CN114645085A - Fragile X syndrome FMR1 gene detection kit - Google Patents
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Abstract
The invention discloses a fragile X syndrome FMR1 gene detection kit, which comprises a specific PCR amplification primer for amplifying a CGG repetitive region of an FMR1 gene, a third primer for randomly anchoring the CGG repetitive region, DNA polymerase with high thermal stability, a reference standard, a high GC reaction buffer solution, dNTPs and Taq Extender Additive. Various sequences with different sizes are amplified through three-primer PCR reaction, the amplified product is subjected to capillary electrophoresis, and the number of CGG repeats is calculated through comparison with a standard product, so that the female gene heterozygosity and homozygosity can be accurately judged. The kit is used for group screening and prenatal diagnosis of fragile X syndrome, can exactly detect normal and premutation carriers, full-mutation patients, female patients and carriers, and has the characteristics of rapidness, high accuracy, high flux, economy and safety. Therefore, the kit has good clinical application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a fragile X syndrome FMR1 gene detection kit.
Background
Fragile X syndrome is an X-linked recessive genetic disease which mainly takes mental retardation as clinical manifestation, has the incidence rate next to trisomy 21 among genetic diseases related to mental retardation, is the most common known X-linked genetic disease, and is also the monogenic genetic disease with the highest incidence rate known to human at present. The occurrence of fragile X syndrome is closely related to abnormal amplification of non-coding region (CGG) n repeat sequence located at 5' end of FMR1 gene of Xq27.3. A non-coding region near the 5' end of the No.1 exon of the FMR1 gene has a trinucleotide repeat sequence mainly consisting of CGG (cytosine-guanine). The (CGG) n repeat copy number shows great heterogeneity in the population. Normal human the sequence has 6-45 (CGG) n; 45-55 CGGs are called the middle region or "gray region"; when (CGG) n reaches 55-200, it is a pre-mutation. When the number of (CGG) n exceeds 200, the FMR1 gene promoter is frequently methylated, so that the FMR1 gene transcription is inhibited or weakened, the protein expression is blocked, the brain development is influenced, and the fragile X syndrome can be clinically caused.
In the incidence of the people reported at present, the incidence of the diseases in males is about 1/4000, the incidence of the diseases in females is about 1/7000-8000, and the clinical manifestations of the patients mainly comprise moderate to severe dysnoesia and language disorder, and are accompanied by different degrees of body type abnormalities: large head, big ear, prominent mandible and forehead, high jaw arch, hyper-extended joints and flat feet, etc., and large testis appears after adolescent development in most male patients; the disease is also accompanied by behavioral disorders: most patients have isolated or hyperactivity of sexual emotion, inattention, hyperexcitability and the like. However, patients with the full mutation do not all show typical clinical symptoms, and female patients have another normal X chromosome, so that the clinical symptoms are not typical and the patients are difficult to distinguish through clinical symptoms. In addition, the FMR1 pre-mutation has a carrying rate of up to 1.7% in the female population, and the pre-mutation is not only related to the recurrence risk of offspring, but also is a pathogenic mutation. Therefore, disease screening through FMR1 gene detection is an important means for solving the problem, has great significance particularly in prenatal diagnosis and genetic counseling, can reduce the birth of neonates, reduce the incidence of the disease and improve the population quality. The existing FMR1 gene detection method comprises methylation specific PCR, PCR amplification detection method, Southern blotting method and the like, but the methods have the defects that the carrier of the pre-mutation can not be detected, the PCR product of the carrier of the pre-mutation or the patient with the full mutation is difficult to amplify, the operation is complicated, and the flux is low, so that the clinical application is limited; the three-primer PCR has an ultra-strong amplification system, and can avoid the problem that the amplification capability of the PCR is reduced along with the increase of the number of (CGG) n repeats because the conventional PCR is difficult to amplify due to the high GC content of the sequence. PCR products are usually difficult to amplify by carriers of pre-mutations or patients with full-mutations, and two X chromosomes exist in women, which further increases the difficulty of PCR detection. Three primer PCR can still pair with the repeat sequence in the template to amplify a series of continuous short PCR products even though the full length of the repeat sequence cannot be amplified.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a fragile X syndrome FMR1 gene detection kit, the copy number of a CGG repetitive region of an FMR1 gene is amplified by three primers, continuous PCR products with different fragment lengths are generated, a continuous trapezoidal peak group is formed, the continuous trapezoidal peak group is used for distinguishing a normal CGG homozygote or a heterozygote of normal CGG and large CGG in a conventional PCR amplification result, the missed detection of the CGG of the large fragment is avoided, whether a detected person is a patient or a carrier of a pre-mutation can be judged quickly and accurately, the diagnosis rate and the detection rate of the carrier of the fragile X syndrome are improved, and great contribution is made for reducing the birth defect rate and relieving the national and social burdens.
