Primer group for detecting Vel blood type characteristic sites, detection product and application thereof
By providing a primer set for detecting characteristic sites of VEL blood type, combined with PCR-SSP technology, the problems of low efficiency and high cost of VEL blood type detection in the prior art are solved, and the safety of rapid and accurate identification and clinical diagnosis of Vel blood type is improved.
Patent Information
- Application Number
- CN202411954949.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-03-25
AI Technical Summary
The prior art lacks detection primers for VEL blood type characteristic sites, resulting in VEL blood type detection relying on antigen typing kits, which are inefficient and cost-effective.
A primer set for detecting characteristic sites of Vel blood type is provided, including primer pairs for detecting c.111-119T>C and c.165C>T sites. It can identify specific gene sequences through PCR-SSP technology to achieve rapid and accurate identification of Vel blood type.
Through the application of this primer set, the rapid and accurate identification of Vel blood type is achieved, the number of detection reaction systems is reduced, the detection efficiency is improved, the time and labor cost is reduced, and the safety of clinical diagnosis is improved.
Smart Images
Figure CN119685489A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical fields of "immunological detection technology", "biotechnology" and "medical equipment", and in particular to a primer set for detecting Vel blood type characteristic sites, a detection product and an application thereof. Background Art
[0002] The VEL blood group system antigen was confirmed as an independent system antigen by the International Society of Blood Transfusion (ISBT) in 2014. The VEL blood group system number is 034. The VEL antigen was discovered by Sussman and Miller in 1952 and was classified as a high-frequency "series" antigen at the time. As of October 2024, it has been confirmed that there is one antigen in this system, named Vel, and numbered 034001. As the research on this antigen gradually deepened, ISBT classified this antigen as a "system" antigen in 2014. This antigen is encoded and expressed by the gene SMIM1, and Vel expression negative (Vel-) is a rare phenotype. Anti-Vel antibodies can cause intravascular hemolysis and neonatal hemolytic disease, which has great clinical significance. In the study of the distribution of the VEL blood group system in the Chinese population, some characteristic sites were found, such as c.111-119T>C and c.165C>T. The US NCBI database accession number (GenBank accession number) is PQ537116. Currently, the detection of VEL blood type relies on antigen typing kits, and there is a lack of detection primers targeting characteristic sites.
[0003] In view of this, the present invention is proposed. Summary of the invention
[0004] The object of the present invention is to provide a primer set for detecting Vel blood type characteristic sites, a detection product and its application to solve the above technical problems.
[0005] The present invention is achieved in that:
[0006] In the first aspect, the present invention provides a primer set for detecting Vel blood type characteristic sites, comprising: a first primer pair for detecting the c.111-119T>C site and / or a second primer pair for detecting the c.165C>T site, the nucleotide sequence of the first primer pair is shown in SEQ ID NO: 1-2, and the nucleotide sequence of the second primer pair is shown in SEQ ID NO: 3-4.
[0007] In a second aspect, the present invention also provides a product for detecting Vel blood type characteristic sites, which includes the above-mentioned primer set for detecting Vel blood type characteristic sites, and the product is a reagent, a kit or a chip.
[0008] In a third aspect, the present invention also provides the use of a primer set for detecting characteristic loci of Vel blood type in the preparation of a product for detecting characteristic loci of Vel blood type, wherein the product is a reagent, a kit or a chip.
[0009] The present invention has the following beneficial effects:
[0010] The present invention uses sequence-specific primer technology (PCR-SSP) to identify specific gene sequences in the characteristic sites of Vel blood type in the Chinese population, thereby realizing rapid and accurate identification of Vel blood type without antigen typing. This greatly reduces the number of reaction systems during detection, improves detection efficiency, and saves time and labor costs.
[0011] The primer set for detecting the Vel blood type characteristic site provided by the present invention has the technical advantages of high detection sensitivity and high accuracy, high clinical diagnostic safety, and is suitable for clinical diagnostic applications.
[0012] In addition, the present invention also provides detection products that match the detection primer set, such as reagents and kits, which automatically detect fluorescent signals through a PCR instrument, reduce the possibility of human errors during operation, and improve the reliability of detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.
