Primer probe composition and application thereof

Abstract

The invention discloses a primer probe composition and application thereof. The primer probe composition disclosed by the invention comprises a forward primer, a reverse primer and a probe, the nucleotide sequence of the forward primer is as shown in SEQ ID NO.1, the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.2, and the nucleotide sequence of the probe is as shown in SEQ ID NO.3. When the primer probe composition is used for testing clinical samples, normal and rapid eye movement sleep behavior disorder patient samples can be well distinguished, and whether the rapid eye movement sleep behavior disorder patient can be converted into Parkinson's disease or not in the later period can be evaluated.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a primer-probe composition and its application. More specifically, it relates to a primer-probe composition and its application in the preparation of products for diagnosing rapid eye movement sleep behavior disorder and / or products for predicting the conversion of rapid eye movement sleep behavior disorder to Parkinson's disease. Background Art

[0002] Rapid eye movement sleep behavior disorder (RBD) is a sleep disorder characterized by muscle relaxation failure during REM sleep. Patients often have shouting, limb twitching or even violent movements in dreams, which may harm themselves or others. RBD is not only an independent sleep disease, but also an important precursor marker of α-synucleinopathies (such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy). Studies have shown that about 80% of RBD patients will progress to Parkinson's disease or other neurodegenerative diseases within 10 - 15 years, making it a key window for predicting neurodegenerative lesions.

[0003] Currently, the diagnosis of RBD mainly relies on polysomnography (PSG), which is confirmed by detecting abnormal electromyogram activity during REM sleep and combining with clinical manifestations. However, the PSG technology is costly, complex to operate, and relies on professional analysis, resulting in difficulty in popularization in primary medical institutions, and many potential patients cannot be identified early. At the same time, the diagnosis of Parkinson's disease (PD) still focuses on motor symptoms (such as tremors, bradykinesia), but at this time, patients often have entered the middle and late stages, and the substantia nigra dopamine neurons have been significantly lost, missing the best intervention opportunity. Therefore, developing highly sensitive and specific RBD diagnostic reagents is crucial for early screening; at the same time, exploring predictive markers for the conversion of RBD to PD helps to intervene in the preclinical stage of neurodegenerative lesions, strive for a key time window for neuroprotective treatment, achieve precise diagnosis and treatment of neurodegenerative diseases, and provide new strategies for the early prevention and control of neurodegenerative diseases. Summary of the Invention

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a primer-probe composition and its application in the preparation of products for diagnosing rapid eye movement sleep behavior disorder and / or products for predicting the conversion of rapid eye movement sleep behavior disorder to Parkinson's disease.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a primer-probe composition.

[0007] Further, the primer-probe composition includes a forward primer, a reverse primer and a probe.

[0008] Further, the nucleotide sequence of the forward primer is as shown in SEQ ID NO.1.

[0009] Further, the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.2.

[0010] Further, the nucleotide sequence of the probe is as shown in SEQ ID NO.3.

[0011] Further, the 5' end of the probe can be labeled with a fluorescent emitting group; the 3' end of the probe can be labeled with a fluorescent quenching group.

[0012] Further, the fluorescent emitting group is selected from one of FAM, VIC, HEX, JOE, Cy3, ROX, and Cy5.

[0013] Further, the fluorescent quenching group is selected from one of BHQ1, BHQ2, BHQ3, TAMRA, and MGB.

[0014] Further, the primer-probe composition further contains primers and probes for detecting reference genes.

[0015] Preferably, the reference genes include but are not limited to GAPDH, β-actin, B2M, ACTB, SDHA, HPRT1, ARBP, etc. In a specific embodiment of the present invention, the reference gene is selected from ACTB.

[0016] The "reference gene" referred to in the present application can be various house-keeping genes, and these house-keeping genes are stably expressed in cells, which helps to maintain the functions of cells. House-keeping genes are expressed in various types of cells in organisms, and their products are genes encoding proteins necessary for maintaining the basic life activities of cells, such as tubulin genes, glycolytic enzyme system genes, and ribosomal protein genes.

[0017] In the present invention, the primer-probe composition is used to detect the DNA methylation level of the SNCA gene. The SNCA gene is located on human chromosome 4q21-23 (NC_000004.12, 89724099-89838324), spanning approximately 121198 bp, Gene ID: 6622, and the research site in this study is located at 7037 bp of the SNCA gene.

[0018] In various aspects of the present invention, the primers and probes used are not limited to the specific sequences described in the present invention, but include those sequences having at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% identity thereto and still maintaining their functions. Those skilled in the art can determine sequence identity through conventional procedures.

