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Detection method of genes in chromosome 21, correlated detection probe combination, detection kit and correlated application

A gene detection and chromosome technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of long detection time, high sample requirements, poor quality of life of patients, etc., to achieve ingenious design, Avoid sample contamination and detect rapid results

Active Publication Date: 2014-05-28
上海春夏正像生物科技有限公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

Trisomy 21 has almost no self-care survivability, and the life expectancy is similar to that of normal people. The patient's quality of life is extremely poor, and it also imposes a heavy burden on the family and society
There is currently no effective treatment for trisomy 21, and preventing the birth of patients is the most effective way to block the inheritance of the disease, so prenatal genetic testing is the best choice to avoid re-birth patients
However, since chromosome detection requires living cell culture and subsequent chromosome banding analysis, it requires high sample requirements (2ml whole blood or 10ml amniotic fluid), and the detection time is long (2 weeks), making it difficult to carry out widely.
Due to complex technical conditions and many influencing factors, chromosome detection has many failure cases even in laboratories with good conditions.
The subsequent development of fluorescence in situ hybridization can directly operate on fixed cells without using living cells, which greatly simplifies the detection process, but the limitation of probe detection range cannot replace chromosome detection
However, including AFP detection and next-generation sequencing detection of pregnant women's plasma, it is far from reaching the accuracy of chromosome detection, and it is only used as a screening method to find high-risk groups

Method used

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  • Detection method of genes in chromosome 21, correlated detection probe combination, detection kit and correlated application
  • Detection method of genes in chromosome 21, correlated detection probe combination, detection kit and correlated application
  • Detection method of genes in chromosome 21, correlated detection probe combination, detection kit and correlated application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: DNA preparation

[0024] 1) Preparation of normal human genomic DNA for testing

[0025] Take normal human blood filter paper, extract and prepare by Chlex100, and adjust the concentration of normal human genomic DNA to 10ng / μl. 2) Normal genotype standard preparation

[0026] Based on human chromosome 21 ADAMTS5 (a disintegrin and metallopeptidase with thrombospondin type1motif5) gene, DSCRA (Down syndrome critical region gene3, Down syndrome key Regional gene 3) gene and internal reference gene β-actin sequence, artificially synthesized ADAMTS5 (SEQ ID NO:1), DSCRA (SEQ ID NO:2) and β-actin (SEQ ID NO:3). The T vectors containing the above fragments were mixed at a ratio of 1:1:1, and the concentration was adjusted to 1ng / ul as a 10-fold normal genotype template.

[0027] 3) Preparation of 21 Chromosomal Abnormal Genotype Standards

[0028] Synthetic ADAMTS5 (SEQ ID NO:1), DSCRA (SEQ ID NO:2) and β-actin (SEQ ID NO:3). The T vectors containing the abov...

Embodiment 2

[0029]Example 2: Design and synthesis of chromosome 21-specific single-copy gene amplification primers and MGB probes

[0030] Synthetic primers were designed according to human chromosome 21 ADAMTS5 and DSCRA, the ADAMTS5 gene and DSCRA gene probes were labeled with Fam, and the internal reference gene probe was labeled with Vic. Verify that fluorescent quantitative PCR primers specifically amplify target gene fragments, and Taqman MGB site-specific probes report corresponding fluorescent signals. Artificially synthesized gene fragment-specific amplification primers and site-specific Taqman MGB reporter probes. See Table 1 and Table 2 for gene fragment-specific primers and site-specific Taqman MGB probe sequences (SEQ ID NO: 4 to SEQ ID NO: 12).

[0031] Table 1

[0032]

[0033] Table 2

[0034]

Embodiment 3

[0035] Example 3: Quantitative PCR detection of 21 chromosome-specific single-copy genes

[0036] 1) ADAMTS5 gene verification

[0037] Reaction system: 2.5 μl of primer-probe mixture, 12.5 μl of 2-fold Multiplex PCR Mix (QIAGEN N.V., Germany), 2.5 μl of standard template, 5 μl of 5-fold Easy Buffer, ddH 2 O2.5 μl.

[0038] Quantitative PCR instrument: ABI7500 (Life Technologies, USA).

[0039] Reaction conditions: 95°C for 5 minutes; 94°C for 30 seconds—60°C for 30 seconds—72°C for 45 seconds, 40 cycles.

[0040] 2) DSCRA genetic verification

[0041] Reaction system: 2.5 μl of primer-probe mixture, 12.5 μl of 2-fold Multiplex PCR Mix (QIAGEN N.V., Germany), 2.5 μl of standard template, 5 μl of 5-fold Easy Buffer, ddH 2 O2.5 μl.

[0042] Quantitative PCR instrument: ABI7500 (Life Technologies, USA).

[0043] Reaction conditions: 95°C for 5 minutes; 94°C for 30 seconds—60°C for 30 seconds—72°C for 45 seconds, 40 cycles.

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Abstract

The invention provides a detection method of genes in chromosome 21. The method comprises the steps as follows: genomic DNA (deoxyribonucleic acid) of a detected object is extracted; two sets of PCR (polymerase chain reaction) primer pairs and Taqman MGB (minor groove binder) fluorescence probes are combined to perform fluorescent quantitative PCR on the genomic DNA respectively, wherein the PCR primer pairs are nucleotide sequences represented by SEQ ID NO: 4 and SEQ ID NO: 5 as well as SEQ ID NO: 7 and SEQ ID NO: 8 respectively, and nucleotide sequences of the Taqman MGB fluorescence probes are represented by SEQ ID NO: 6 and SEQ ID NO: 9 respectively; and according to a real-time amplification curve formed by fluorescence signals collected in the fluorescent quantitative PCR, whether trisomy probability exists in the chromosome 21 of genomic DNA is analyzed. The invention further relates to correlated detection probe combination, a detection kit and a correlated application. According to the invention, the design is ingenious, sample contamination can be effectively prevented through closed tube detection, the detection is rapid, accurate, simple and convenient, and the method can be taken as reference for doctor diagnosis and medication and is suitable for large-scale popularization and application.

Description

technical field [0001] The present invention relates to the technical field of disease-related detection, more specifically, to the technical field of gene detection in chromosome 21, in particular to a gene detection method in chromosome 21, a related detection probe composition, a detection kit, and related applications. Background technique [0002] Trisomy 21 (Down's syndrome, Down's syndrome) is a common autosomal disease, the main reason is that the severe genetic material imbalance caused by the trisomy of chromosome 21 leads to severe mental retardation (IQ50-70), special face (international face), high rate of various body deformities and high incidence of tumors and other series of disease characteristics. The incidence of S21 trisomy is about 1 / 500, and the incidence increases significantly with the age of pregnant women. Trisomy 21 has almost no self-care survivability, and the life expectancy is similar to that of normal people. The patient's quality of life is...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/686C12Q2561/101C12Q2561/113C12Q2545/101
Inventor 陈金中赵翊均
Owner 上海春夏正像生物科技有限公司
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