[0014] Figure 1 This is the solubility curve when the characteristic locus c.111-119T>C genotype is positive;
[0015] Figure 2 This is the solubility curve when the characteristic locus c.165C>T genotype is positive;
[0016] Figure 3 Melting curve plot of negative sample. DETAILED DESCRIPTION
[0017] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.
[0018] In the first aspect, the present invention provides a primer set for detecting Vel blood type characteristic sites, comprising: a first primer pair for detecting the c.111-119T>C site and / or a second primer pair for detecting the c.165C>T site, the nucleotide sequence of the first primer pair is shown in SEQ ID NO: 1-2, and the nucleotide sequence of the second primer pair is shown in SEQ ID NO: 3-4.
[0019] The location of the c.111-119T>C locus on the chromosome is 1p36.32, and the location of the c.165C>T locus on the chromosome is 1p36.32.
[0020] Upstream primer SEQ ID NO: 1: 5′-GTGGGCGGGGAGAGCTTCCTCTC-3′;
[0021] Downstream primer SEQ ID NO: 2: 5′-AGAACCAACTGGAGGTGA-3′;
[0022] Upstream primer SEQ ID NO: 3: 5′-GCCATGAAGGTGCTGGGT-3′;
[0023] Downstream primer SEQ ID NO: 4: 5'-AGCATTTATTTGCACTTGTGC-3'.
[0024] When detecting the characteristic site of Vel blood type, the first primer pair or the second primer pair is mixed with the internal reference primer and the DNA of the sample to be tested, and a PCR reaction system including the first primer pair and / or a PCR reaction system including the second primer pair is prepared; PCR amplification is performed, and a melting curve test is performed on the PCR product. According to the fluorescence PCR results, the Vel blood type genotype measured is determined. Specifically, if the fluorescence signal of a certain site has a peak value, the genotype corresponding to the site is positive (i.e., a site mutation occurs, (Vel-)), otherwise it is negative.
[0025] In a second aspect, the present invention also provides a product for detecting Vel blood type characteristic sites, which includes the above-mentioned primer set for detecting Vel blood type characteristic sites, and the product is a reagent, a kit or a chip.
[0026] The form of the reagent includes but is not limited to: solid, liquid or semi-solid.
[0027] The kit also includes qPCR Mix.
[0028] The reagents, for example, are a PCR premix, including the primer set for detecting the characteristic loci of the Vel blood type. In other embodiments, the PCR premix also includes: DNA polymerase, dNTPs, Mg2+ and Buffer.
[0029] In one embodiment, the kit further includes: a positive standard, a fluorescent dye and water, and the fluorescent dye includes but is not limited to: SYBR Green, EVA Green, etc.
[0030] In a preferred embodiment of the present invention, the product also includes internal reference primers for detecting internal reference genes.
[0031] In a preferred embodiment of the present invention, the internal reference gene is selected from at least one of GAPDH, β-Actin, U6, HMBS, B2M, TUBB, SDHA, 18S rRNA, ACTB, RPL4, PPIA, HPRT1, YWHAZ, RPP30 and ERG.
[0032] In a preferred embodiment of the present invention, the internal reference gene is GAPDH.
[0033] In a preferred embodiment of the present invention, the nucleotide sequence of the internal reference primer for detecting the GAPDH gene is shown in SEQ ID NO: 5-6.
[0034] In a third aspect, the present invention also provides the use of a primer set for detecting characteristic loci of Vel blood type in the preparation of a product for detecting characteristic loci of Vel blood type, wherein the product is a reagent, a kit or a chip.
[0035] In a preferred embodiment of the present invention, the product detects the Vel blood type characteristic site by the following method:
[0036] The primer set for detecting the characteristic locus of Vel blood type is mixed with the internal reference primer and the DNA of the sample to be tested to prepare a PCR reaction system including the first primer pair and / or a PCR reaction system including the second primer pair; PCR amplification is performed, and a melting curve test is performed on the PCR product.