[0019] In some embodiments, the primers can be labeled with a labeling substance. The labeling substances include but are not limited to fluorescent substances, radioisotopes or enzymes. Among them, the fluorescent substances include but are not limited to TAMRATTM, Alexa555, Alexa647, Cy3, Cy5 of the cyanine dye series, fluorescein. The radioisotopes include but are not limited to 32P, 33P, 35S. The enzymes include but are not limited to alkaline phosphatase, horseradish peroxidase.

[0020] In some embodiments, a fluorescent labeling group and a fluorescent quenching group are provided at both ends of the probe.

[0021] The fluorescent labeling group is selected from one of FAM, VIC, HEX, JOE, Cy3, ROX and Cy5.

[0022] The fluorescent quenching group is selected from one of BHQ1, BHQ2, BHQ3, TAMRA and MGB.

[0023] The second aspect of the present invention provides a kit for diagnosing rapid eye movement sleep behavior disorder and / or predicting the conversion of rapid eye movement sleep behavior disorder to Parkinson's disease.

[0024] Furthermore, the kit contains the primer-probe composition described in the first aspect of the present invention.

[0025] Furthermore, the kit further includes a bisulfite modification reagent and a conventional PCR amplification reagent.

[0026] Furthermore, the conventional PCR amplification reagent includes dNTP, DNA polymerase, and amplification buffer.

[0027] Preferably, the kit further includes an instruction manual.

[0028] In the present invention, a suitable amount of one or more primers or probes is provided in one or more containers or immobilized on a matrix. The primers can be provided as suspended in an aqueous solution or, for example, as a lyophilized or freeze-dried powder. Among them, the container containing the nucleic acid can be any conventional container capable of accommodating the provided form, such as a microcentrifuge tube, an ampoule or a bottle.

[0029] In certain applications, one or more primers or probes (as described above) can be provided in pre-measured single-use amounts in separate, typically disposable tubes or equivalent containers. Using such an arrangement, a sample for testing the presence of DNA methylation of the SNCA gene can be added to a separate tube and directly amplified.

[0030] In certain embodiments, the kit can contain the reaction reagents necessary to perform a PCR amplification reaction, including DNA sample preparation reagents, enzymes for PCR, buffers, Mg 2+ and deoxynucleotides (dNTPs).

[0031] Among them, the enzymes for PCR include DNA polymerase and / or RNA polymerase.

[0032] dNTP is a nucleoside source for PCR-based DNA amplification, and the four types of dATP, dGTP, dCTP, and dTTP are necessary. In addition, for dNTP, a chemically modified substance for use in the hot start method can be used. For example, CleanAmpTM dNTP manufactured by TriLink BioTechnologies, Inc. can be used.

[0033] Preferably, the kit can also include a positive control and a negative control. Specifically, the positive control is human methylated standard DNA, and the negative control is human unmethylated standard DNA.

[0034] In the present invention, the "bisulfite modification reagent" refers to a reagent that contains bisulfite, disulfite, hydrogen sulfite, or a combination thereof in some embodiments. After treatment with the bisulfite reagent, the cytosine nucleotides that are not methylated in the DNA will be converted to uracil, while the methylated cytosine and other bases remain unchanged. Therefore, methylation and unmethylated cytidine in, for example, CpG dinucleotide sequences can be distinguished.

[0035] Preferably, the kit can also include DNA purification reagents, DNA extraction reagents, etc. Specifically, the DNA extraction reagents can include a lysis buffer, a binding buffer, a washing buffer, and an elution buffer. The lysis buffer is usually composed of a protein denaturant, a detergent, a pH buffer, and a nuclease inhibitor.

[0036] More preferably, the kit can also include an instruction manual for indicating the detection operation steps and the result judgment criteria.

[0037] In some of these embodiments, the kit can be used for the following detection platforms: PCR amplification method, fluorescence quantitative PCR method, digital PCR method, liquid phase chip method, third-generation sequencing method, second-generation sequencing method, pyrosequencing method, bisulfite conversion sequencing method, methylation chip method, reduced representation bisulfite sequencing technology, or a combination thereof.

[0038] When implementing the present invention, as additional necessary implements, implements widely used in molecular biology experiments such as pipettes, pipette tips, 1.5 ml microtubes, etc. can be cited. As devices, instruments widely used in molecular biology experiments such as PCR, purification benches, tube centrifuges, etc. can be cited.

[0039] The third aspect of the present invention provides a method for detecting the DNA methylation level at the SNCA gene locus.

[0040] Furthermore, the method includes performing PCR amplification on the sample DNA using the primer-probe composition described in the first aspect of the present invention.

[0041] Furthermore, the SNCA gene locus is located at 7037 bp of the SNCA gene.

[0042] Furthermore, the sample DNA is bisulfite-treated human genomic DNA.

[0043] Furthermore, the human genomic DNA is obtained from a human whole blood sample.

[0044] Furthermore, the PCR amplification is carried out under similar amplification conditions.

