CN115478101A - Kit and method for detecting multiple gene site copy number variation of autism patient - Google Patents
Kit and method for detecting multiple gene site copy number variation of autism patient Download PDFInfo
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Abstract
The invention discloses a kit and a method for detecting multiple gene locus copy number variation of an autism patient, wherein multiple genes comprise SHANK3, CHRNA7 and GABRB3 genes and probes shown as SEQ ID No. 1-SEQ ID No.20. The probe set and the kit provided by the invention can be used for simultaneously detecting copy number variation of a plurality of sites of autism, and have the characteristics of high sensitivity, short detection time and low cost.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a kit and a method for detecting copy number variation of gene loci of patients with autism.
Background
Autism (ASD), a common type of neurodevelopmental disease, occurs at a rate of 1% of the general population. ASD is clinically manifested as impaired interpersonal communication, stereotype repetition of behavioral patterns, and narrow interests. The human brain comprises billions of neurons, each neuron can form thousands of synapses, and the synapses are used as functional structures for communication among the neurons to construct a neural network in which the neurons are mutually communicated, so that a billion-level neural loop network is formed.
The deletions and duplications of the SHANK3, GABRB3, CHRNA7 genes are common genetic mutations leading to ASD. The SHANK3 gene is located on chromosome 22q13.3. The SHANK3 protein is a postsynaptic framework protein that links neurotransmitter receptors, ion channels, and other membrane proteins to the cytoskeleton and G-protein coupled signaling pathways. The SHANK3 protein is also involved in synapse formation and in the maturation of dendritic spines. The CHRNA7 gene is located on chromosome 15q13.3.CThe HRNA7 gene contains 10 exons and spans more than 75 kb. In humans, the CHRNA7 gene encodes neuronal acetylcholine receptor alpha subunit-7. Nicotinic acetylcholine receptors (nAChRs) are members of the superfamily of ligand-gated ion channels that mediate rapid signaling at synapses. The GABRB3 gene is located on chromosome 15q12, and contains 10 exons and spans 250 kb. GABRB3 gene encoding GABA A The receptor β 3 subunit, the major inhibitory neurotransmitter in the mammalian brain, GABA, acts through it. These 3 genes are involved in synaptic function, formation of neural connections and synaptic transmission. It is known that neurodevelopmental diseases such as autism may occur due to abnormal neural circuits caused by gene mutation that destroys synaptic plasticity of neurons.
The traditional autism screening method mainly observes the expressions of interpersonal relationship, language communication, perception and the like of a person to be detected according to the autism diagnosis standard and determines diagnosis according to the standard. The methods have the defects that only the patients can be subjected to preliminary autism screening, the chromosome variation of the patients cannot be diagnosed, missed diagnosis and misdiagnosis are easy to occur during diagnosis, the treatment of the patients is delayed, and the serious autism children are caused to appear. The clinical auxiliary detection of the related gene mutation of the patient can more clearly lead to the genetic factors of the patient who self-closes.
At present, various methods are used for detecting the mutation of the gene related to the autism q, including second-generation sequencing, gene chip or microarray comparative genome hybridization, fluorescence in situ hybridization and the like, and the methods are complex to operate, high in cost and long in time consumption. Therefore, an accurate, rapid and low-cost method for detecting copy number variation of autism-related genes is urgently needed clinically.
Disclosure of Invention
The invention aims to provide a kit for detecting genomic copy number variation of an autistic patient aiming at the defects in the prior art.
It is a further object of the present invention to provide a method for detecting genomic copy number variation in patients with autism.
In order to achieve the purpose, the invention adopts the technical scheme that: a kit for detecting the copy number variation of multiple gene loci of an autism patient comprises a probe set, ligase, hybridization solution and Taq enzyme, wherein genes detected by the probe set consist of SHANK3, CHRNA7 and GABRB 3. The probe is a sequence shown in SEQ ID NO. 1-SEQ ID NO. 18.
Further, the corresponding relationship between the probe set and the detection gene is as follows: the genes of SHANK3 are detected by SEQ ID NO. 1-SEQ ID NO.6, CHRNA7 is detected by SEQ ID NO. 7-SEQ ID NO. 12, and GABRB3 is detected by SEQ ID NO. 13-SEQ ID NO. 18.