The invention adopts the following technical scheme to realize the purpose:
a fragile X syndrome FMR1 gene detection kit is characterized in that: comprises a primer pair FMR1-F, FMR1-R for amplifying a region comprising a CGG repeat region, a 5 'flanking region and a 3' flanking region; a third primer FMR1-CGG for random anchoring of CGG repeat regions and for combination with specific primer pairs to generate amplicons of different sizes, having the sequence:
FMR1-F：GCCCGCACTT CCACCACCAG CTCCTCCA；
FMR1-R：TCTGGACCCT GAAGTGTGCC GTTG；
FMR1-CGG：TGAAGTGTGC CGTTGATACG GCGGCGGCGG CGG。
further, the kit also comprises 10 XPCR Buffer, 5 XPQ-solution, dNTP Mix (25mM), PCR reaction Taq enzyme and Taq Extender Additive.
Further, the kit comprises the following amplification systems: 10 XPCR Buffer 1uL, 5 XQ-solution 5uL, FMR1-F/R primer pair 0.5uL, FMR1-CGG 0.5uL, dNTPs 0.25 uL, HotStarTaq DNA polymerase0.33 uL, Taq Extender Additive 0.67 uL, DNA Sample 1uL, PCR deionized water 0.75 uL.
The fragile X syndrome FMR1 gene detection method comprises the following steps:
(1) and designing an amplification primer aiming at the CGG repetitive region of the FMR1 gene.
The amplification primer consists of SEQ ID NO. 1-SEQ ID NO. 2.
(2) Third primers are designed which, in combination with the specific primer pair, are capable of generating amplicons of different sizes.
The third primer of the invention consists of SEQ ID NO. 3.
(3) Preparation of a detection template: and extracting the DNA of the sample to be detected.
(4) Performing PCR amplification by using the DNA extracted in the step (3) as a template and using the amplification primers and the third primer in the step (1) and the step (2) to obtain a PCR product of a target sequence;
and (4) detecting the PCR product obtained in the step (3) by adopting capillary electrophoresis, analyzing and outputting the result by using GeneMaker software, and interpreting the result of the examined person through a peak pattern diagram of sample electrophoresis.
Drawings
FIG. 1 shows the capillary electrophoresis peak of the standard at 46 CGG repeats.
FIG. 2 shows the capillary electrophoresis peak for the standard at 54 CGG repeats.
FIG. 3 shows the capillary electrophoresis peak for the standard 100 CGG repeats.
FIG. 4 shows a peak of a sample obtained by capillary electrophoresis using DNA extracted from a clinical sample as a template, and this data shows that the sample is a normal female peak when it is compared with a standard 54 peak.
Detailed Description
The invention is described in further detail below with reference to the figures and the detailed description.
In the following examples, unless otherwise specified, all the methods were conventional, and all the reagents used were commercially available.
Example 1
(1) PCR amplification primer design
A pair of specific PCR amplification primers are designed according to 150bp in the upstream 5 'end and the downstream 3' end of a CGG repetitive sequence of an FMR1 gene, an upstream primer FMR1-F and a downstream primer FMR1-R are designed, and an amplified FMR1 gene region comprises a CGG repetitive region, a 5 'flanking region and a 3' flanking region. The length of the amplified fragment is (221+3xn) bp, and n represents the number of CGG repeats.
FMR1-F：GCCCGCACTT CCACCACCAG CTCCTCCA；
FMR1-R：TCTGGACCCT GAAGTGTGCC GTTG；
(2) Third primer design
A third primer FMR1-CGG which is randomly anchored to the CGG repetitive sequence region and combined with a specific amplification primer pair to generate amplicons of different sizes is designed in the CGG repetitive sequence region of the FMR1 gene.
FMR1-CGG：TGAAGTGTGC CGTTGATACG GCGGCGGCGG CGG。
Example 2 preparation of assay template
The high-quality and high-purity genomic DNA is extracted by adopting a MagaBio dry blood spot genomic DNA purification kit (Hangzhou Bori science and technology Co., Ltd.), and the specific operation process is carried out according to the specification requirement.
EXAMPLE 3 preparation of PCR reaction Components
The PCR reaction components were as follows: 10 XPCR Buffer comprises Tris-HCl with a concentration of 100mmol/L, pH value of 8.3 and 500mmol/L KCl; MgCl2 at a concentration of 25 mmol/L; 5 × Q-solution (QIAGE); dNTP with concentration of 10 mmol/L; the upstream primer FMR1-F with the concentration of 10 mu mol/L; the downstream primer FMR1-R with the concentration of 10 mu mol/L; a third primer FMR1-CGG with the concentration of 10 mu mol/L; DNA Polymerase HotStarTaq DNA Polymerase (Thermo Scientific) with an activity of 5U/. mu.L; 1 XTaq Extender Additive (Merck Co.); deionized water.
Example 4 Fragile X syndrome FMR1 Gene detection
Using the DNA extracted in example 2 as a template, PCR amplification primers FMR1-F/R and FMR1-CGG and the PCR components of example 3, and after loading, obtaining the target region amplification product by PCR amplification procedure.
