Primer pair and kit for detecting CYP2D6 deletion type and application of primer pair and kit
Through a kit based on capillary electrophoresis and specific primer probes, rapid and accurate detection of CYP2D6 gene deletion type is achieved, solving the complex and time-consuming problems of existing detection methods and meeting the clinical rapid detection needs.
Patent Information
- Application Number
- CN202510190729.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-20
- Publication Date
- 2025-04-25
AI Technical Summary
The existing CYP2D6 gene deletion detection methods are complex, time-consuming, costly and have high operating requirements, making it difficult to meet the clinical fast, simple and accurate detection needs.
Using a kit based on capillary electrophoresis, including PCR amplification system and endonuclease, the rapid detection of CYP2D6 deletion type using specific CYP2D6 deletion primers and probes was achieved through PCR reaction, enzyme cleavage and fragment analysis.
This method has high sensitivity, simple operation, short detection time, clear and accurate results, and is suitable for large-scale clinical applications.
Smart Images
Figure CN119876381A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of gene detection technology, and in particular to a primer pair, a kit and an application thereof for detecting CYP2D6 deficiency type. Background Art
[0002] The cytochrome P450 family is an important enzyme system in drug-phase metabolizing enzymes, playing an important role in the metabolism of endogenous substances and exogenous drugs in organisms. The product encoded by the CYP2D6 gene is one of the important members of the cytochrome P450 family. The human CYP2D6 gene is mainly expressed in the liver, accounting for 2% to 9% of liver enzymes, but is involved in the metabolism of more than 20% of commonly used clinical drugs, such as cancer treatment drugs, antipsychotic drugs, analgesics, anti-cardiovascular disease drugs, etc. The CYP2D6 gene is highly polymorphic, which leads to individual differences in drug metabolism, thereby affecting the efficacy of drugs and even the production of toxic side effects. Based on the metabolic ability of CYP2D6 for drugs, it can be divided into ultra-fast metabolizers, normal metabolizers, intermediate metabolizers, and slow metabolizers. Among them, the CYP2D6 gene has a mutation type CYP2D6*5, which is a deletion of the entire CYP2D6 gene. This gene deletion leads to the inactivation of drug metabolizing enzymes, affecting clinical medication. The probability of this mutation type in East Asian populations is 3% to 7%. Therefore, special attention should be paid to the CYP2D6*5 genotype in clinical medication. At present, the following methods are commonly used to detect CYP2D6 gene deletion.
[0003] Direct sequencing is to first amplify the target gene through PCR reaction, then purify the PCR product, and then perform a round of single-stranded amplification for sequencing. Finally, the single-stranded amplification product is purified and sequenced. Although direct sequencing is the gold standard for genetic testing, it has complicated steps, long time consumption, high cost, easy contamination when opening the cover, and low sensitivity.
[0004] The second generation sequencing method sets multiple detection regions in the CYP2D6 gene deletion fragment region, designs primers for simultaneous amplification, and then uses sequencing to obtain the sequencing depth of each region, and uses reference sets and weighted analysis to obtain the deletion type of the sample to be tested. However, this method is complicated, time-consuming, costly, and requires high professional skills.
[0005] Multiplex ligation probe amplification technology includes DNA denaturation, hybridization, ligation reaction, PCR amplification, fragment separation, and data analysis steps. This technology is complex and cumbersome, involving multiple instruments and multiple technologies, time-consuming and labor-intensive, and has high requirements for operators, and is not suitable for large-scale application in clinical testing.
[0006] In summary, there is an urgent need for a CYP2D6*5 genotyping test product that is simple to operate, fast to detect, clear and accurate in interpretation, and highly sensitive. Summary of the invention
[0007] In order to solve the above technical problems, the present invention provides a primer pair, a kit and applications thereof for detecting CYP2D6 deletion type.
[0008] Specifically, the present invention provides a kit for detecting CYP2D6 deletion type based on capillary electrophoresis, comprising a PCR amplification system and an endonuclease;
[0009] The PCR amplification system comprises CYP2D6 deletion primers and probes.
[0010] In some specific embodiments, the primer is:
[0011] A CYP2D6 deletion upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1;
[0012] The nucleotide sequence of the CYP2D6 deletion type downstream primer is shown in SEQ ID NO: 2.
[0013] In some embodiments, the endonuclease is selected from one or more of NdeI endonuclease, KpnI endonuclease, BsoBI endonuclease, AvaI endonuclease and PvuII endonuclease.
[0014] In some specific embodiments, the endonuclease is NdeI endonuclease, KpnI endonuclease or BsoBI endonuclease.
