Primer and probe for detecting CSF1R microglial cell encephalopathy pathogenic mutant gene

Abstract

The invention relates to the technical field of gene detection, in particular to primers and probes for detecting CSF1R microglial cell encephalopathy pathogenic mutant genes, the primers are as shown in SEQ ID NO: 1-SEQ ID NO: 90, and the probes are as shown in SEQ ID NO: 91-SEQ ID NO: 331. The primer and the probe provided by the invention can efficiently and specifically detect the CSF1R microglial cell encephalopathy pathogenic mutant gene, are high in sensitivity and strong in specificity, can be used for detecting the mutation site of the exon of the CSF1R gene, can also be used for detecting the mutation site of the intron of the CSF1R gene, are more comprehensive and accurate in detection, are simple to operate, are high in detection efficiency, and can be widely applied to detection of CSF1R microglial cell encephalopathy. The method can solve the problems of long time-consuming period, high cost, missing detection and the like in current clinical detection of CSF1R microglial cell encephalopathy pathogenic mutant genes, and is convenient for clinical popularization.

Description

Technical Field

[0001] The invention relates to the technical field of gene detection, in particular to primers and probes for detecting CSF1R microglial encephalopathy pathogenic mutation genes. Background Art

[0002] Colony stimulating factor-1 receptor (CSF1R) microglial encephalopathy is a rare, rapidly progressive dementia caused by mutations in the CSF1R gene. The clinical manifestations of CSF1R microglial encephalopathy are complex and diverse, with progressive cognitive decline, movement disorders, mental and behavioral abnormalities, and epilepsy as the main symptoms. The time from diagnosis to death is only about 6 years, which seriously affects the quality of life and prognosis of patients and also brings heavy economic and care burdens to their families. One of the main reasons for the poor prognosis of CSF1R microglial encephalopathy is the untimely diagnosis and lack of simple and easy detection methods. Clinically, it is often necessary to complete whole genome or whole exome testing, which is time-consuming and expensive. In addition, because there are still many new mutation sites reported for this disease, some mutation sites are in deep introns, so sometimes exon testing is easy to miss the diagnosis, and whole genome detection of this gene is often mistaken as variants of uncertain significance (VUS) and not reported. In addition, polymorphisms of the CSF1R gene can affect the expression of CSF1R, thereby affecting its normal biological effect function. For example, a deletion in exon 13 can lead to a continuous deletion of nearly 40 amino acid sites in the tyrosine kinase region of the CSF1R gene.

[0003] Although the emerging Amplification Refractory Mutation System (ARMS) PCR method can also detect gene loci, its false positive rate is still high, which limits the further improvement of its mutation detection sensitivity and makes it difficult to detect mutations below 1%. In addition, ARMS-PCR requires strict optimization of reaction conditions in the early stage of use, including the screening of ARMS primers, the selection of primers, probes, and Mg. 2+The selection of the optimal concentration of Taq enzyme, the exploration of annealing temperature, etc. are not convenient for clinical testing. Although digital PCR has higher sensitivity and specificity, its template quality requirements are high. If the amount of template exceeds the amount of the microsystem, it will lead to inability to quantify. If it is too little, the quantitative accuracy will be reduced. It is also expensive, complicated and time-consuming to operate. It is currently not suitable for clinical testing. Although multiplex PCR can theoretically detect mutation sites under different fluorescent channels at the same time and improve detection efficiency, fluorescent groups in different spectral ranges interfere with each other. In addition, with the increase in the number of multiplex PCR detection, the possibility of forming primer dimers or even polymers also increases significantly, which can lead to the occurrence of nonspecific amplification, a decrease in target amplification efficiency, and a decrease in detection sensitivity and specificity.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide primers and probes for detecting CSF1R microglial encephalopathy pathogenic mutation genes, which can efficiently and specifically detect CSF1R microglial encephalopathy pathogenic mutation genes, have high sensitivity and strong specificity, and can be used not only to detect mutation sites in exons of CSF1R genes, but also to detect mutation sites in introns of CSF1R genes, so that the detection is more comprehensive and accurate, and the operation is simple and the detection efficiency is high.

[0006] In a first aspect of the present invention, a CSF1R microglial encephalopathy pathogenic mutant gene is provided, wherein the mutant gene comprises: mutation sites in exons and introns of the CSF1R gene; the mutation sites comprise: mutation site 1 to mutation site 149 (see Table 1 for details).

[0007] In a second aspect of the present invention, primers and probes for detecting pathogenic mutation genes of CSF1R microglial encephalopathy are provided. Specific primers and probes are designed based on the above-mentioned mutant genes. The primers are shown in SEQ ID NO:1-SEQ ID NO:90, and the probes are shown in SEQ ID NO:91-SEQ ID NO:331.