The PCR amplification procedure was:
example 5 capillary electrophoresis detection
The amplified products of example 4 were used to prepare a sample loading system for capillary electrophoresis, followed by on-machine detection.
Table 1: capillary electrophoresis reaction system
| Reagent | Volume (ul)/reaction |
| Deionization formamide (HiDi) | 8.5 |
| Molecular internal standard (LIZ1200) | 0.5 |
| PCR amplification productArticle (A) | 1 |
| Total volume | 10.0 |
Note: the PCR amplification product can be diluted according to the sensitivity of the detection instrument.
FIG. 1 is a diagram of the capillary electrophoresis peaks for the standard at 46 CGG repeats.
FIG. 2 is a diagram of capillary electrophoresis peaks for the standard at 54 CGG repeats.
FIG. 3 is a diagram of capillary electrophoresis peaks for 100 CGG repeats of the standard.
FIG. 4 is a peak diagram of capillary electrophoresis performed on DNA extracted from a clinical sample as a template, and the comparison with a peak diagram of a standard 54 shows that the sample is a peak diagram of a normal female.
Sequence listing
<110> Zhejiang Bosheng Biotechnology Ltd
Children's Hospital Affiliated to Medical College of Zhejiang University
<120> fragile X syndrome FMR1 gene detection kit
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213> Unknown (Unknown)
<400> 1
gcccgcactt ccaccaccag ctcctcca 28
<210> 2
<211> 24
<212> DNA
<213> Unknown (Unknown)
<400> 2
tctggaccct gaagtgtgcc gttg 24
<210> 3
<211> 33
<212> DNA
<213> Unknown (Unknown)
<400> 3
tgaagtgtgc cgttgatacg gcggcggcgg cgg 33
Claims (3)
1. A fragile X syndrome FMR1 gene detection kit is characterized in that: comprises a primer pair FMR1-F, FMR1-R for amplifying a nucleic acid comprising a CGG repeat region, a 5 'flanking region and a 3' flanking region; a third primer FMR1-CGG for random anchoring of CGG repeat regions and for combination with specific primer pairs to generate amplicons of different sizes, having the sequence:
FMR1-F：GCCCGCACTT CCACCACCAG CTCCTCCA；
FMR1-R：TCTGGACCCT GAAGTGTGCC GTTG；
FMR1-CGG：TGAAGTGTGC CGTTGATACG GCGGCGGCGG CGG。
2. the kit of claim 1, wherein said kit further comprises 10 XPCR Buffer, 5 XPQ-solution, dNTP Mix (25mM), PCR reaction Taq enzyme and Taq Extender Additive.
3. The kit of claim 1, wherein the kit comprises the following amplification systems: 10 XPCR Buffer 1uL, 5 XQ-solution 5uL, FMR1-F/R primer pair 0.5uL, FMR1-CGG 0.5uL, dNTPs 0.25 uL, HotStarTaq DNA Polymerase0.33 uL, Taq Extender Additive 0.67 uL, DNA Sample 1uL, PCR deionized water 0.75 uL.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130115595A1 (en) * | 2010-02-05 | 2013-05-09 | Quest Diagnostics Investments Incorporated | Method to detect repeat sequence motifs in nucleic acid |
| CN109355376A (en) * | 2018-12-04 | 2019-02-19 | 深圳会众生物技术有限公司 | Fragile X mental retardation FMR1 genetic test primer, kit and detection method |
| CN111100924A (en) * | 2018-10-26 | 2020-05-05 | 上海晶准生物医药有限公司 | Quality control product for detecting CGG (CGG repeat number) of FMR1 gene, application thereof and kit containing quality control product |
-
2020
- 2020-12-17 CN CN202011495895.2A patent/CN114645085A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130115595A1 (en) * | 2010-02-05 | 2013-05-09 | Quest Diagnostics Investments Incorporated | Method to detect repeat sequence motifs in nucleic acid |
| CN111100924A (en) * | 2018-10-26 | 2020-05-05 | 上海晶准生物医药有限公司 | Quality control product for detecting CGG (CGG repeat number) of FMR1 gene, application thereof and kit containing quality control product |
| CN109355376A (en) * | 2018-12-04 | 2019-02-19 | 深圳会众生物技术有限公司 | Fragile X mental retardation FMR1 genetic test primer, kit and detection method |
Non-Patent Citations (3)
| Title |
|---|
| JIN‑YU ZHANG等: "FMR1 allele frequencies in 51, 000 newborns: a large‑scale population study in China", 《WORLD JOURNAL OF PEDIATRICS》, vol. 17, pages 653, XP037633763, DOI: 10.1007/s12519-021-00473-6 * |
| 孙莉等: "基于三引物荧光 PCR-毛细管电泳法的 FMR1 基因突变检测技术建立及其在自闭症辅助诊断中的应用", 《分子诊断与治疗杂志》, vol. 9, no. 5, pages 319 - 324 * |
| 林海等: "《环境工程微生物学实验教程》", vol. 1, 31 October 2020, 冶金工业出版社, pages: 119 * |
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