Further, the kit also comprises a probe sequence for detecting a control gene NODAL: SEQ ID NO.19, SEQ ID NO.20.
Further, the kit also comprises a pair of universal primers. The universal primer is a sequence shown in SEQ ID NO.21 and SEQ ID NO 22.
In order to achieve the second object, the invention adopts the technical scheme that: a method for detecting multiple gene locus copy number variation in an autistic patient using the kit of any one of claims 1 to 4, the method comprising the steps of:
(1) Extracting the genome DNA of a sample to be detected, and performing high-temperature denaturation treatment;
(2) Carrying out hybridization reaction on the probe set or the probe set in the kit and the denatured genomic DNA to obtain a hybridization product;
(3) Completely pairing and hybridizing the left probe 3 'end and the right probe 5' end which are adjacent to each other and are hybridized with the genome DNA by using the ligase to form a complete probe, and obtaining a ligation product;
(4) Performing PCR reaction on the connection product by using a universal primer, and performing capillary electrophoresis on the PCR product;
(5) Analyzing the result of capillary electrophoresis to obtain the variation information of the copy numbers of SHANK3, CHRNA7 and GABRB3 related to the autism.
Further, the hybridization reaction conditions are as follows: hybridization was carried out at 95 ℃ for 1min and at 60 ℃ for 16h.
The beneficial effects of the invention include:
1. the kit and the detection method of the invention combine the advantages of the multiplex ligation dependent probe amplification technology, can detect and identify the copy number variation of multiple gene loci of autism patients, and have the obvious advantages of high sensitivity and strong specificity.
2. The probe of the invention is hybridized and then PCR amplified, and combines with capillary electrophoresis with high resolution, thus ensuring the characteristics of high flux, high resolution and low cost of detection, and the whole detection process can be completed within 24 hours.
Drawings
FIG. 1 is a diagram of the peak shape of capillary electrophoresis according to the present invention
Detailed Description
The invention relates to a kit and a method for detecting multiple gene locus copy number variation of an autism patient. The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.
Example 1 Probe and kit
1. Probe pin
Based on the sequence information of the internal reference gene NODAL and the target genes SHANK3, CHRNA7 and GABRB3, the part of the probe which is combined with the template DNA is designed, and the Tm value calculation and the secondary structure evaluation are carried out on the sequence. Finally, a binding sequence with high specificity and sensitivity is obtained, and the copy number variation of the autism related gene of the sample to be detected can be detected under the same reaction condition.
The Tm value of the sequence bound to the template DNA can be evaluated in a manner conventional in the art.
Preferably, the Tm value is evaluated by using OligoAnalyzer 3.1 (https:// sg. Idtdna. Com/calc/Analyzer).
The above primers are combined with the universal primer sequence to form a probe. Wherein, the sequence of the universal primer used by the probe of the invention is shown in SEQ ID NO. 21-22.
The secondary structure of the probe was evaluated. Its secondary structure was evaluated using UNAfold (https:// www.idtdna.com/Unnafold/Home/Index).
TABLE 1 probes and generic primer sequence Listing for detection of SHANK3, CHRNA7, GABRB3 and NODAL genes
2. Reagent kit
The kit comprises the probe in step 1.
Wherein, the kit also comprises reaction liquid of hybridization reaction, ligation reaction and PCR reaction.
Wherein, the reaction solution used for the hybridization reaction is a hybridization buffer solution which is conventionally used for the probe hybridization ligation reaction in the field; the reaction solution used in the ligation reaction is a ligation buffer solution and ligase which are conventionally used in probe hybridization ligation reaction in the field; the PCR reaction solution is a buffer solution (containing dNTP and Mg) which is conventionally used in PCR in the field 2+ ) Universal primer, taq enzyme and ultrapure water.
The conditions used in the kit of the present invention include hybridization reaction conditions, ligation reaction conditions, and PCR reaction conditions.
Wherein, the hybridization reaction conditions are 1min at 95 ℃ and 16h at 60 ℃. The reaction conditions of the ligation reaction are: 15min at 54 ℃, 5min at 98 ℃ and 5min at 20 ℃. The reaction conditions of the PCR reaction are as follows: 30s at 95 ℃, 30s at 60 ℃ and 60s at 72 ℃;35 cycles; and the temperature is 72 ℃ for 20min.