[0015] In some embodiments, the CYP2D6 deletion primer comprises a fluorescent label, the fluorescent label is selected from FAM, VIC, HEX, JOE, ROX, TAMRA and CY5 fluorescent labels, and the fluorescent label is located at the 5' end of the CYP2D6 deletion upstream primer.
[0016] In some embodiments, the PCR amplification system further comprises one or more of DNA polymerase, dNTP, MgCl2, enhancer, deionized formamide, and fluorescent internal standard dye TK500.
[0017] In some specific embodiments, the DNA polymerase is pfu DNA polymerase.
[0018] In some specific embodiments, the enhancers are tetramethylammonium chloride and BSA.
[0019] In some specific embodiments, the PCR amplification system includes 20mM Tris-HCl at pH 8.8, 20mM KCl, 10mM (NH4)2SO4, 2mM MgCl2, 200µM dNTPs, 50mM tetramethylammonium chloride, 300µg / mL BSA and 0.1U / μL pfu DNA polymerase.
[0020] In some specific embodiments, the kit further comprises one or more of a nucleic acid extraction reagent, a CYP2D6 deficiency negative quality control product, and a CYP2D6 deficiency positive quality control product.
[0021] The present invention also provides a primer for detecting the CYP2D6 deletion type, wherein the primer is:
[0022] A CYP2D6 deletion upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1;
[0023] The nucleotide sequence of the CYP2D6 deletion type downstream primer is shown in SEQ ID NO: 2.
[0024] In some specific embodiments, the CYP2D6 deletion primer comprises a fluorescent label, and the fluorescent label is selected from FAM, VIC, HEX, JOE, ROX, TAMRA and CY5 fluorescent labels, and the fluorescent label is located at the 5' end of the CYP2D6 deletion upstream primer.
[0025] The present invention also provides a method for detecting CYP2D6 deficiency type, the method comprising the following steps:
[0026] (1) performing a PCR reaction on the sample DNA using the PCR amplification system in the kit described in the present disclosure;
[0027] (2) digesting the PCR product obtained in step 1 with an endonuclease in the kit described in the present disclosure;
[0028] (3) The digested product obtained in step 2 is subjected to fragment analysis by a gene analyzer;
[0029] (4) Interpretation of results;
[0030] The method is for non-diagnostic purposes.
[0031] In some specific embodiments, the method further comprises extracting sample DNA.
[0032] In some specific embodiments, the conditions of the PCR reaction in step (1) of the method are: pre-denaturation at 98°C for 10 min; denaturation at 98°C for 10 s, annealing at 60°C for 20 s, extension at 72°C for 1 min 30 s, for a total of 32 cycles; extension at 72°C for 10 min.
[0033] In some specific embodiments, the reaction conditions of the enzyme digestion in step (2) of the method are: digestion at 37° C. for 30-60 min.
[0034] In some specific embodiments, the method for interpreting the result in step (4) of the method is that in the following cases, it is a CYP2D6 deficiency type:
[0035] If KpnI endonuclease is used for digestion, there will be a FAM, VIC / HEX / JOE, ROX, TAMRA or CY5 fluorescent labeling peak signal near 140bp;
[0036] If PvuII endonuclease is used for digestion, there will be a FAM, VIC / HEX / JOE, ROX, TAMRA or CY5 fluorescent labeling peak signal near 253bp;
[0037] If AvaI / BsoBI endonuclease is used for digestion, there will be FAM, VIC / HEX / JOE, ROX, TAMRA or CY5 fluorescent labeling peak signals near 308bp;
[0038] If NdeI endonuclease is used for digestion, there will be a FAM, VIC / HEX / JOE, ROX, TAMRA or CY5 fluorescent labeling peak signal near 554bp.
[0039] The present invention also provides the use of the kit described in the present disclosure or the primers and probe described in the present disclosure in the detection of CYP2D6 deficiency type; the use is for non-diagnostic purposes.
[0040] In this article, non-diagnostic application scenarios include, for example, detecting the CYP2D6 gene in an in vitro sample for research and development purposes.
[0041] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.
[0042] The reagents and raw materials used in the present invention are commercially available.
[0043] The positive and progressive effects of the present invention are:
[0044] Compared with the prior art, the method of the present invention has the advantages of higher sensitivity, simple operation, shorter detection time, and clear and accurate detection results that are easy to interpret. BRIEF DESCRIPTION OF THE DRAWINGS
[0045] Figure 1 This is the capillary electrophoresis fragment analysis diagram of the non-deletion type and deletion type of the CYP2D6 gene when cut with KpnI.