[0045] Furthermore, based on a total volume of 25 μL, the system for the PCR amplification includes: 12.5 μL of 2×PCR reaction buffer, 1 μL each of the forward and reverse primers, 1 μL of the probe, 5 μL of the sample DNA, and the balance is water.

[0046] Preferably, the reaction conditions for the PCR amplification are:

[0047] 1) Pre-denaturation at 95°C for 30 seconds;

[0048] 2) Denaturation at 95°C for 20 seconds, annealing at 50°C for 20 seconds, extension at 72°C for 30 seconds, for 10 cycles;

[0049] 3) Denaturation at 95°C for 5 seconds, annealing at 53°C for 30 seconds, for 35 cycles.

[0050] Exemplary embodiments of amplification conditions are provided in the embodiments of the present invention. However, as used herein, the term "amplification conditions" refers to the temperature and / or incubation time suitable for obtaining a detectable amount of the target. Thus, the term "similar amplification conditions" means that, if desired, assays can be performed on each target at a similar temperature. The term "similar amplification conditions" also means that, if desired, assays can be performed on each target for a similar incubation time. In some cases, the term "similar amplification conditions" also relates to the number of amplification cycles. However, it is well known in the art that the number of cycles is not always strict. For example, some samples may be removed before or left for additional amplification cycles compared to other samples. In other cases, the term "similar amplification conditions" also relates to the nature of the buffers and amplification reagents (enzymes, nucleotides, salts, etc.) used. The term "similar amplification conditions" also means that the conditions (e.g., time, buffer, number of cycles, temperature, etc.) can be slightly altered or can be the same.

[0051] The fourth aspect of the present invention provides the use of the primer-probe composition described in the first aspect of the present invention in the preparation of a product for detecting the DNA methylation level at the SNCA gene locus.

[0052] Furthermore, the SNCA gene locus is located at 7037 bp of the SNCA gene.

[0053] The fifth aspect of the present invention provides the use of the primer-probe composition described in the first aspect of the present invention in the preparation of a product for the auxiliary diagnosis of rapid eye movement sleep behavior disorder.

[0054] The sixth aspect of the present invention provides the use of the primer-probe composition described in the first aspect of the present invention in the preparation of a product for predicting the conversion of rapid eye movement sleep behavior disorder to Parkinson's disease.

[0055] The terms "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain cases. However, in the same or other cases, other embodiments may also be preferred. In addition, the recitation of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of the present invention.

[0056] In the description of the present invention, the term "and / or" includes all and any combinations of one or more of the related listed items.

[0057] In the description of the present invention, the descriptions of terms such as "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0058] Advantages and beneficial effects of the present invention over the prior art:

[0059] By means of fluorescence quantitative PCR, we designed specific primer-probes at the 7037bp of the SNCA gene and tested clinical samples. The results showed that it could very well distinguish between normal and rapid eye movement sleep behavior disorder patient samples and could evaluate whether RBD patients could be converted into PD in the later stage. Description of the Drawings

[0060] Figure 1 It is the amplification curve graph of the methylation site of the SNCA gene for RBD patients. Detailed Embodiments

[0061] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.

[0062] Unless otherwise specified, the experimental methods used in the following embodiments are generally in accordance with conventional conditions for the experimental methods without specific conditions noted in the embodiments. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0063] Embodiment

[0064] I. Experimental Materials

[0065] Samples: Whole blood samples were used, and the samples were sourced from patients with rapid eye movement sleep behavior disorder (RBD) and healthy control groups.

[0066] Reagents: Whole blood nucleic acid extraction kit, bisulfite conversion reagent, QPCR-related reagents (including methylation site-specific primers and probes)

[0067] Instruments: Fluorescence quantitative PCR instrument

[0068] II. Experimental Methods

[0069] Extraction and purification of whole blood nucleic acid DNA: Standard nucleic acid extraction kits or commercial automated extraction instruments were used to extract nucleic acid DNA from the whole blood samples of patients and perform purification treatment to ensure high-quality DNA samples were obtained.

[0070] Bisulfite conversion treatment: The extracted whole blood nucleic acid DNA was subjected to bisulfite conversion treatment to convert unmethylated cytosine (C) to uracil (U), while methylated cytosine remained unchanged. This treatment step is crucial for methylation detection.

[0071] Detection of the methylation status of the SNCA gene: Real-time fluorescence quantitative PCR (QPCR) technology was used. Through methylation site-specific primers and probes, the DNA fragments after bisulfite treatment were amplified to detect the proportion of methylated cytosine at specific sites of the SNCA gene in patient samples, thereby reflecting the methylation status of this site.

[0072] The reaction system for PCR amplification was 25 μL, including 12.5 μL of 2×PCR reaction Mix (including reaction solution and Taq enzyme), 1 μL each of the forward and reverse primers in the 10 μM primer, 1 μL of the 10 μM methylation fluorescence probe, 5 μL of the product after bisulfite conversion, and H2O was added to make up to 25 μL.

