Kit, detection device and detection method for ddPCR detection of ApoE genotype
By using ddPCR technology and specially designed primer probe sets in ApoE genotype detection, the problems of low sensitivity and inability to quantify existing detection methods are solved, and the detection effect of high sensitivity and absolute quantification is achieved.
Patent Information
- Application Number
- CN202411826330.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-04-11
AI Technical Summary
The existing ApoE genotype detection methods have problems such as low sensitivity and inability to achieve absolute quantification, which is difficult to meet the needs of quantitative detection.
The ddPCR technology combined with a specially designed primer probe set is used to achieve absolute quantification of the ApoE genotype by dividing the sample into thousands of tiny droplets for amplification and detection.
It realizes high sensitivity detection and absolute quantification of ApoE genotype, can accurately detect low abundance targets, and has the advantages of good specificity and accurate signal.
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Figure CN119799873A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of ApoE typing detection, and in particular to a kit, a detection device and a detection method for ddPCR detection of ApoE genotype. Background Art
[0002] Apolipoprotein E (ApoE) is a major cholesterol carrier that regulates lipid metabolism in the body by mediating cellular cholesterol uptake. The two single nucleotide polymorphisms of ApoE, 526C>T (rs429358) and 388T>C (rs7412), constitute three genotypes: E2 (388T-526T), E3 (388T-526C), and E4 (388C-526C), of which E3 is wild type, and E2 and E4 are mutant types, resulting in three homozygous (E2 / 2, E3 / 3, E4 / 4) and three heterozygous (E2 / 3, E2 / 4, E3 / 4). APOE genotyping is of great significance in the risk assessment and personalized treatment of cardiovascular diseases and Alzheimer's disease. People carrying the E4 allele have an increased risk of cardiovascular and cerebrovascular diseases, people carrying the E2 allele have an increased risk of hyperlipidemia, and people carrying the E4 allele have a significantly increased risk of Alzheimer's disease. Many studies have shown that ApoE gene polymorphism is one of the important reasons for the individual differences in the lipid-lowering efficacy of statins, and the efficacy of statins in E2 allele carriers is better than that of E3 and E4 allele carriers. In summary, the ApoE gene is an important risk factor for cardiovascular and cerebrovascular diseases and an important predictor of the lipid-lowering efficacy of statins. Genotyping of the ApoE gene can help assess whether the population is susceptible to hyperlipidemia or Alzheimer's disease, and thus can prevent, diagnose and treat it early, help improve prognosis, and assist in predicting the lipid-lowering efficacy of statins, thereby helping to provide personalized treatment.
[0003] At present, the commonly used detection methods for ApoE genotyping include TaqMan probe method, gene chip hybridization method and sequencing method. The TaqMan probe method is to add a pair of primers during PCR amplification, and then add one or two specific fluorescent probes at the same time. The two ends of the probe are respectively labeled with a reporter fluorescent group and a quencher fluorescent group. Although this method is simple to operate and has a short detection cycle, it is difficult to design specific probes for single-base mutations and the cost is high, and it is impossible to achieve absolute quantification without a standard curve. The gene chip hybridization method requires PCR amplification of the corresponding ApoE gene fragment, and then hybridization with the specific nucleic acid probe on the chip to distinguish and determine the genotype of ApoE. This detection method has cumbersome operation steps, prone to errors, and takes a long time to experiment. After the PCR is completed, the tube needs to be opened for operation, which is easy to cause contamination; in addition, this detection method cannot perform quantitative detection. The sequencing method has a high accuracy rate, but it has the disadvantages of long cycle, high cost, limited sensitivity and inability to perform absolute quantification.
[0004] Quantitative testing is very necessary for quality control (including testing of reference materials, repeatability testing within batches and between batches, testing of standard materials, etc.) and metrological traceability (certification of standard materials, instrument calibration, etc.) of ApoE testing. ddPCR (Droplet Digital PCR) is an advanced PCR technology that achieves absolute quantification of target nucleic acids by dividing samples into thousands of tiny droplets for amplification and detection. ddPCR technology overcomes some limitations of traditional quantitative PCR (qPCR) and is particularly suitable for the detection and quantification of low-abundance targets.