[0008] Specifically, the probes include: a wild-type probe and a mutant probe, wherein the 5' ends of the wild-type probe and the mutant probe are both connected with fluorescent reporter groups which are different from each other, and the 3' ends of the wild-type probe and the mutant probe are both connected with fluorescent quenching groups.

[0009] Preferably, the fluorescent reporter group is selected from FAM, TET, VIC, HEX, JOE and the like, and the fluorescent quencher group is selected from MGB, Eclipse and the like.

[0010] The third aspect of the present invention provides a kit for detecting CSF1R microglial encephalopathy pathogenic mutation gene, comprising the above-mentioned primers and probes for detecting CSF1R microglial encephalopathy pathogenic mutation gene.

[0011] Preferably, the kit further comprises: an internal reference primer, the sequence of the internal reference primer being shown as SEQ ID NO:332-SEQ ID NO:333.

[0012] Preferably, the kit further comprises: a DNA extraction reagent, a PCR reaction reagent, a positive control substance and a negative control substance.

[0013] The fourth aspect of the present invention provides the use of the above-mentioned CSF1R microglial encephalopathy pathogenic mutation gene in the diagnosis of CSF1R microglial encephalopathy.

[0014] The fifth aspect of the present invention provides the use of the above-mentioned CSF1R microglial encephalopathy pathogenic mutation gene in the preparation of a diagnostic product for detecting CSF1R microglial encephalopathy.

[0015] Preferably, the diagnostic product includes: reagents and kits.

[0016] Preferably, the reagents or kits include: specific primers for one or more mutation sites mentioned above, and specific probes for one or more mutation sites mentioned above; more preferably, the reagents or kits include: specific primers for 149 mutation sites, the primers are shown in SEQ ID NO:1-SEQ ID NO:90, and specific probes for 149 mutation sites, the probes are shown in SEQ ID NO:91-SEQ ID NO:331.

[0017] Beneficial effects:

[0018] The primers and probes for detecting the CSF1R microglial encephalopathy pathogenic gene provided by the present invention can be used to efficiently and specifically detect the CSF1R microglial encephalopathy pathogenic gene, have high sensitivity and strong specificity, can be used not only to detect mutation sites in exons of the CSF1R gene, but also to detect mutation sites in introns of the CSF1R gene, and the detection is more comprehensive and accurate, and the operation is simple and the detection efficiency is high. The current clinical detection of the CSF1R microglial encephalopathy pathogenic gene, which is time-consuming, expensive, and missed, can be solved, and is convenient for clinical promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is an exemplary result diagram of the clustering map provided by the present invention.

[0021] Figure 2 This is a sequencing peak diagram of the sample provided by the present invention.

[0022] Figure 3 A clustering map of samples provided by the present invention.

[0023] Figure 4 This is the peak diagram of the sample binary sequencing provided by the present invention.

[0024] Figure 5 This is the sample di-clustering map provided by the present invention.

[0025] Figure 6 This is the three sequencing peak diagrams of the samples provided by the present invention.

[0026] Figure 7 This is the sample three-cluster map provided by the present invention.

[0027] Figure 8 This is the sequencing peak diagram of sample four provided by the present invention.

[0028] Fig. 9 This is the four-cluster map of samples provided by the present invention. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0030] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] This example provides a CSF1R microglial encephalopathy pathogenic mutation gene, primers and probes for detecting the CSF1R microglial encephalopathy pathogenic mutation gene, and the specific design steps are as follows:

[0034] 1. Selection of single nucleotide polymorphism (SNP) sites.

[0035] The pathogenic mutation gene of human CSF1R microglial encephalopathy was screened out by SNP retrieval, including the mutation sites in the exons and introns of the CSF1R gene. The single nucleotide polymorphism sites (SNPs) are shown in Table 1 (mutation site 1 to mutation site 149).

[0036] Table 1

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044] 2. Design specific primers and probes for amplifying polymorphic sites of human CSF1R gene.

[0045] 2.1 Use the genome viewer on the NCBI website to retrieve sequences of approximately 175 bp before and after the mutation site.

[0046] 2.2 Obtain CSF1R sequence information from the NCBI website, open the web search box, and enter the sequence near the mutation site obtained in step 2.1.

[0047] 2.3 Reference sequence position information Select all mutation points between 120bp-150bp in length of amplified fragments, select fragments that need to span introns, and adjust the sequence interval required for designing primers. If the amplified fragment is too short, it lacks specificity; if the amplified fragment is too long, the ability to bind to the primer will decrease, affecting the amplification efficiency; the amplified fragment length of 120-150bp has both high amplification specificity and ensures high amplification efficiency.

