Clinic rapid PCR detection kit of fragile X syndrome

A detection kit and kit technology, applied in the field of bioengineering, can solve the problems of time-consuming, cumbersome steps, and high consumption, and achieve the effect of reducing the incidence of disease

Active Publication Date: 2014-08-13
江苏佰龄全基因生物医学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Currently published detection methods such as methylation PCR, MLPA, Southern Blot, Real Time PCR and other methods are used to detect Fragile X Syndrome, but their application is limited due to the shortcomings of long time consumption, high consumption and cumbersome steps. scope, and the present invention makes up for these shortcomings well, and has carried out innovation on the basis of prior art, makes the detection method of fragile X syndrome more perfect

Method used

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  • Clinic rapid PCR detection kit of fragile X syndrome
  • Clinic rapid PCR detection kit of fragile X syndrome
  • Clinic rapid PCR detection kit of fragile X syndrome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of a PCR detection kit for detecting Fragile X syndrome

[0028] 1. Primer design

[0029] Design a set of specific primers in the -600-+733 region of the FRM-1 gene, including primers F and R for amplifying the target fragment (including the CGG repeat region); and primer F for amplifying the internal reference fragment , M.

[0030] The sequences of F, R and M in the primer group are selected from any one of groups 1-6 in Table 1, and the primer sequences are shown in Attached Table 1.

[0031] 2. Prepare PCR reaction solution

[0032] The components of the PCR reaction solution are as follows: 1) 10× buffer solution, the buffer solution includes a concentration of 500mM Tris-HCl (pH9.0) and 20mM MgCl 2 . 2) dNTPs at a concentration of 2.5mmol / L; 3) the upstream primer F of the target gene primer at a concentration of 10 μmol / L; 4) the downstream primer R of the target gene primer at a concentration of 10 μmol / L; 5) the concentration of 10 μm...

Embodiment 2

[0033] Embodiment 2 application kit carries out negative sample detection

[0034] 1) DNA extraction: Peripheral blood was collected with the consent of the subject or the knowledge of his guardian. A commercially available full-type gold blood DNA extraction kit was used to extract, and the concentration was calibrated to be 100 ng / μl. The sample was clinically negative.

[0035]2) Prepare a PCR amplification system using the kit of Example 1;

[0036] PCR amplification system:

[0037]

[0038] in,

[0039] The sequence of the primer group is shown in Group 1 in Table 1,

[0040] Composition of PCR enhancer: 6 μl of 2.5mol / L betaine, 4 μl of DMSO.

[0041] (3) PCR amplification conditions: pre-denaturation at 95°C for 8 min, suspend adding 1ul of Taq enzyme; denaturation at 95°C for 45 s, annealing at 60°C for 45 s, extension at 72°C for 1.5 min, and final extension for 10 min after 30 cycles.

[0042] (4) After the reaction, 1.5% agarose gel electrophoresis was use...

Embodiment 3

[0043] Embodiment 3 application kit carries out positive sample detection

[0044] (1) DNA extraction: Peripheral blood was collected with the consent of the patient or the knowledge of the guardian. A commercially available full-type gold blood DNA extraction kit was used to extract, and the concentration was calibrated to be 100 ng / μl. The sample tested positive for Fragile X syndrome clinically.

[0045] (2) Prepare a PCR amplification system using the kit of Example 1;

[0046] PCR amplification system:

[0047]

[0048] Wherein, the sequence of the primer group is shown in Group 1 in Table 1,

[0049] Composition of PCR enhancer: 6 μl of 2.5mol / L betaine, 4 μl of DMSO.

[0050] (3) PCR amplification conditions: pre-denaturation at 95°C for 8 minutes, suspend adding 1 μl of Taq enzyme; denaturation at 95°C for 45 seconds, annealing at 60°C for 45 seconds, extension at 72°C for 1.5 minutes, and final extension for 10 minutes after 30 cycles.

[0051] (4) After the r...

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Abstract

The invention provides a clinic rapid PCR detection kit of a fragile X syndrome. The kit comprises DNA polymerase, dNTPs, a primer group and a buffer system. The kit is used for detecting the copy number of the FMR-1(CGG)n fragment of the virulence gene of the fragile X syndrome, so the content range of the copy number can be deducted, and the carriers and patients of the virulence gene can be fast found; and the kit can be used in antenatal diagnosis and infant patient birth prevention in order to reduce the incidence of the fragile X syndrome.

Description

technical field [0001] The invention relates to a rapid clinical PCR detection kit for fragile X syndrome, which belongs to the technical field of bioengineering. Background technique [0002] Fragile X syndrome is a hereditary mental retardation syndrome second only to Down syndrome in incidence. It is X-linked inheritance and accounts for 40% of X-linked mental retardation. The penetrance rate is significantly different for men and women, 80% for men and 30% for women. The incidence of Fragile X Syndrome also varies according to gender, 1 / 4000 for men and 1 / 6000 for women. [0003] More than 95% of patients with Fragile X Syndrome are based on molecular genetics caused by the mutation of the CGG repeat region structure expansion on the FMR1 gene, and less than 5% are due to missense mutations and deletion mutations of the FMR1 gene that affect the FMR1 gene. due to normal structure. [0004] The FMR1 gene is located on chromosome Xq27.3, contains 17 exons, and has a tot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2531/113C12Q2525/151C12Q2537/16
Inventor 潘海波谢阳邢楠楠华琴
Owner 江苏佰龄全基因生物医学技术有限公司
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