[0005] In summary, the current methods for detecting ApoE genotypes have problems such as low sensitivity and inability to achieve absolute quantification. Therefore, there is an urgent need for a product or method for quantitatively detecting ApoE genotypes.
[0006] Currently, the needle Summary of the invention
[0007] The purpose of the present application is to provide a kit, a detection device and a detection method for ddPCR detection of ApoE genotype.
[0008] This application adopts the following technical solutions:
[0009] The first aspect of the present application discloses a kit for ddPCR detection of ApoE genotype, comprising a first primer probe group for detecting ApoE388T>C and a second primer probe group for detecting ApoE 526C>T; the first primer probe group comprises: forward primer 388T-F, the sequence of which is shown in SEQ ID NO.1; forward primer 388C-F, the sequence of which is shown in SEQ ID NO.2; reverse primer 388-R, the sequence of which is shown in SEQ ID NO.3; probe 388-P, the sequence of which is shown in SEQ ID NO.4; the second primer probe group comprises: forward primer 526C-F, the sequence of which is shown in SEQ ID NO.5; reverse primer 526C-R, the sequence of which is shown in SEQ ID NO.6; forward primer 526T-F, the sequence of which is shown in SEQ ID NO.7; reverse primer 526T-R, the sequence of which is shown in SEQID NO.8; and probe 526-P, the sequence of which is shown in SEQ ID NO.9.
[0010] It should be noted that in the kit of the present application, the 3' ends of forward primer 388T-F and forward primer 388C-F are located at the polymorphic site, and the last base of the sequence of forward primer 388T-F (the base closest to the 3' end) matches ApoE 388T, and the last base of the sequence of forward primer 388C-F matches ApoE 388C. DNA polymerase is strict with base pairing at the 3' end of the primer. When a single base mismatch occurs at the 3' end of the primer, the DNA polymerase cannot effectively extend from this position, which manifests as a blocked amplification, thereby inhibiting nonspecific amplification. Similarly, forward primer 526C-F and forward primer 526T-F also have similar special designs. In addition, the present application designs and tests a large number of specific primers for different forward primers and reverse primers to screen out primer probe sets with good specificity and accurate signals (clear division of positive droplets and negative droplets, less diffusion of droplet clusters, and higher number of droplets). In addition, in the present application, one probe is shared for ApoE388 wild type and mutant, and one probe is shared for ApoE 526 wild type and mutant, which can reduce the cost of probes, and the probes of the present application have good specificity and can produce accurate signals. In summary, the ddPCR detection kit of the present application is used to detect the ApoE genotype, which has the advantages of good specificity, high sensitivity, and high accuracy of the detection results, and can achieve absolute quantification of the ApoE gene amplification target without a standard curve.
[0011] In one implementation of the present application, the fluorescent groups of the probe 388-P and the probe 526-P are independently selected from FAM, FITC, HEX, VIC, TET, JOE, ROX, CY3, CY5, CY5.5, CY7, ATTO 425 and SF670, and the quenching groups of the probe 388-P and the probe 526-P are independently selected from BHQ1, BHQ2, BHQ3, MGB and DABCYL.
[0012] In one implementation of the present application, the fluorescent group of the probe 388-P is different from the fluorescent group of the probe 526-P.
[0013] In one implementation of the present application, the kit further includes at least one of the following: ddPCR premix, droplet generation oil, positive control, and negative control.
[0014] In one implementation of the present application, the ddPCR premix comprises at least one of the following: PCR buffer, Taq DNA polymerase, Mg 2+ and dNTPs.
[0015] The second aspect of the present application discloses an ApoE genotype detection device, comprising: an amplification module: using the kit as described in the first aspect of the present application to perform ddPCR detection on the nucleic acid sample to be tested; a judgment module: judging the ApoE genotype and content according to the results of the ddPCR detection.
[0016] The third aspect of the present application discloses a method for detecting ApoE genotype for non-diagnostic and therapeutic purposes, characterized in that it includes: obtaining a nucleic acid sample to be tested; configuring a ddPCR reaction system using the kit described in the first aspect of the present application, and performing a reaction according to the reaction procedure; and determining the genotype and content of ApoE.
[0017] In one implementation of the present application, in the ddPCR reaction system, the concentration of forward primer 388T-F, forward primer 388C-F, reverse primer 388-R, forward primer 526C-F, reverse primer 526C-R, forward primer 526T-F or reverse primer 526T-R is 300nM to 500nM.