[0037] In real-time fluorescence quantitative PCR, when double-stranded DNA is heated, the hydrogen bonds between its complementary bases will gradually break, causing the double strand to separate into two single strands. This process is called DNA "melting". The temperature at which half of the total DNA double helix structure is degraded is called the melting temperature (Tm). DNA of different sequences has different GC content and distribution, and the Tm value is also different. As the temperature rises, the fluorescence intensity of DNA will change. By monitoring this change, the graph drawn is the melting curve of DNA.
[0038] The melting curve in the present invention can detect the difference of a single base, and has low cost, high throughput, fast speed, accurate results, and is not limited by the detection site, thus realizing a true closed-tube operation. The principle is to obtain a large number of target sequences through PCR amplification, and then directly run the melting program on them. In the process of temperature increase, the double-stranded DNA gradually melts, and the dye embedded in the double-stranded DNA also falls off, and the fluorescent signal weakens until the double-stranded DNA is completely melted and the fluorescent signal is weakened to the minimum. The DNA sequence is analyzed by monitoring this process.
[0039] In a preferred embodiment of the present invention, the final concentrations of the upstream and downstream primers in the PCR reaction system including the first primer pair are 0.1-1 μmol / L respectively; the final concentrations of the upstream and downstream primers in the PCR reaction system including the second primer pair are 0.1-1 μmol / L respectively; and the final concentration of the internal reference primer in the PCR reaction system is 0.1-1 μmol / L.
[0040] Under the above PCR reaction system, the detection accuracy is higher.
[0041] In an optional embodiment, the final concentrations of the upstream and downstream primers in the first primer pair in the PCR reaction system including the first primer pair are 0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L or 1 μmol / L, respectively.
[0042] The final concentrations of the upstream and downstream primers in the PCR reaction system including the second primer pair are 0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L or 1 μmol / L, respectively.
[0043] The final concentration of the internal reference primer in the PCR reaction system is 0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L or 1 μmol / L.
[0044] In a preferred embodiment of the present invention, the PCR amplification program is: 95°C-100°C, 3-5 minutes; 95°C-95.5°C, 30 seconds, 60°C-61°C, 30 seconds, 70°C-72°C, 30-60 seconds, a total of 30-35 cycles; 72°C, 5-10 minutes. Through repeated experiments, the reaction steps are optimized, and the PCR instrument is automatically operated after the program is set, and the number of cycles is shortened, thereby improving the detection efficiency.
[0045] The conditions for melting curve detection include: heating at 4°C per second, collecting fluorescence values every 0.5°C, and collecting fluorescence signals at a temperature range of 60°C to 95°C. Under the above melting program, the melting process of double-stranded DNA can be monitored quickly and accurately.
[0046] In a preferred embodiment of the present invention, the sample to be tested is blood, serum, plasma or oral mucosa.
[0047] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.
[0048] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.
[0049] Example 1
[0050] This embodiment designs detection primers P1 and P2 for the characteristic sites c.111-119T>C and c.165C>T of Vel blood type, respectively, and provides a method and a kit for detecting the characteristic sites of Vel blood type.
[0051] The target site of the P1 primer is c.111-119T>C
[0052] P1F 5'-GTGGGCGGGGGAGAGCTTCCTCTC-3'; SEQ ID NO: 1;
[0053] P1R 5'-AGAACCAACTGGAGGTGA-3'; SEQ ID NO: 2;
[0054] The target site of the P2 primer is c.165C>T.
[0055] P2F 5'-GCCATGAAGGTGCTGGGT-3'; SEQ ID NO: 3
[0056] P2R 5'-AGCATTTATTTGCACTTGTGC-3'; SEQ ID NO: 4.
[0057] The kit consists of:
[0058] 96-well plate (each well contains two sets of primers (internal reference primers and P1 or P2 primers), each plate can be used for 48 people; each well has internal reference primers; each person has two wells, containing a pair of P1 primers and a pair of P2 primers respectively); PCR buffer; fluorescent dye SYBR Green I; other auxiliary reagents.
[0059] Prepare PCR fluorescence buffer according to the following ratio:
[0060] Serial number Product Name volume 1 10X TE buffer 30ml 2 dNTPs(10mM) 8ml 3 Cresol red sodium solution (10mmol / L) 6ml 4 SYBR Green I 2ml 5 water 4ml
[0061] Mix the above PCR fluorescent buffer with Taq enzyme and water to prepare PCR working solution. Prepare PCR working solution (in the reagent preparation area), prepare 1 ml according to the proportion in the table below, and then divide and store. Add DNA and primers before use.
