obtain the original capillary electrophoresis data.

[0058] 2. Data analysis

[0059] 1) Open GeneMarker 2.2.0 and import the original capillary electrophoresis data;

[0060] 2) Click Project in the upper left corner and select Run;

[0061] 3) Select Panel, Size Standard, Standard Color in the Run wizard interface to define;

[0062] 4) Set the range of the X-axis in the menu to 150-1000;

[0063] 5) Use GeneMarker to read the data to generate a map, and obtain the size of the full-length fragment. The maps...

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Abstract

The invention provides a detection kit and system for detecting the fragile X chromosome syndrome. The detection kit comprises a Q amplification system agent and a TP amplification system agent, wherein the Q amplification system agent comprises amplification primers FMR1-F and FMR1-QR, and a TP amplification system comprises amplification primers TP and TP-R. By means of the detection kit and system, Q amplification and IP amplification can be conducted, the different primers are used for detecting templates with high GC multiplicity, thus the amplification system is optimized, and high-sensibility capillary electrophoresis is combined and used, so that 200 or more CGG repeats can be detected, and thus the accuracy is high.

Description

technical field [0001] The invention belongs to the field of biology and medical treatment, and relates to the detection of fragile X chromosome. Background technique [0002] Fragile X Syndrome (FXS) was first discovered by Martin and Bell in 1943, so it is also called Martin-Bell syndrome. It mainly manifests as moderate or severe mental retardation, language expression impairment, behavioral attention impairment, and epileptic seizures. Its incidence rate is about 1 / 2500, among which, the male incidence rate is 1 / 1000, and the female incidence rate is 1 / 2000. About 15% of men with mental retardation are caused by this disease. [0003] It has been confirmed that its molecular pathogenic gene is FMR1 (fragile X mental retardation 1). The gene is numbered 3129728 in Genbank, located on chromosome Xq27.3, and its 5-terminal untranslated region has a CGG trinucleotide repeat sequence and has polymorphism. ACMG guidelines launched the detection of FMR1 guidelines in 2006 a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6883C12Q2525/151C12Q2565/125
Inventor 王明旭王冬石旺
Owner 北京信诺佰世医学检验所有限公司
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