[0046] Figure 2 This is the capillary electrophoresis fragment analysis diagram of the non-deletion type and deletion type of the CYP2D6 gene when digested with BsoBI.
[0047] Figure 3 This is the capillary electrophoresis fragment analysis diagram of the non-deletion type and deletion type of the CYP2D6 gene when cut with NdeI.
[0048] Figure 4 This is a capillary electrophoresis fragment analysis diagram of different primer-probe combinations used for CYP2D6 gene deletion type in Example 1.
[0049] Figure 5 This is a capillary electrophoresis fragment analysis diagram of different PCR reaction solutions used for CYP2D6 gene deletion in Example 2.
[0050] Figure 6 This is the capillary electrophoresis fragment analysis diagram of the established method for detecting CYP2D6 gene deletion samples and corresponding positive and negative controls. DETAILED DESCRIPTION
[0051] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.
[0052] Example 1 Design of detection primers and probes
[0053] Since the upstream and downstream of the CYP2D6 gene have homology arm sequences of up to 2,800 bases, it is very difficult to screen out suitable detection primers and probes. To this end, a large number of primer-probe sets were designed for testing. Due to the large number of combinations, 3 sets were selected as examples to demonstrate the differences in detection effects.
[0054] Different primer-probe combinations and corresponding detection effect differences, primer-probe combinations are shown in Table 1
[0055] Table 1 Different primer-probe combinations
[0056]
[0057] Among them, the final concentration of the CYP2D6 gene deletion primer probe is 0.3 μM, the PCR reaction solution is 2 × Hotstart Flash Pfu Master Mix of Suzhou Yuanqi Biotechnology Co., Ltd., the polymerase contained is pfu DNA polymerase with a final concentration of 0.1 U / μL, and the endonuclease contained is NdeI enzyme with a final concentration of 0.5 U / μL. Amplification is performed according to the PCR reaction system in Table 2 and the PCR reaction amplification program in Table 3.
[0058] Table 2 PCR reaction system preparation
[0059]
[0060] Table 3 PCR amplification program
[0061]
[0062] After amplification, add 0.5 μL of NdeI (20 U / μL) endonuclease to the PCR reaction tube and digest at 37°C for 60 min. After digestion, take 1 μL of the enzymatic solution, mix it with 8.5 μL of deionized formamide and 0.5 μL of TK500 internal standard, and select the fragment analysis program on the gene analyzer for capillary electrophoresis.
[0063] The differences in the test results of different primer-probe combinations for detecting CYP2D6 gene deletion samples are shown in Figure 4 , wherein the probe 5' is fluorescently labeled with TAMRA.
[0064] The results are as follows Figure 4 As shown, the amplification results of primer-probe combination 2 were poor, while combinations 1 and 3
[0065] The amplification efficiency was high, but combination 3 had a high background and many miscellaneous bands. Finally, 2D6de1-F and 2D6del-R were selected as the primer-probe pairs for subsequent implementation.
[0066] Example 2 Screening of PCR reaction system
[0067] Since the specificity and efficiency of long-fragment amplification are very difficult, a large number of PCR reaction system tests were conducted. Due to the large number of tests, 3 groups were selected as examples to demonstrate the differences in detection effects.
[0068] Different PCR reaction solutions and corresponding detection effects are different. The PCR reaction solution combination is shown in Table 4
[0069] Table 4 Combinations of different PCR reaction solutions
[0070]
[0071] The primer probes for the CYP2D6 gene deletion type are 2D6de1-F and 2D6de1-R with a final concentration of 0.3 μM. The polymerase contained is pfu DNA polymerase with a final concentration of 0.1 U / μL, and the endonuclease contained is KpnI enzyme with a final concentration of 0.5 U / μL. Amplification is performed according to the PCR reaction system in Table 5 and the PCR reaction amplification program in Table 6.
[0072] Table 5 PCR reaction system preparation
[0073]
[0074] Table 6 PCR reaction amplification program
[0075]
[0076] After amplification, add 0.5 μL of KpnI (20 U / μL) endonuclease to the PCR reaction tube and digest at 37°C for 60 min. After digestion, take 1 μL of enzymatic solution, mix it with 8.5 μL of deionized formamide and 0.5 μL of TK500 internal standard, and select the fragment analysis program on the gene analyzer for capillary electrophoresis.
[0077] The difference in the test results of different PCR reaction solution combinations for detecting CYP2D6 gene deletion samples is shown in the figure below, where the probe 5' is labeled with TAMRA fluorescence.