[0018] In one implementation of the present application, in the ddPCR reaction system, the concentration of forward primer 388T-F, forward primer 388C-F, reverse primer 388-R, forward primer 526C-F, reverse primer 526C-R, forward primer 526T-F or reverse primer 526T-R is 400 nM.
[0019] In one implementation of the present application, in the ddPCR reaction system, the concentration of probe 388-P or probe 526-P is 150 nM to 250 nM.
[0020] In one implementation of the present application, in the ddPCR reaction system, the concentration of probe 388-P or probe 526-P is 200 nM.
[0021] In one implementation of the present application, in the reaction procedure, the annealing temperature is 59° C. to 62° C.
[0022] In one implementation of the present application, in the reaction procedure, the annealing temperature is 60°C.
[0023] The beneficial effects of this application are:
[0024] The ddPCR detection kit, detection device and detection method of the present application detect ApoE genotype and have the advantages of good specificity, high sensitivity and high accuracy of detection results, and can achieve absolute quantification of ApoE gene amplification targets without the need for a standard curve. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a graph of electrophoresis results of PCR products for detecting ApoE 388 and 526 sites using different forward primers involved in Example 1 of the present application.
[0026] Figure 2 This is a graph of the ddPCR detection results of detecting the ApoE 388 site using different reverse primers involved in Example 1 of the present application.
[0027] Figure 3 This is a graph of the ddPCR detection results of detecting the ApoE 526 site using different reverse primers involved in Example 1 of the present application.
[0028] Figure 4 This is a graph of ddPCR detection results involving Example 2 of the present application using different primer probe concentrations for detection of the ApoE 388 wild-type site, the ApoE 388 mutant site, the ApoE526 wild-type site, and the ApoE 526 mutant site.
[0029] Figure 5 This is a graph of ddPCR detection results involving ApoE 388 wild-type site, ApoE 388 mutant site, ApoE526 wild-type site, and ApoE 526 mutant site in Example 2 of the present application using different annealing temperatures for detection.
[0030] Figure 6 This is a linear relationship diagram for detecting various ApoE genotypes involved in Example 3 of the present application.
[0031] Figure 7 This is a diagram of the ddPCR detection results and sanger sequencing results of the ApoE E3 / 3 cell line genomic sample involved in Example 4 of the present application.
[0032] Figure 8 This is the ddPCR detection result diagram and Sanger sequencing result diagram for the ApoE E2 / 2 type cell line genome sample involved in Example 4 of the present application.
[0033] Fig. 9 This is a diagram of the ddPCR detection results and sanger sequencing results of the ApoE E3 / 4 cell line genomic sample involved in Example 4 of the present application.
[0034] Fig.10 This is a diagram of the ddPCR detection results and sanger sequencing results of the ApoE E2 / 3 cell line genomic sample involved in Example 4 of the present application.
[0035] Fig.11 This is a graph of ddPCR detection results involving the comparative example of the present application using 388 mutation probes, 388 wild probes, 526 mutation probes, and 526 wild probes for detection.
[0036] Fig.12 This is a graph of ddPCR test results involving the comparative example of the present application, in which 388 mutation probes, 388 wild probes, 526 mutation probes, and 526 wild probes are used to further test mutant samples and wild samples. DETAILED DESCRIPTION
[0037] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can easily recognize that some of the features can be omitted in different situations, or can be replaced by other materials and methods. In some cases, some operations related to the present application are not shown or described in the specification, in order to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and the related operations can be fully understood according to the description in the specification and the general technical knowledge in the art.
[0038] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.
[0039] The purpose of this application is to overcome the shortcomings of the prior art and provide an ApoE gene polymorphism detection method to solve the problems of low sensitivity and inability to achieve absolute quantification when detecting ApoE gene polymorphism in DNA samples in the prior art, while having universality, to obtain an accurate, stable and reliable ApoE genotyping nucleic acid quantitative detection method.