[0062]
[0063] For each sample, the concentration was adjusted to 50 ng / μl, and 6 μl of DNA sample was aspirated to form a reaction mixture with 60 μl of PCR working solution prepared above. 10 μl of reaction mixture was added to each well for PCR amplification, and the PCR reaction volume was set to 10 μl. The final concentrations of P1F, P1R, InF and InR in the reaction wells for detecting c.111-119T>C were 0.5 μmol / L respectively; the final concentrations of P2F, P2R, InF and InR in the reaction wells for detecting c.165C>T were 0.5 μmol / L respectively.
[0064] During preparation, shake and mix for a few seconds, and centrifuge at 800 rpm for a few seconds. Prepare other portions according to the above proportions.
[0065] The target gene of the internal reference primer is GAPDH, and its sequence is as follows:
[0066] InF 5'-GATTCCACCCATGGCAAATTC-3'; SEQ ID NO: 5;
[0067] InR 5'-CTGGAAGATGGTGATGGGATT-3'; SEQ ID NO: 6.
[0068] The detection method is as follows:
[0069] Step 1: Sample collection and processing: Collect an appropriate amount of blood sample from the individual to be tested, collect the blood using an EDTA anticoagulant tube, and store it in a refrigerator for no more than seven days.
[0070] Step 2: DNA extraction. Use Hangzhou Bioer Technology DNA extraction kit to extract genomic DNA from whole blood samples. Specific steps are carried out according to the kit instructions. The extracted genomic DNA is dissolved in sterile deionized water and the concentration is adjusted to 50ng / μl.
[0071] Step 3: PCR amplification: Use specific primers and Taq DNA polymerase to perform PCR amplification on genomic DNA.
[0072] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 30 cycles; and final extension at 72°C for 5 minutes.
[0073] Step 4: Fluorescence signal collection. The PCR product is tested for melting curve in the PCR instrument, the temperature is increased by 4°C per second, and the fluorescence value is collected once every 0.5°C. The collection temperature range is 60-95°C, and the fluorescence signal intensity is automatically recorded.
[0074] Step 5: Result analysis. According to the fluorescence PCR results, determine the tested Vel blood type genotype. Specifically, if the fluorescence signal of a certain site has a peak, the genotype corresponding to the site is positive, otherwise it is negative.
[0075] The genotype of the characteristic locus is positive: there are two peaks, one peak is the internal reference, and the other peak is the specific amplification product; the genotype of the characteristic locus is negative: there is only one internal reference peak.
[0076] Figure 1 This is the melting curve for the positive genotype of the characteristic site c.111-119T>C in the population; Figure 2 This is the melting curve for the positive genotype of the characteristic site c.165C>T in the population. Figure 3 This is the melting curve for the negative genotype of the characteristic site c.111-119T>C in the population.
[0077] The results show that the detection primers and kit provided by the present invention can quickly and accurately identify the characteristic sites of the Vel blood type in the Chinese population, thereby assisting clinical diagnosis.
[0078] Example 2
[0079] Sensitivity test: 20 genotype-positive clinical experimental samples (DNA extracted from peripheral blood) were detected according to the method of Example 1, including 10 samples with positive c.111-119T>C genotype and 10 samples with positive c.165C>T genotype.
[0080] The nucleic acid concentrations of 10 samples with positive c.111-119T>C genotype and 10 samples with positive c.165C>T genotype were diluted to 10 ng / μL respectively, and PCR amplification was performed according to the conditions of Example 1, and the melting curve was detected to test the minimum detection limit of the test reagent.
[0081] Experimental results: At a concentration of 10ng / μL, the target gene can be detected. The minimum detection limit of the reagent is 10ng. The specific test results are shown in the table:
[0082]
[0083] Example 3
[0084] Perform reproducibility test of the kit.
[0085] Experimental process: The 10 samples (concentration of 10 ng / μL) of Example 2 were repeatedly tested 10 times using the kit provided in Example 1, and the test results were analyzed. The results showed that at a concentration of 10 ng / μL, 10 repeated experiments could accurately detect the 10 samples.