[0078] The results are as follows Figure 5 As shown, PCR reaction solution combination 3 has a higher peak height and fewer impurity peaks, and the amplification effect is better than PCR reaction solution combinations 1 and 2. The PCR reaction solution with a final concentration of 20mM Tris-HCl (pH8.8), 20mM KCl, 10mM (NH4)2SO4, 2mM MgCl2, 200µM dNTPs, 50mM tetramethylammonium chloride, 300µg / mLBSA, and 0.1U / μL pfu DNA polymerase was selected as the subsequent PCR reaction solution.
[0079] Example 3
[0080] A kit for detecting CYP2D6 gene deletion type. The composition of the kit is shown in Table 7.
[0081] Table 7 Composition of CYP2D6 gene deletion test kit (48 servings / box)
[0082]
[0083] The 2D6de1 PCR reaction solution contained a final concentration of 20 mM Tris-HCl (pH 8.8), 20 mM KCl, 10 mM (NH4)2SO4, 200 μM dNTPs, 2 mM MgCl2, 50 mM tetramethylammonium chloride, 300 μg / mL BSA and 0.1 U / μL pfu DNA polymerase.
[0084] The 2D6de1 primer-probe mixture contains primer probes (2D6de1-F and 2D6del-R) at a final concentration of 0.3 μM, wherein the 5' of the probe is fluorescently labeled with TAMRA.
[0085] Among them, 2D6de1 negative quality control product is 10 6 / mL CYP2D6 gene wild-type cell culture (MOLT-4, purchased from Beina Biotechnology) extracted genomic DNA.
[0086] Among them, 2D6de1 positive quality control product is 10 6 / mL CYP2D6 gene-deficient cell culture (THP-1, purchased from Beina Biotechnology) extracted genomic DNA.
[0087] The 2D6de1 endonuclease was 20 U / μL of KpnI endonuclease (NEB).
[0088] Among them, the 2D6de1 buffer is deionized formamide.
[0089] The internal standard of 2D6de1 is TK500 fluorescent internal standard dye.
[0090] Example 4: Establishment of detection method
[0091] (1) Sample processing
[0092] Take 200 μL of each of the sample to be tested, negative quality control product, and positive quality control product for nucleic acid extraction simultaneously. Use the nucleic acid extraction or purification reagent of Anhui Tongke Biotechnology Co., Ltd. for nucleic acid extraction. Please strictly follow the product instructions during the extraction process.
[0093] (2) Preparation of PCR reaction system
[0094] According to the proportions in Table 4, the corresponding components in the CYP2D6 gene deletion detection kit of Example 3 were drawn, fully mixed and briefly centrifuged, and then dispensed into PCR reaction tubes at 18 μL / tube.
[0095] Table 8 PCR reaction system preparation
[0096]
[0097] N = number of samples to be tested (n) + number of quality control products (2) + 1
[0098] (3) Sample addition
[0099] Take 2 µL of the nucleic acid extract of 2D6de1 negative control, 2D6de1 positive control and the sample to be tested and add them to the corresponding PCR reaction tubes. Close the tube caps tightly, centrifuge for a moment, and immediately perform PCR reaction.
[0100] (4) Amplification
[0101] Place each reaction tube in the PCR amplification instrument in order, and perform the PCR reaction according to the following procedure. The reaction volume is set to 20 μL. Please refer to the operating instructions of each applicable instrument for settings.
[0102] Table 9 PCR reaction amplification program
[0103]
[0104] (5) Enzyme digestion
[0105] Add 0.5 μL BsoBI endonuclease (NEB, final concentration 0.5 U / μL) directly to the PCR product and digest at 37°C for 30 min.
[0106] (6) 8.5 μL deionized formamide + 0.5 μL TK500 + 1 μL enzyme solution were run on a 3500 genetic analyzer (LifeTechnologies Holdings Pte Ltd) or Classic 108 / 116 genetic analyzer (Nanjing Suyuan Gene Technology Co., Ltd.) in fragment analysis mode.
[0107] (7) Results analysis
[0108] After the reaction is completed, the experimental results are analyzed using the software supporting the gene analyzer.
[0109] (8) Quality control:
[0110] All quality control product test results should meet the following conditions:
[0111] Table 10 Quality control product result interpretation criteria
[0112]
[0113] (9) Result judgment:
[0114] ①The test results are interpreted according to the following table:
[0115] Table 11 Sample result interpretation criteria
[0116]
[0117] ② If the internal standard fails to run or the peak shape and position are abnormal, it means that the test result of the sample is invalid. The cause should be found and eliminated, and the sample should be tested again.
