Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a primer probe composition and application thereof in preparing a product for diagnosing rapid eye movement sleep disorder and/or a product for predicting the conversion of the rapid eye movement sleep disorder to Parkinson's disease.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The first aspect of the present invention provides a primer probe composition.
Further, the primer probe composition includes a forward primer, a reverse primer, and a probe.
Further, the nucleotide sequence of the forward primer is shown as SEQ ID NO. 1.
Further, the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 2.
Further, the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
Further, the 5 'end of the probe can be marked by adopting a fluorescent luminous group, and the 3' end of the probe can be marked by adopting a fluorescent quenching group.
Further, the fluorescent chromophore is selected from one of FAM, VIC, HEX, JOE, cy, ROX, and Cy 5.
Further, the fluorescence quenching group is selected from one of BHQ1, BHQ2, BHQ3, TAMRA and MGB.
Further, the primer probe composition also comprises a primer and a probe for detecting an internal reference gene.
Preferably, the reference genes include, but are not limited to, GAPDH, beta-actin, B2M, ACTB, SDHA, HPRT1, ARBP, and the like. In a specific embodiment of the invention, the reference gene is selected from ACTB.
The "reference gene" according to the present application may be various housekeeping genes (house-KEEPING GENES) which are constitutively expressed in cells and contribute to maintaining the functions of the cells. The housekeeping gene is expressed in various cells of organism, and the product is the gene coded by protein necessary for maintaining basic life activity of the cells, such as tubulin gene, glycolytic enzyme system gene, ribosomal protein gene, etc.
In the present invention, the primer probe composition is used to detect the DNA methylation level of SNCA gene, which is located on human chromosome 4q21-23 (NC_ 000004.12,89724099-89838324), span about 121198bp, gene ID 6622, and the research site is located at 7037bp of gene SNCA gene.
In various aspects of the invention, the primers and probes used are not limited to the specific sequences recited herein, but include those sequences that are at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% identical thereto, and yet retain their function. The person skilled in the art can determine the sequence identity by routine procedures.
In some embodiments, the primer may be labeled with a labeling substance. The labeling substance includes, but is not limited to, a fluorescent substance, a radioisotope, or an enzyme. Among them, the fluorescent substances include, but are not limited to TAMRATTM, alexa555, alexa647, cy3, cy5 of cyanine dye series, fluorescein. Radioisotopes include, but are not limited to, 32P, 33P, 35S. Enzymes include, but are not limited to, alkaline phosphatase, horseradish peroxidase.
In some embodiments, both ends of the probe are provided with a fluorescent labeling group and a fluorescent quenching group.
The fluorescent labeling group is selected from one of FAM, VIC, HEX, JOE, cy, ROX and Cy 5.
The fluorescence quenching group is selected from one of BHQ1, BHQ2, BHQ3, TAMRA and MGB.
In a second aspect, the invention provides a kit for diagnosing and/or predicting the conversion of rapid eye movement sleep disorders to parkinson's disease.
Further, the kit comprises the primer probe composition according to the first aspect of the present invention.
Further, the kit also comprises bisulphite modification reagents and conventional PCR amplification reagents.
Further, the conventional PCR amplification reagents comprise dNTPs, DNA polymerase and amplification buffer.
Preferably, the kit further comprises instructions.
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 substrate, the primers may be provided suspended in an aqueous solution, or e.g. as a freeze-dried or lyophilized powder. The container in which the nucleic acid is provided may be any conventional container capable of holding the provided form, such as a microcentrifuge tube, ampoule or bottle.
In some applications, one or more primers or probes (as described above) may be provided in a separate, typically disposable tube or equivalent container in a pre-measured single use amount. With this arrangement, samples for testing for the presence of DNA methylation of the SNCA gene can be added to separate tubes for amplification directly.
In certain embodiments, the kit may contain the reagents necessary to perform a PCR amplification reaction, including DNA sample preparation reagents, enzymes for PCR, buffers, mg2+, and deoxyribonucleotides (dNTPs).
Wherein the enzyme of PCR comprises DNA polymerase and/or RNA polymerase.
DNTPs are nucleoside sources, dATP, dGTP, dCTP, dTTP, which are necessary for PCR-based DNA amplification. As dntps, those chemically modified for use in the hot start method can be used, and TriLink BioTechnologies, inc. CLEANAMPTM DNTP, for example, can be used.
Preferably, the kit can also comprise a positive quality control and a negative quality control. Specifically, the positive quality control substance is human methylation standard DNA, and the negative quality control substance is human non-methylation standard DNA.
In the present invention, the "bisulfite-modifying reagent" refers to a reagent comprising bisulfite (disulfite), bisulfite (hydrogen sulfite), or a combination thereof in some embodiments, bisulfite reagent treated DNA that converts unmethylated cytosine nucleotides to uracil while methylated cytosines and other bases remain unchanged, thus distinguishing between methylated and unmethylated cytosines in, for example, cpG dinucleotide sequences.
Preferably, the kit may further include a DNA purification reagent, a DNA extraction reagent, and the like. In particular, the DNA extraction reagent may include a lysis buffer, a binding buffer, a wash buffer, and an elution buffer. The lysis buffer typically consists of a protein denaturing agent, a detergent, a pH buffer and a nuclease inhibitor.
More preferably, the kit may further comprise instructions for indicating the detection procedure and the result determination criteria.
In some of these embodiments, the kit may be used in an assay platform such as a PCR amplification method, a fluorescent quantitative PCR method, a digital PCR method, a liquid chip method, a third generation sequencing method, a second generation sequencing method, a pyrophosphate sequencing method, a bisulfite conversion sequencing method, a methylation chip method, a reduced bisulfite sequencing technique, or a combination thereof.