EXAMPLE 2 detection of Normal samples
1. Processing of samples
The kit of example 1 was used to detect normal human DNA by the following procedure:
1. sample collection, DNA extraction and processing
Peripheral blood of healthy people is collected, and the genome DNA of a sample is extracted by adopting a conventional DNA extraction kit. The DNA sample concentration and purity were measured with a Denovix DS-11 UV spectrophotometer and diluted to 20 ng/. Mu.L and stored below-18 ℃ until use.
mu.L of the diluted genomic DNA was aspirated into a PCR reaction tube, and denatured at 98 ℃ for 5 minutes to obtain denatured genomic DNA.
Hybridization reaction
And (3) taking a hybridization buffer solution and a probe mixture in the kit to prepare a hybridization reaction solution.
And (3) adding 3 mu L of hybridization reaction liquid into the PCR reaction tube in the step 1, blowing and uniformly mixing by using a liquid transfer gun, and hybridizing at 95 ℃ for 1min and 60 ℃ for 16h to obtain a hybridization product.
Ligation reaction
Preparing a connecting reaction liquid, and uniformly mixing by blowing with a liquid transfer gun.
And (3) adding 32 mu L of the ligation reaction solution into the PCR reaction tube in the step (2), and blowing, beating and uniformly mixing. At 54 ℃ 15min,98 ℃ 5min,20 ℃ pause. The ligation product is obtained.
Reaction of
Preparing PCR reaction liquid, and uniformly mixing by blowing with a pipette.
| Components | 1 part dosage (mu L) |
| Ultrapure water | 6.5 |
| PCR buffer solution (containing dNTP and Mg) 2+ Etc.) | 1 |
| General primer F1 | 1 |
| General primer R1 | 1 |
| Taq enzyme | 0.5 |
| Total volume | 10 |
And (4) taking 10 mu L of PCR reaction solution, adding the PCR reaction solution into the PCR reaction tube in the step (3), and blowing, beating and uniformly mixing. 30s at 95 ℃, 30s at 60 ℃ and 60s at 72 ℃;35 cycles; reacting at 72 ℃ for 20min to obtain an amplification product.
Data analysis
The resulting amplification product was subjected to capillary electrophoresis on an ABI 3730XL sequencer.
And analyzing the acquired fluorescence data result by using GeneMarker V2.4.0 software to obtain the information of the size, the peak height and the peak area of the fragment with specific fluorescence.
The peak area ratio (R) of each gene was calculated from the peak areas.
Peak area ratio (R) = (area of gene peak to be detected of sample/area of internal reference gene peak of sample)/(area of gene peak to be detected of reference sample/area of internal reference gene peak of reference sample)
And finally, judging the copy number of the gene locus according to the peak area ratio, wherein the corresponding relation between the peak area ratio and the gene copy number is as follows:
| peak area ratio (R) | Number of copies |
| R=0 | 0 |
| 0.40＜R＜0.65 | 1 |
| 0.80＜R＜1.20 | 2 |
| 1.30＜R＜1.65 | 3 |
| 1.75＜R＜2.15 | 4 |
| R is other value | Ambiguous results |
As can be seen from the capillary electrophoresis chart shown in FIG. 1, the kit of the present invention comprising a plurality of pairs of probes has clear peak shape chart and no noise when detecting copy numbers of a plurality of sites on a genome.
Therefore, the probe and the kit provided by the invention have high specificity and sensitivity, can detect a plurality of sites in one tube simultaneously, and have high detection efficiency and low cost.
Example 3 autistic patient sample testing
Whole blood samples were taken from 3 patients with autism and 3 normal persons. And extracting genome DNA by a conventional method to obtain 6 samples to be detected. The detection is carried out by using Affymetrix Cytoscan gene chips and the kit and the method of the invention described in the embodiment 1 and the embodiment 2, respectively, and the detection results shown in the table 1 and the table 2 are obtained.