[0048] 2.4 Use NCBI BLAST's Primer-BLAST to design primers for the required interval.

[0049] 2.5 The probe design is as follows:

[0050] (1) Select target SNP sites

[0051] Determine the SNP site: Determine the specific sequence and position of the SNP site to be detected.

[0052] Sequence acquisition: Obtain sequence information of the target SNP site from a public database (such as dbSNP).

[0053] (2) Probe design principles

[0054] Specificity: The probe should be highly specific to the target sequence and avoid cross-hybridization with other sequences.

[0055] Length: Generally, the probe length is between 18-25 nucleotides to ensure specificity.

[0056] GC content: GC content is usually between 40%-60% to ensure probe stability and hybridization efficiency.

[0057] Tm value: The melting temperature (Tm) of the probe is about 5-10°C higher than the Tm value of the primer to ensure that the probe can stably bind to the target DNA during PCR amplification.

[0058] (3) Probe design steps

[0059] Primer design software: Use specialized software (such as Primer Express, Primer 5.0, OLI GO 6.0, etc.) to design probes.

[0060] Input sequence: Input the target sequence and upstream and downstream sequences into the software.

[0061] Set parameters: Set probe length, GC content, Tm value and other parameters.

[0062] Position selection: Make sure the probe covers the SNP site and is located in the center or 3' end of the SNP site as much as possible.

[0063] Modification: The 5' end of the wild-type probe sequence is connected to the FAM fluorescent reporter group, the 5' end of the mutant probe sequence is connected to the VIC fluorescent reporter group, and the 3' ends of the wild-type probe and the mutant probe sequences are connected to the MGB fluorescent quencher group.

[0064] (4) Probe optimization

[0065] Avoid secondary structures: The probes themselves should not form secondary structures such as hairpins or dimers.

[0066] Avoid consecutive GC: Avoid consecutive GC pairs in the probe, which may affect hybridization efficiency.

[0067] (5) Probe Verification

[0068] BLAST search: Use tools such as BLAST to verify the specificity of the probe sequence and ensure that it hybridizes only to the target sequence.

[0069] The primer and probe sequences are shown in Table 2 , where the sites corresponding to mutation sites 13, 53, 54, 72, 76, 84, 85, 94, 101, 102, 111, 128, 137, 138, 145, 146, and 147 are CSF1R intron mutation sites.

[0070] Table 2

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] Example 2

[0082] This embodiment provides a kit for detecting a CSF1R microglial encephalopathy pathogenic mutation gene and its detection steps:

[0083] 1. Some reagents are shown in Table 3.

[0084] Table 3

[0085]

[0086] 2. The sequences of internal reference primers are shown in Table 4.

[0087] Table 4

[0088]

[0089] 3. Control reagent.

[0090] Positive control: solution containing CSF1R genome;

[0091] Negative control: nuclease-free water, TE Buffer and other template-free controls.

[0092] 4. Detection steps.

[0093] 4.1 Genomic DNA extraction

[0094] 4.1.1 Use the Ezup column blood genomic DNA extraction kit, the reagents are shown in Table 5.

[0095] Table 5

[0096]

[0097] 4.1.2 Blood sample processing

[0098] a. Blood samples were anticoagulated with ACD (0.48% Citric Acid, 1.23% Sodium Citrate, 1.47% Glucose) or EDTA (pH 8.0).

[0099] b. When the blood collection volume is ≤200μl, take the blood sample and add it to a 1.5ml centrifuge tube, then add PBSSolution to make the total volume balanced to 200μl and mix well.

[0100] c. When the blood collection volume is >200μl, add 2 times the volume of Buffer TBP, mix thoroughly, and place at room temperature for 1 minute until the red blood cells are completely lysed. Centrifuge at 8,000rpm for 1 minute and discard the supernatant. Resuspend the precipitate with 500μl TE Buffer, centrifuge at 8,000rpm for 1 minute, discard the supernatant, and wash once more with TE Buffer until the precipitate is white, and then add 200μl PBS solution.

[0101] 4.1.3 Add 20 μl Proteinase K to the centrifuge tube and mix well. Then add 200 μl Buffer DL, shake and mix well, and place in a 56℃ water bath for 10 min.

[0102] a. When the mixed solution becomes clear and transparent, the lysis is complete. If the solution does not become clear, it means that the cell lysis is not complete, and the water bath time should be appropriately extended, otherwise the DNA acquisition rate may be reduced or the extracted DNA may be impure.