[0040] The present application specially designs specific forward primers, and the 3' ends of forward primer 388T-F and forward primer 388C-F are located at the polymorphic site. The last base of the sequence of forward primer 388T-F (the base closest to the 3' end) matches ApoE 388T, and the last base of the sequence of forward primer 388C-F matches ApoE 388C. Based on the strict base pairing of DNA polymerase on the 3' end of the primer, when a single base mismatch occurs at the 3' end of the primer, the DNA polymerase cannot effectively extend from this position, which manifests as amplification obstruction, thereby inhibiting nonspecific amplification. Similarly, forward primer 526C-F and forward primer 526T-F also have similar special designs.
[0041] In addition, the present application screened out a primer probe set with good specificity and accurate signal (clear division of positive droplets and negative droplets, less diffuse droplet clusters, and higher droplet number) by designing and testing a large number of specific primers on different forward primers and reverse primers.
[0042] In addition, in the present application, one probe is shared for the wild type and mutant type of ApoE 388, and one probe is shared for the wild type and mutant type of ApoE 526, which can reduce the probe cost, and the probe of the present application has good specificity and can generate accurate signals. When using the primer probe set of the present application for detection, for samples of unknown genotype, since the wild type and mutant type share one probe, two tubes are required for detection, one tube using the upstream primer and downstream primer corresponding to the wild type, and the other tube using the upstream primer and downstream primer corresponding to the mutant type.
[0043] The ddPCR detection kit of the present application is used to detect the ApoE genotype, which has the advantages of good specificity, high sensitivity, and high accuracy of the detection result, and can achieve absolute quantification of the ApoE gene amplification target without the need for a standard curve.
[0044] The kit of the present application can detect ApoE gene at a level as low as 4.4 copies / reaction.
[0045] It should be noted that the relationship between ApoE genotype and disease or drug efficacy is the increased or decreased possibility of disease risk or good or bad efficacy. ApoE genotype cannot be used directly to diagnose or treat disease, and must be combined with clinical information or other diagnostic methods to make a judgment.
[0046] The present application is further described in detail below by specific examples. The following examples are only further described for the present application and should not be construed as limiting the present application. In the present example, unless otherwise specified, the reagents and instruments used are all commercially available, and the experimental operations are carried out in accordance with the product instructions and conventional experimental specifications.
[0047] Example 1: Primer set optimization
[0048] 1) Specific primers and universal probes were designed for the ApoE gene 388T>C and 526C>T mutation sites, respectively, as shown in the following table:
[0049]
[0050] In the above table, lowercase letters represent mismatched bases, and the last base of each upstream primer (F primer) matches the base at the mutation site. Among them, 388-T-F1, 388-T-F3, 388-T-F5, 526-C-F1, 526-C-F3, and 526-C-F5 are wild primers, and 388-C-F2, 388-C-F4, 388-C-F6, 526-T-F2, 526-T-F4, and 526-T-F6 are mutant primers; in other words, 388-T-F1, 388-T-F3, and 388-T-F5 can match ApoE 388T, 388-C-F2, 388-C-F4, and 388-C-F6 can match ApoE 388C, and 526-C-F1, 526-C-F3, and 526-C-F5 can match ApoE 526-T-F2, 526-T-F4, 526-T-F6 can match ApoE 526T.
[0051] 2) The upstream primers 388-T-F1, 388-C-F2, 388-T-F3, 388-C-F4, 388-T-F5 and 388-C-F6 were combined with the downstream primer 388-R1, and PCR amplification was performed using the 388T>C site wild-type and mutant plasmids, respectively.
[0052] The PCR amplification system is as follows:
[0053] Components volume 2×MaxBuffer 12.5μL dNTP 0.5μL DNAase 0.5μL Upstream primer (10 μM) 1μL Downstream primer (10 μM) 1μL <![CDATA[Template DNA (plasmid, 5×10 3 copies / μL)]]> 5μL <![CDATA[ddH2O]]> up to 25 μL
[0054] The PCR amplification program is as follows:
[0055]
[0056] 3) Detect the amplified product by gel electrophoresis.