In carrying out the present invention, examples of other necessary devices include devices widely used in experiments in molecular biology, such as a pipette, a pipette tip, and a 1.5ml microtube (microtube), and examples of devices widely used in experiments in molecular biology, such as a PCR, a purification stage, and a tube centrifuge.
In a third aspect, the invention provides a method of detecting the level of DNA methylation at the SNCA gene locus.
Further, the method comprises PCR amplification of sample DNA using the primer probe composition of the first aspect of the invention.
Further, the SNCA gene locus is located at 7037bp of the SNCA gene.
Further, the sample DNA is sulfite-treated human genomic DNA.
Further, the human genomic DNA is obtained from a human whole blood sample.
Further, the PCR amplification is performed under similar amplification conditions.
Further, the PCR amplification system comprises, in a total volume of 25. Mu.L, 12.5. Mu.L of 2 XPCR reaction buffer, 1. Mu.L of each of the forward and reverse primers, 1. Mu.L of probe, 5. Mu.L of sample DNA, and the balance of water.
Preferably, the reaction conditions for the PCR amplification are:
1) Pre-denaturation at 95 ℃ for 30 seconds;
2) Denaturation at 95 ℃ for 20 seconds, annealing at 50 ℃ for 20 seconds, elongation at 72 ℃ for 30 seconds, 10 cycles;
3) Denaturation at 95 ℃ for 5 seconds, annealing at 53 ℃ for 30 seconds, 35 cycles.
The examples section of the present invention provides exemplary embodiments of amplification conditions. However, the term "amplification conditions" as used herein relates to a temperature and/or incubation time suitable for obtaining a detectable amount of target. Thus, the term "similar amplification conditions" means that each target can be assayed at a similar temperature, if desired. The term "similar amplification conditions" also means that each target can be assayed at similar incubation times, if desired. 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 critical. For example, some samples may be removed or left for additional amplification cycles before 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.) may vary slightly or may be the same.
In a fourth aspect, the invention provides the use of a primer probe composition according to the first aspect of the invention in the preparation of a product for detecting the DNA methylation level of a SNCA gene locus.
Further, the SNCA gene locus is located at 7037bp of the SNCA gene.
In a fifth aspect, the invention provides the use of a primer probe composition according to the first aspect of the invention for the preparation of a product for aiding in the diagnosis of rapid eye movement sleep disorders.
In a sixth aspect, the invention provides the use of a primer probe composition according to the first aspect of the invention in the preparation of a product for predicting the conversion of rapid eye movement sleep disorders to parkinson's disease.
The words "preferably," "more preferably," and the like in the present invention refer to embodiments of the invention that may provide certain benefits in some instances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
In the description of the present invention, the term "and/or" includes all and any combination of one or more of the associated listed items.
In the description of the present invention, the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc. describe mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Compared with the prior art, the invention has the advantages and beneficial effects that:
The specific primer probe at 7037bp of SNCA gene is designed by a fluorescence quantitative PCR method, and clinical samples are tested, so that the results show that normal and rapid eye movement sleep behavior disorder patient samples can be well distinguished, and whether the RBD patient can be converted into PD in the later period can be evaluated.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Examples
1. Experimental materials
Sample using whole blood samples derived from patients with rapid eye movement sleep disorder (RBD) and healthy control populations.
Reagent Whole blood nucleic acid extraction kit, sulfite conversion reagent, QPCR related reagent (including methylation site specific primer and probe)
Instrument, fluorescent quantitative PCR instrument
2. Experimental method
Extraction and purification of whole blood nucleic acid DNA by extracting nucleic acid DNA from whole blood sample of patient with standard nucleic acid extraction kit or commercial automatic extraction instrument, and purifying to obtain high quality DNA sample.
Sulfite conversion treatment the extracted whole blood nucleic acid DNA is subjected to sulfite conversion treatment to convert unmethylated cytosine (C) into uracil (U) while methylated cytosine remains unchanged. This processing step is critical for methylation detection.
And detecting the methylation state of the SNCA gene, namely detecting the proportion of methylated cytosine at a specific site of the SNCA gene in a patient sample by adopting a real-time fluorescent Quantitative PCR (QPCR) technology and amplifying a DNA fragment subjected to bisulfite treatment through a methylation site specific primer and a probe, so as to reflect the methylation state of the site.
The PCR amplification reaction system was 25. Mu.L, and contained 12.5. Mu.L of 2 XPCR reaction Mix (including reaction solution and Taq enzyme), 1. Mu.L of each of 10. Mu.M forward and reverse primers in the primers, 1. Mu.L of 10. Mu.M methylation fluorescent probe, 5. Mu.L of sulfite-converted product, and H2O up to 25. Mu.L.
The reaction conditions for PCR amplification were 1) 95℃for 30 seconds, 2) 95℃for 20 seconds, 50℃for 20 seconds, 72℃for 30 seconds, step 2) 10 cycles, 3) 95℃for 5 seconds, 53℃for 30 seconds (collecting fluorescent signals), and 35 cycles.
In the primer probe composition used in the 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), and the sequence of the probe is 5'-CGGTTCGCGAGTGTGAGCGGCGTTT-3' (SEQ ID NO. 3).
3. Experimental results
The left plot in fig. 1 shows an amplification curve of the reference ACTB, showing that the amplified CT values of the samples are substantially identical, with a CT value of 27. The right graph shows the amplification curve of the methylation site, the amplification is carried out through a primer probe specific to methylation of the site, the result shows that the CT value of a normal human sample is 20, the CT value of RBD patients is between 24 and 28, two RBD patients with lower methylation level are converted into PD after follow-up, the site can distinguish normal people and RBD patients, whether the RBD patients can be converted into PD later or not can be evaluated, the RBD patients with lower methylation level are also identified, and the risk of the later conversion into PD is increased.