[0103] b. When the blood sample exceeds 500 μl, the amount of Proteinase K and lysis buffer can be appropriately increased and the water bath time can be extended.

[0104] 4.1.4 Add another 200 μl of anhydrous ethanol to the above centrifuge tube and mix thoroughly by inversion (Note: adding anhydrous ethanol may produce translucent fibrous suspension, which will not affect the extraction and application of DNA).

[0105] 4.1.5 Place the adsorption column in the collection tube, use a pipette to add all the solution and translucent fibrous suspension into the adsorption column, let it stand for 2 minutes, then centrifuge at 10,000 rpm at room temperature for 1 minute, and discard the waste liquid in the collection tube.

[0106] 4.1.6 Place the adsorption column back into the collection tube, add 500 μl GW Solution to the adsorption column, centrifuge at 10,000 rpm for 30 seconds and discard the waste liquid.

[0107] 4.1.7 Place the adsorption column back into the collection tube, add 700 μl Wash Solution to the adsorption column, centrifuge at 10,000 rpm for 30 seconds and discard the waste liquid. This step needs to be repeated once.

[0108] 4.1.8 Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 minutes at room temperature to remove the residual Wash Solution. In this step, the cover of the adsorption column needs to be opened and placed at room temperature for several minutes to completely dry the residual Wash Solution in the adsorption material. The residual Wash Solution will affect the yield of genomic DNA and subsequent experiments.

[0109] 4.1.9 Take out the adsorption column, put it into a new 1.5ml centrifuge tube, add 50μl CE Buffer and let it stand for 3min, centrifuge at 12,000rpm for 2min at room temperature, and collect the DNA solution. The extracted DNA can be used for the next experiment immediately or stored at -20℃. The DNA acquisition rate can be increased by repeating this step or preheating the CE Buffer to 60℃.

[0110] 4.2 Reagent preparation

[0111] 4.2.1 Take out the required components from the kit 30 minutes in advance, place them at room temperature, wait for the temperature to equilibrate to room temperature, oscillate to mix, and centrifuge for later use.

[0112] 4.2.2 Prepare the number of test reagents according to the number of samples to be tested and quality control. Add 10μl 2×Taq master mix, 1μl of upstream and downstream primers with a concentration of 10μM, and 0.4μl of probe with a concentration of 10μM to each reaction well in turn, and then transfer the reaction tube to the sample processing area.

[0113] 4.3 Sample addition

[0114] Add 1μl to 7.6μl of nucleic acid template to the reaction tube. The template volume can be adjusted according to actual needs, and the volume of nuclease-free water can be adjusted accordingly, so that the total volume of template and nuclease-free water is 7.6μl. Finally, oscillate to mix, and centrifuge briefly (the recommended template input amount is ≥10ng).

[0115] 4.4 Nucleic Acid Amplification

[0116] 4.4.1 Mix the reaction tube thoroughly. After mixing, centrifuge the reaction solution quickly and briefly to the bottom of the reaction tube.

[0117] 4.4.2 Place the reaction tube into the fluorescence detection device. Select “Genotyping” for the experiment type. The wild-type target is labeled with FAM, the mutant target is labeled with VIC, the negative control is “Negative Control”, and the positive control is “Positive Control Allele1 / Allele2”.

[0118] 4.5 Reaction system configuration

[0119] In a 20μL system, add 1μL template, 10μL 2×Taq master mix, 1μL 10μM forward primer, 1μL 10μM reverse primer, 0.4μL 10μM probe, and make up the rest with dd H2O. (Note: The amount of template input can be adjusted according to actual needs.)

[0120] 4.6 Fluorescence PCR reaction

[0121] The instruments include ABI 7500 (Applied Biosystems, USA), ABI-QSDX and other fluorescence quantitative PCR instruments and fluorescence detection equipment of most brands on the market. The reaction procedure is shown in Table 6.

[0122] Table 6

[0123]

[0124] * Fluorescence signal collection

[0125] 4.7 Quality Control

[0126] The experiment needs to set up positive and negative controls (nuclease-free water, TE Buffer and other template-free controls). The cluster diagram of the heterozygous typing results of the positive control is in the middle; if the negative control amplification curve is not detected and the test result is in the lower left corner of the cluster diagram, the experiment is established, otherwise the experiment is considered invalid.

[0127] 4.8 Results Analysis

[0128] 1. Positive: The homozygous type is distributed in the upper left corner and lower right corner of the cluster diagram, and the heterozygous type is in the middle of the cluster diagram;

[0129] 2. Negative: The amplification curve was not detected and the test result is in the lower left corner of the cluster diagram.