[0057] Figure 1 1 is a graph showing the electrophoresis results of PCR products for detecting ApoE 388 and 526 sites using different forward primers as described in Example 1 of the present application. Figure 1 Channels 1 to 6 in the figure represent the results of primers F1 to F6, respectively. Figure 1 In parts a and b of the results, the primer sets 388-T-F1 and 388-T-F3 had PCR amplification products in both wild-type and mutant samples, indicating nonspecific amplification, and the primer set 388-T-F5 had PCR amplification products only in wild-type samples; the primer sets 388-C-F2 and 388-C-F4 had PCR amplification products in both wild-type and mutant samples, indicating nonspecific amplification, and the primer set 388-C-F6 had PCR amplification products only in mutant samples. The above results indicate that the primer sets 388-T-F5 and 388-C-F6 upstream of the 388T>C site have the best specificity and are the best choice.
[0058] 4) Similarly, the upstream primers 526-C-F1, 526-T-F2, 526-C-F3, 526-T-F4, 526-C-F5, and 526-T-F6 were combined with the downstream primer 526-R1, and PCR amplification was performed using the 526C>T site wild-type and mutant plasmids, respectively, and the amplified products were detected by gel electrophoresis.
[0059] according to Figure 1In the c and d parts, the primers 526-C-F1 and 526-C-F5 had no PCR amplification products in both the wild-type sample and the mutant sample, and the primer 526-C-F3 had a PCR amplification product in the wild-type sample; the primers 526-T-F4 and 526-T-F6 had no PCR amplification products in both the wild-type sample and the mutant sample, and the primer 526-T-F2 had a PCR amplification product only in the mutant sample. In summary, the primer set 526-C-F3 and 526-T-F2 upstream of the 526C>T site had the best specificity and was the best choice.
[0060] 5) The upstream specific primers (388-T-F5, 388-C-F6, 526-T-F2, 526-C-F3) of the 388T>C and 526C>T sites screened above were combined with downstream primers (388-R1, 388-R2, 526-R1, 526-R2, 526-R3) to prepare the ddPCR reaction system, including: 2×ddPCR Mix buffer 10μL, Primer (10μm) 0.8μL, Probe (10μm) 0.4μL, plasmid DNA 2μL, and ddH2O was added to make the final volume 20μL. The ddPCR reaction conditions were 95℃5min, followed by 45 cycles of 95℃15s, 60℃45s.
[0061] The ddPCR method was used to specifically detect the mutant and wild-type plasmids at the APOE 388T>C site. Figure 2 This is a graph of the ddPCR detection results of detecting the ApoE 388 site using different reverse primers involved in Example 1 of the present application. Figure 2 Part a is a graph showing the ddPCR detection results of ApoE 388 site using 388-T-F5 and 388-R1 involved in Example 1 of the present application. Figure 2 Part b is a graph showing the ddPCR detection results of ApoE 388 site using 388-C-F6 and 388-R1 involved in Example 1 of the present application. Figure 2 Part c is a graph showing the ddPCR detection results of the ApoE388 site using 388-T-F5 and 388-R2 as described in Example 1 of the present application. Figure 2Part d is a diagram of the ddPCR detection results of the ApoE388 site using 388-C-F6 and 388-R2 involved in Example 1 of the present application. The detection results show that when the 388T>C site-specific upstream primer is combined with the 388-R1 downstream primer, the wild primer detects positive droplets in the mutant sample, and the mutant primer detects positive droplets in the wild-type sample; when combined with the 388-R2 downstream primer, the wild primer does not detect wild-type droplets in the mutant sample, and the mutant primer does not detect mutant droplets in the wild-type sample. The detection result of the combination of the 388T>C site-specific upstream primer and the 388-R2 downstream primer has good specificity.