[0118] The results of the established method for detecting CYP2D6 gene deletion samples and the corresponding positive and negative controls are shown in Figure 6 .
[0119] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. A kit for detecting CYP2D6 deficiency type based on capillary electrophoresis, characterized in that: Including PCR amplification system and endonuclease; The PCR amplification system includes CYP2D6 deletion primers; the primers are: A CYP2D6 deletion upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1; The nucleotide sequence of the CYP2D6 deletion type downstream primer is shown in SEQ ID NO:
2.
2. The kit according to claim 1, characterized in that The endonuclease is selected from one or more of NdeI endonuclease, KpnI endonuclease, BsoBI endonuclease, AvaI endonuclease and PvuII endonuclease; preferably, the endonuclease is NdeI endonuclease, KpnI endonuclease or BsoBI endonuclease; And / or, the CYP2D6 deletion primer comprises a fluorescent marker, the fluorescent marker is selected from FAM, VIC, HEX, JOE, ROX, TAMRA and CY5 fluorescent markers, and the fluorescent marker is located at the 5' end of the CYP2D6 deletion upstream primer.
3. The kit according to claim 1 or 2, characterized in that The PCR amplification system further comprises one or more of DNA polymerase, dNTP, MgCl2, enhancer, deionized formamide, and fluorescent internal standard dye TK500; Preferably, the DNA polymerase is pfu DNA polymerase; and / or, the enhancer is tetramethylammonium chloride and BSA; More preferably, the PCR amplification system comprises 20 mM Tris-HCl at pH 8.8, 20 mM KCl, 10 mM (NH4)2SO4, 2 mM MgCl2, 200 µM dNTPs, 50 mM tetramethylammonium chloride, 300 µg / mL BSA and 0.1 U / µL pfu DNA polymerase.
4. The kit according to any one of claims 1 to 3, characterized in that The kit also includes one or more of a nucleic acid extraction reagent, a CYP2D6 deficiency type negative quality control product, and a CYP2D6 deficiency type positive quality control product.
5. A primer for detecting CYP2D6 deletion type, characterized in that: The primers are: A CYP2D6 deletion upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1; A CYP2D6 deletion type downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2; Preferably, the CYP2D6 deletion primer comprises a fluorescent marker, the fluorescent marker is selected from FAM, VIC, HEX, JOE, ROX, TAMRA and CY5 fluorescent markers, and the fluorescent marker is located at the 5' end of the CYP2D6 deletion upstream primer.
6. A method for detecting CYP2D6 deficiency type, characterized in that: The method comprises the following steps: (1) performing a PCR reaction on the sample DNA using the PCR amplification system in the kit according to any one of claims 1 to 4; (2) digesting the PCR product obtained in step 1 with an endonuclease in the kit according to any one of claims 1 to 4; (3) The digested product obtained in step 2 is subjected to fragment analysis by a gene analyzer; (4) Interpretation of results; The method is for non-diagnostic purposes.
7. The method according to claim 6, characterized in that The method also includes extracting sample DNA.
8. The method according to claim 6 or 7, characterized in that The conditions of the PCR reaction in step (1) of the method are: pre-denaturation at 98°C for 10 min; denaturation at 98°C for 10 s, annealing at 60°C for 20 s, extension at 72°C for 1 min 30 s, for a total of 32 cycles; extension at 72°C for 10 min; And / or, the reaction conditions of the enzyme digestion in step (2) of the method are: enzyme digestion at 37° C. for 30-60 min.
9. The method according to any one of claims 6 to 8, characterized in that The result interpretation method in step (4) of the method is that in the following cases, it is CYP2D6 deficiency type: If KpnI endonuclease is used for digestion, there will be a FAM, VIC / HEX / JOE, ROX, TAMRA or CY5 fluorescent labeling peak signal near 140bp; If PvuII endonuclease is used for digestion, there will be a FAM, VIC / HEX / JOE, ROX, TAMRA or CY5 fluorescent labeling peak signal near 253bp; If AvaI / BsoBI endonuclease is used for digestion, there will be FAM, VIC / HEX / JOE, ROX, TAMRA or CY5 fluorescent labeling peak signals near 308bp; If NdeI endonuclease is used for digestion, there will be a FAM, VIC / HEX / JOE, ROX, TAMRA or CY5 fluorescent labeling peak signal near 554bp.
10. Use of the kit according to any one of claims 1 to 4 or the primer according to claim 5 in the detection of CYP2D6 deficiency type; the use is for non-diagnostic purposes.