TABLE 1 Gene chip test results
| Sample numbering | The result of the detection |
| A1 | 22q13.3 microdeletions |
| A2 | 15q13.3 micro-repeat |
| A3 | 15q12 microdeletion |
| C1 | Is normal and normal |
| C2 | Is normal |
| C3 | Is normal |
TABLE 2 test results of the present invention
| Sample numbering | SHANK3 | CHRNA7 | GABRB3 |
| A1 | 0.58 | 1.13 | 1.06 |
| A2 | 1.00 | 1.42 | 0.95 |
| A3 | 0.89 | 0.91 | 0.56 |
| C1 | 0.96 | 1.05 | 0.89 |
| C2 | 1.12 | 0.87 | 1.01 |
| C3 | 1.03 | 1.13 | 0.95 |
The gene chip method and the method of the invention are compared and detected, and the obtained detection results are consistent. The conventional gene chip method has complex operation and high cost, the detection time is more than 2 days, and the detection resolution is more than kb. The method has the advantages of simpler operation, low cost, detection time which can be finished within 24 hours, direct detection aiming at specific genes and higher detection resolution.
The foregoing is a further detailed description of the invention in connection with specific/preferred embodiments and it is not intended to limit the invention to the specific embodiments described. It will be apparent to those skilled in the art from this disclosure that various alternatives and modifications can be made to the described embodiments without departing from the spirit of the invention and these alternatives and modifications are to be considered as within the scope of the invention.
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<400> 21
gtgccagcaa gatccaatct aga 23
Claims (6)
1. A kit for detecting the copy number variation of multiple gene loci of an autism patient is characterized by comprising a probe set, ligase, hybridization solution and Taq enzyme, wherein genes detected by the probe set consist of SHANK3, CHRNA7 and GABRB 3. The probe is a sequence shown in SEQ ID NO. 1-SEQ ID NO. 18.
2. The kit of claim 1, wherein the probe set and the detection gene are related by: the genes of SHANK3 are detected by SEQ ID NO. 1-SEQ ID NO.6, CHRNA7 is detected by SEQ ID NO. 7-SEQ ID NO. 12, and GABRB3 is detected by SEQ ID NO. 13-SEQ ID NO. 18.
3. The kit of claim 1, wherein the kit further comprises a probe sequence for detecting the control gene NODAL: SEQ ID NO.19, SEQ ID NO.20.
4. The kit of claim 1, further comprising a pair of universal primers. The universal primer is a sequence shown in SEQ ID NO.21 and SEQ ID NO 22.
5. A method for detecting multiple gene locus copy number variation in patients with autism using the kit of any one of claims 1-4, the method comprising the steps of:
(1) Extracting the genome DNA of a sample to be detected, and performing high-temperature denaturation treatment;
(2) Carrying out hybridization reaction on the probe set or the probe set in the kit and the denatured genomic DNA to obtain a hybridization product;
(3) Completely pairing and hybridizing the left probe 3 'end and the right probe 5' end which are adjacent to each other and are hybridized with the genome DNA by using the ligase to form a complete probe, and obtaining a ligation product;
(4) Performing PCR reaction on the connection product by using a universal primer, and performing capillary electrophoresis on the PCR product;
(5) Analyzing the capillary electrophoresis result to obtain the variation information of the copy numbers of SHANK3, CHRNA7 and GABRB3 related to the autism.
6. The method of claim 5, wherein the hybridization reaction conditions are: hybridization was carried out at 95 ℃ for 1min and at 60 ℃ for 16h.
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| CN202110595474.5A CN115478101A (en) | 2021-05-31 | 2021-05-31 | Kit and method for detecting multiple gene site copy number variation of autism patient |
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| CN202110595474.5A CN115478101A (en) | 2021-05-31 | 2021-05-31 | Kit and method for detecting multiple gene site copy number variation of autism patient |
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| CN115478101A true CN115478101A (en) | 2022-12-16 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117487904A (en) * | 2023-12-28 | 2024-02-02 | 湖南家辉生物技术有限公司 | GABRB3 gene mutant, mutant protein, reagent, kit and application |
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2021
- 2021-05-31 CN CN202110595474.5A patent/CN115478101A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117487904A (en) * | 2023-12-28 | 2024-02-02 | 湖南家辉生物技术有限公司 | GABRB3 gene mutant, mutant protein, reagent, kit and application |
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