[0062] The ddPCR method was used to specifically detect the mutant and wild-type plasmids at the APOE 526C>T site. Figure 3 This is a graph of the ddPCR detection results of the ApoE 526 site using different reverse primers involved in Example 1 of the present application, Figure 3 Part a of the present application is a graph showing the ddPCR detection results of the ApoE 526 site using 526-C-F3 and 526-R1 involved in Example 1. Figure 3 Part b is a graph showing the ddPCR detection results of ApoE 526 locus using 526-C-F3 and 526-R2 involved in Example 1 of the present application. Figure 3 Part c is a graph showing the ddPCR detection results of the ApoE526 site using 526-C-F3 and 526-R3 involved in Example 1 of the present application. Figure 3 Part d is a diagram showing the ddPCR detection results of the ApoE526 site using 526-T-F2 and 526-R1 involved in Example 1 of the present application. Figure 3 Part e is a graph showing the ddPCR detection results of ApoE 526 locus using 526-T-F2 and 526-R2 involved in Example 1 of the present application. Figure 3Part f is a ddPCR test result diagram of the ApoE 526 site using 526-T-F2 and 526-R3 involved in Example 1 of the present application. The test results show that the 526C>T site-specific upstream wild primer 526-C-F3 is combined with 526-R1, 526-R2 and 526-R3 downstream primers. Positive droplets are detected in both wild samples and mutant samples by the 526-R2 group and the 526-R3 group. The 526-R1 group wild primer only detects positive droplets in wild-type samples, indicating that the 526-R1 group has good wild-type detection specificity. The test results showed that the 526C>T site-specific upstream mutation primer 526-T-F2 was combined with the 526-R1, 526-R2 and 526-R3 downstream primers. The 526-R1 and 526-R3 groups detected positive droplets in both wild-type and mutant samples, while the 526-R2 group mutation primers only detected positive droplets in mutant samples, indicating that the 526-R2 group mutation detection specificity was good.
[0063] In summary, for ApoE 388T>C, the upstream primers are 388-T-F5 and 388-C-F6, the downstream primer is 388-R2, and the probe is 388-TP. For ApoE 526C>T, for ApoE 526 wild type (i.e. ApoE 526C), the upstream primer is 526-C-F3, the downstream primer is 526-R1; for ApoE 526 mutant (i.e. ApoE 526T), the upstream primer is 526-T-F2, the downstream primer is 526-R2; the probe is 526-TP.
[0064] Example 2: Reaction system and process optimization
[0065] Example 1 optimized the primer set, and this example further optimized the detection method. The primer probe set in Example 1 (for ApoE 388T>C, the upstream primers are 388-T-F5 and 388-C-F6, the downstream primer is 388-R2, and the probe is 388-TP. For ApoE 526C>T, for ApoE 526 wild type (i.e., ApoE 526C), the upstream primer is 526-C-F3, and the downstream primer is 526-R1; for ApoE 526 mutant (i.e., ApoE 526T), the upstream primer is 526-T-F2, and the downstream primer is 526-R2; the probe is 526-TP) is used to optimize the primer / probe concentration and annealing temperature.
[0066] (1) Primer / probe concentration optimization:
[0067] The ddPCR reaction system included: 2×ddPCRMixbuffer 10μL, Primer (10μm) 0.4-1.2μL, Probe (10μm) 0.2-0.4μL, ApoE plasmid DNA 2μL, and ddH2O to make up the final volume to 20μL. The reaction conditions were: 95℃5min, followed by 45 cycles: 95℃15s, 60℃45s.
[0068] Different amounts of primers and probes were set, so that the final concentration of primers in the ddPCR reaction system ranged from 200nM to 600nM, and the final concentration of probes was 100nM and 200nM. The dPCR method was used for verification, and the optimal conditions were clear division between positive and negative droplets, less diffusion of droplet clusters, higher number of droplets, and accurate copy number.
[0069] Figure 4 This is a ddPCR test result diagram of the ApoE 388 wild-type site, ApoE 388 mutant site, ApoE526 wild-type site, and ApoE 526 mutant site involved in Example 2 of the present application using different primer probe concentrations for detection. Figure 4 In part a, the primers refer to 388-T-F5 and 388-R2, and the probe refers to 388-TP. To illustrate schematically, the primer concentration of 200 nM in the figure means that the concentrations of 388-T-F5 and 388-R2 are both 200 nM. Figure 4 In part b, the primers refer to 388-C-F6 and 388-R2, and the probe refers to 388-TP. For schematic illustration, the primer concentration of 200nM in the figure means that the concentrations of 388-C-F6 and 388-R2 are both 200nM. According to the test results, taking the optimal conditions as the standard, the concentrations of wild primers and mutant primers at the ApoE388T>C site were finally determined to be 400nM, and the probe concentration was 200nM.
[0070] Figure 4 In part c, the primers refer to 526-C-F3 and 526-R1, and the probe refers to 526-TP. To illustrate schematically, the primer concentration of 200 nM in the figure means that the concentrations of 526-C-F3 and 526-R1 are both 200 nM. Figure 4 In the d part, the primers refer to 526-T-F2 and 526-R2, and the probe refers to 526-TP. For schematic illustration, the primer concentration of 200nM in the figure means that the concentrations of 526-T-F2 and 526-R2 are both 200nM. According to the test results, taking the optimal conditions as the standard, the concentrations of wild primers and mutant primers at the ApoE526C>T site were finally determined to be 400nM, and the probe concentration was 200nM.