[0086] In the field of immunological detection technology, the present invention provides a primer set and detection kit for detecting Vel blood type characteristic sites based on sequence-specific primer technology. The characteristic sites of Vel blood type in the Chinese population can be directly identified through two sets of PCR reactions without antigen typing, thereby improving detection efficiency and reducing detection costs. This will greatly promote the development of immunological detection technology, improve the accuracy and efficiency of detection, and provide a more reliable basis for clinical diagnosis and treatment.
[0087] In the field of biotechnology, the technology of the present invention can be applied to gene research and bioengineering. By identifying the characteristic sites of the Vel blood type in the Chinese population, the polymorphic characteristics of the Vel blood type genotype distribution in the region can be better understood, which is of great significance for studying the characteristics of the region, the association of disease occurrence and the development of new biotechnology products.
[0088] In the field of medical devices, the detection kit of the present invention can be used as a new medical device for clinical diagnosis, which will greatly improve the quality and safety of medical services.
[0089] In general, the technology of the present invention has broad application prospects and a large market demand, and is expected to play an important role in the fields of immunological detection technology, biotechnology and medical devices.
[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A primer set for detecting Vel blood type characteristic sites, characterized in that: It includes: a first primer pair for detecting the c.111-119T>C site and / or a second primer pair for detecting the c.165C>T site, the nucleotide sequence of the first primer pair is shown in SEQ ID NO: 1-2, and the nucleotide sequence of the second primer pair is shown in SEQ ID NO: 3-4.
2. A product for detecting characteristic loci of Vel blood type, characterized in that: It comprises the primer set for detecting the characteristic site of Vel blood type as claimed in claim 1, and the product is a reagent, a kit or a chip.
3. The product for detecting the characteristic loci of Vel blood type according to claim 2, characterized in that: The product also includes internal reference primers for detecting internal reference genes.
4. The product for detecting the characteristic loci of Vel blood type according to claim 3, characterized in that: The internal reference gene is selected from at least one of GAPDH, β-Actin, U6, HMBS, B2M, TUBB, SDHA, 18S rRNA, ACTB, RPL4, PPIA, HPRT1, YWHAZ, RPP30 and ERG.
5. The product for detecting the characteristic loci of Vel blood type according to claim 4, characterized in that: The internal reference gene is GAPDH.
6. The product for detecting the characteristic loci of Vel blood type according to claim 5, characterized in that: The nucleotide sequences of the internal reference primers for detecting the GAPDH gene are shown in SEQ ID NOs: 5-6.
7. Use of the primer set for detecting the characteristic locus of Vel blood type according to claim 1 in preparing a product for detecting the characteristic locus of Vel blood type, characterized in that: The product is a reagent, a kit or a chip.
8. The use according to claim 7, characterized in that: The product detects the Vel blood type characteristic loci by the following method: The primer set for detecting the characteristic locus of Vel blood type is mixed with the internal reference primer and the DNA of the sample to be tested to prepare a PCR reaction system including the first primer pair and / or a PCR reaction system including the second primer pair; PCR amplification is performed, and a melting curve detection is performed on the PCR product.
9. The use according to claim 8, characterized in that: In the PCR reaction system including the first primer pair, the final concentrations of the upstream and downstream primers in the first primer pair are 0.1-1 μmol / L respectively; the final concentrations of the upstream and downstream primers in the PCR reaction system including the second primer pair are 0.1-1 μmol / L respectively; the final concentration of the internal reference primer in the PCR reaction system is 0.1-1 μmol / L.
10. The use according to claim 8, characterized in that: The PCR amplification program is: 95°C-100°C, 3-5 minutes; 95°C-95.5°C, 30 seconds, 60°C-61°C, 30 seconds, 70°C-72°C, 30-60 seconds, a total of 30-35 cycles; 72°C, 5-10 minutes; The conditions for the melting curve detection include: increasing the temperature by 4°C per second, collecting the fluorescence value once every 0.5°C, and collecting the fluorescence signal at a temperature range of 60°C to 95°C; Preferably, the sample to be tested is blood, serum, plasma or oral mucosa.