[0073] The reaction conditions for PCR amplification were: 1) Pre-denaturation at 95°C for 30 seconds; 2) Denaturation at 95°C for 20 seconds, annealing at 50°C for 20 seconds, extension at 72°C for 30 seconds, step 2) was repeated for 10 cycles; 3) Denaturation at 95°C for 5 seconds, annealing at 53°C for 30 seconds (fluorescence signal collection), repeated for 35 cycles.

[0074] In the primer-probe composition used in the present invention, the sequence of the forward primer is 5’-GGGGAGAACGTTTTTTCGGGTGGTTGGCGC-3’ (SEQ ID NO.1); the sequence of the reverse primer is 5’-CCCCCAACAAACCCAAATATAATAATTC-3’ (SEQ ID NO.2); the sequence of the probe is 5’-CGGTTCGCGAGTGTGAGCGGCGTTT-3’ (SEQ ID NO.3).

[0075] III. Experimental Results

[0076] Figure 1The left figure in it is the amplification curve of internal reference ACTB, showing that the amplification CT values of the samples are basically the same, and the CT value is 27. The right figure is the amplification curve of this methylation site, which is amplified by primers and probes specific for methylation at this site. The results show that the CT value of the normal human sample is 20, while the CT values of RBD patients are between 24 and 28. Among them, two RBD patients with lower methylation levels have been converted to PD after follow-up, indicating that this site can not only distinguish normal people from RBD patients, but also evaluate whether RBD patients can be converted to PD in the later stage. RBD patients with lower methylation levels have an increased risk of being converted to PD in the later stage.

Claims

1. A primer-probe composition, characterized in that, The primer-probe composition includes a forward primer, a reverse primer, and a probe; The nucleotide sequence of the forward primer is as shown in SEQ ID NO.1; The nucleotide sequence of the reverse primer is as shown in SEQ ID NO.2; The nucleotide sequence of the probe is as shown in SEQ ID NO.

3.

2. The primer-probe composition according to claim 1, wherein The 5'-end of the probe can be labeled with a fluorescent emitting group; the 3'-end of the probe can be labeled with a fluorescent quenching group; Preferably, the fluorescent emitting group is selected from one of FAM, VIC, HEX, JOE, Cy3, ROX, and Cy5; Preferably, the fluorescent quenching group is selected from one of BHQ1, BHQ2, BHQ3, TAMRA, and MGB.

3. A kit for diagnosing rapid eye movement sleep behavior disorder and / or predicting the conversion of rapid eye movement sleep behavior disorder to Parkinson's disease, characterized in that, The kit contains the primer-probe composition according to claim 1 or 2.

4. The kit according to claim 3, characterized in that, The kit further includes a bisulfite modification reagent and a conventional PCR amplification reagent; Preferably, the conventional PCR amplification reagent includes dNTP, DNA polymerase, and an amplification buffer; Preferably, the kit further includes an instruction manual.

5. A method for detecting the DNA methylation level of the SNCA gene locus, characterized in that, The method includes performing PCR amplification on the sample DNA using the primer-probe composition according to claim 1 or 2.

6. The method according to claim 5, characterized in that, The SNCA gene locus is located at 7037bp of the SNCA gene; Preferably, the sample DNA is human genomic DNA after bisulfite treatment; Preferably, the human genomic DNA is obtained from a human whole blood sample; Preferably, the PCR amplification is performed under similar amplification conditions.

7. The method according to claim 6, wherein Based on a total volume of 25 μL, the system for the PCR amplification includes: 12.5 μL of 2×PCR reaction buffer, 1 μL each of the forward and reverse primers, 1 μL of the probe, 5 μL of the sample DNA, and the balance is water; Preferably, the reaction conditions for the PCR amplification are: 1) Pre-denaturation at 95°C for 30 seconds; 2) Denaturation at 95°C for 20 seconds, annealing at 50°C for 20 seconds, extension at 72°C for 30 seconds, for 10 cycles; 3) Denaturation at 95°C for 5 seconds, annealing at 53°C for 30 seconds, for 35 cycles.

8. Use of the primer-probe composition according to any one of claims 1-2 in the preparation of a product for detecting the DNA methylation level of the SNCA gene locus; Preferably, the SNCA gene locus is located at 7037bp of the SNCA gene.

9. Use of the primer-probe composition according to any one of claims 1-2 in the preparation of a product for assisting in the diagnosis of rapid eye movement sleep behavior disorder.

10. Use of the primer-probe composition according to any one of claims 1-2 in the preparation of a product for predicting the conversion of rapid eye movement sleep behavior disorder to Parkinson's disease.

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