[0071] (2) Annealing temperature optimization:
[0072] The ddPCR reaction system included: 2×ddPCR Mix buffer 10μL, Primer (10μm) 0.8μL, Probe (10μm) 0.4μL, ApoE plasmid DNA 2μL, and ddH2O to make up the final volume to 20μL. The reaction conditions were: 95℃5min, followed by 45 cycles: 95℃15s, 56-60℃45s.
[0073] Different annealing temperatures of 56°C, 58°C, and 60°C were set, and ddPCR was used for verification, with clear division between positive and negative droplets, less diffuse droplet clusters, higher droplet numbers, and accurate copy numbers as the optimal conditions.
[0074] Figure 5 This is a graph of ddPCR detection results involving ApoE 388 wild-type site, ApoE 388 mutant site, ApoE526 wild-type site, and ApoE 526 mutant site in Example 2 of the present application using different annealing temperatures for detection.
[0075] According to the test results and taking the optimal conditions as the standard, the annealing temperature of the wild primer and mutant primer at the ApoE 388T>C site was finally determined to be 60°C, and the annealing temperature of the wild primer and mutant primer at the ApoE 526C>T site was determined to be 60°C.
[0076] Example 3: Detection method validation
[0077] According to Example 1 and Example 2, the optimal primer probe and detection method were determined, and the performance of the method was verified.
[0078] ApoE mutant plasmid and wild-type plasmid samples were serially diluted to a concentration of 5.08 × 10 4 copies / μL, 5.08×10 3 copies / μL, 5.08×10 2 copies / μL, 5.08×10 1 1 copies / μL, 1.02×10 1 copies / μL, 2.03×10 0 Then, the primer probe and detection method determined in Example 1 and Example 2 were used (the ddPCR reaction system included: 2×ddPCR Mixbuffer 10μL, Prime r(10μm) 0.8μL, Probe (10μm) 0.4μL, ApoE plasmid DNA 2μL, add ddH2O to make up the final volume to 20μL. The reaction conditions are: 95℃5min, then enter 45 cycles: 95℃15s, 60℃45s; the reaction program is: 95℃5min, then enter 45 cycles: 95℃15s, 60℃45s) to verify the detection method, and draw the linear relationship curve of the method according to the detection results, with R 2 ≥0.98 is the verification standard. The test results are shown in the following table:
[0079]
[0080] Figure 6 This is a linear relationship diagram for the detection of each ApoE genotype involved in Example 3 of the present application, which is a linear diagram of ApoE388 wild type, ApoE 388 mutant, ApoE 526 wild type and ApoE 526 mutant, in which the horizontal axis is the sample number and the vertical axis is the content (copies / reaction). Figure 6 It can be seen that in 10 1 ~10 5 copies / reaction concentration range, R 2 >0.98, the linear relationship is good, and the detection method is stable. The method of this embodiment can detect a minimum of 4.4 copies / reaction of the ApoE gene.
[0081] Example 4: Actual sample testing
[0082] (1) ApoE gene E3 / 3 cell line genomic DNA samples 01, 02, and 03 were used for detection. The ddPCR reaction system included: 2×ddPCR Mix buffer 10μL, Primer (10μm) 0.8μL, Probe (10μm) 0.4μL, E3 / 3 genomic DNA 2μL, and ddH2O was added to make the final volume 20μL. The ddPCR reaction conditions were 95℃ for 5min, followed by 45 cycles of 95℃ for 15s and 60℃ for 45s.
[0083] Figure 7 This is the ddPCR test result diagram and Sanger sequencing result diagram for the ApoE E3 / 3 cell line genome sample involved in Example 4 of the present application, Figure 7 Part a of the present application is a ddPCR test result diagram of the ApoE 388 site of the ApoE E3 / 3 cell line genome sample involved in Example 4, Figure 7Part b is a diagram showing the ddPCR detection results of the ApoE 526 site of the ApoE E3 / 3 cell line genomic sample involved in Example 4 of the present application, Figure 7 Parts c to d are the Sanger sequencing result diagrams of ApoE 388 and 526 sites of the ApoE E3 / 3 type cell line genomic sample involved in Example 4 of the present application.