[0130] Example results are as follows Figure 1 As shown: the black square in the lower left corner is negative, the blue dot in the upper left corner and the red dot in the lower right corner are homozygous types, and the green dot in the middle position represents heterozygous types.

[0131] Test example

[0132] Taking the detection of mutation sites in 4 patients with clinically suspected CSF1R microglial encephalopathy as an example, according to the steps of Example 2, the results show that sample 1 (number T0015) is determined to be mutation site 147, and the sequencing peak diagram ( Figure 2 ) and clustering graph ( Figure 3 ) Check the product specificity and interpret the results. The genotype is heterozygous AG; Sample 2 (No. B0471) is determined to have mutation site 148. The sequencing peak diagram ( Figure 4 ) and clustering graph ( Figure 5 ) Check the product specificity and interpret the results. The genotype is heterozygous AG; Sample 3 (No. T0593) is determined to be mutation site 146. The sequencing peak diagram ( Figure 6 ) and clustering graph ( Figure 7 ) Check the product specificity and interpret the results. The genotype is heterozygous AG; Sample 4 (No. T0014-2) is determined to be mutation site 147. The sequencing peak diagram ( Figure 8 ) and clustering graph ( Fig. 9 ) Check the product specificity and interpret the results. The genotype is heterozygous AG.

[0133] A large number of clinical statistical results show that the primers and probes provided by the present invention can efficiently and specifically detect the CSF1R microglial encephalopathy pathogenic mutation gene, with high sensitivity and strong specificity. They can be used not only to detect the mutation sites of the exons of the CSF1R gene, but also to detect the mutation sites of the introns of the CSF1R gene. The detection is more comprehensive and accurate, and the operation is simple and the detection efficiency is high. The current clinical detection of the CSF1R microglial encephalopathy pathogenic mutation gene is time-consuming, expensive, missed, and other problems. The method is convenient for clinical promotion and is expected to improve the detection rate of CSF1R microglial encephalopathy in my country, promote early diagnosis and treatment, and reduce the mortality and disability rates of the disease.

[0134] In summary, the present invention utilizes specific primers and probes to competitively amplify and detect target genes, and uses fluorescence monitoring equipment to achieve real-time monitoring of the target fragment amplification process. The genotype of the gene in the sample can be determined through the allele cluster distribution map. The detection process does not require opening the lid to avoid the generation of aerosols, and the reliability of the results is greatly improved.

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. CSF1R microglial encephalopathy pathogenic mutation gene, characterized in that The mutant gene includes: mutation sites in exons and introns of the CSF1R gene; the mutation sites include: mutation site 1 to mutation site 149.

2. Primers and probes for detecting CSF1R microglial encephalopathy pathogenic mutation gene, characterized in that: The specific primers and probes designed for the mutant gene according to claim 1, wherein the primers are shown in SEQ ID NO:1-SEQ ID NO:90, and the probes are shown in SEQ ID NO:91-SEQ ID NO:

331.

3. The primers and probes for detecting the CSF1R microglial encephalopathy pathogenic mutation gene according to claim 2, characterized in that: The probes include: a wild-type probe and a mutant-type probe. The 5' ends of the wild-type probe and the mutant-type probe are both connected with fluorescent reporter groups, and the fluorescent reporter groups are different from each other.

4. A kit for detecting CSF1R microglial encephalopathy pathogenic mutation gene, characterized in that: The invention comprises the primers and probes for detecting the CSF1R microglial encephalopathy pathogenic mutation gene as described in any one of claims 2 to 3.

5. The kit for detecting CSF1R microglial encephalopathy pathogenic mutation gene according to claim 4, characterized in that: Also includes: An internal reference primer, the sequence of which is shown in SEQ ID NO:332-SEQ ID NO:

333.

6. The kit for detecting the CSF1R microglial encephalopathy pathogenic mutation gene according to claim 4, characterized in that: Also includes: DNA extraction reagents, PCR reaction reagents, positive control substances and negative control substances.

7. Use of the CSF1R microglial encephalopathy pathogenic mutation gene according to claim 1 in the diagnosis of CSF1R microglial encephalopathy.

8. Use of the CSF1R microglial encephalopathy pathogenic mutation gene according to claim 1 in the preparation of a diagnostic product for detecting CSF1R microglial encephalopathy.

9. The use according to claim 8, wherein the diagnostic product comprises: Reagents and test kits.

10. The use according to claim 9, characterized in that: The reagents and kits include: specific primers for one or more mutation sites described in claim 1, and specific probes for one or more mutation sites described in claim 1.

Images