[0084] Test results see Figure 7 The results show that the detection results of samples 01, 02, and 03 are wild-type at 388T>C and 526C>T, which are consistent with the results of Sanger sequencing.
[0085] Sample No. 388T>C site wild type 526C>T site wild type 01 1.09E+04 1.10E+04 02 1.07E+04 1.10E+04 03 1.09E+04 1.15E+04
[0086] (2) Genomic DNA samples 04, 05, and 06 of the cell line with ApoE gene E2 / 2 were used for detection. The ddPCR reaction system included: 2×ddPCRMixbuffer 10μL, Primer (10μm) 0.8μL, Probe (10μm) 0.4μL, E2 / 2 genomic DNA 2μL, and ddH2O was added to make the final volume 20μL. The ddPCR reaction conditions were 95℃ for 5min, followed by 45 cycles of 95℃ for 15s and 60℃ for 45s.
[0087] Figure 8 This is the ddPCR test result diagram and Sanger sequencing result diagram for the ApoE E2 / 2 type cell line genome sample involved in Example 4 of the present application, Figure 8 Part a of the present application is a ddPCR detection result diagram of the ApoE 388 site of the ApoE E2 / 2 type cell line genome sample involved in Example 4, Figure 8 Part b is a diagram showing the ddPCR detection results of the ApoE 526 site of the ApoE E2 / 2 type cell line genome sample involved in Example 4 of the present application, Figure 8 Parts c to d are the Sanger sequencing result diagrams of ApoE 388 and 526 sites of the ApoE E2 / 2 type cell line genomic sample involved in Example 4 of the present application.
[0088] Test results such as Figure 8 The results show that the detection results of samples 04, 05, and 06 are wild type at 388T>C and mutant at 526C>T, which are consistent with the results of Sanger sequencing.
[0089] Sample No. 388T>C site mutation 526C>T site mutation 04 1.06E+04 1.05E+04 05 1.07E+04 1.07E+04 06 1.05E+04 9.90E+03
[0090] (3) ApoE gene E3 / 4 cell line genomic DNA samples 07, 08, and 09 were used for detection. The ddPCR reaction system included: 2×ddPCRMixbuffer 10μL, Primer (10μm) 0.8μL, Probe (10μm) 0.4μL, E3 / 4 type genomic DNA 2μL, and ddH2O was added to make up the final volume to 20μL. The ddPCR reaction conditions were 95℃5min, followed by 45 cycles: 95℃15s, 60℃45s.
[0091] Fig. 9 This is the ddPCR test result diagram and Sanger sequencing result diagram for the ApoE E3 / 4 type cell line genome sample involved in Example 4 of the present application, Fig. 9 Part a of the present application is a diagram of the ddPCR detection results of ApoE 388 wild type for ApoE E3 / 4 type cell line genome samples involved in Example 4, Fig. 9 Part b is a diagram showing the ddPCR detection results of the ApoE 388 mutant in the ApoE E3 / 4 cell line genome sample involved in Example 4 of the present application. Fig. 9 Part c is a diagram showing the ddPCR detection results of the ApoE 526 site of the ApoE E3 / 4 cell line genomic sample involved in Example 4 of the present application. Fig. 9 Part d is a diagram of the Sanger sequencing results of the ApoE388 site and the 526 site of the ApoE E3 / 4 type cell line genomic sample involved in Example 4 of the present application.
[0092] Test results such as Fig. 9 The results show that the detection results of samples 07, 08, and 09 are heterozygous mutations at 388T>C and wild-type at 526C>T, which are consistent with the results of Sanger sequencing.
[0093]
[0094] (4) ApoE gene E2 / 3 cell line genomic DNA samples 10, 11, and 12 were used for detection. The ddPCR reaction system included: 2×ddPCR Mix buffer 10μL, Primer (10μm) 0.8μL, Probe (10μm) 0.4μL, E2 / 3 type genomic DNA 2μL, and ddH2O was added to make the final volume 20μL. The ddPCR reaction conditions were 95℃ for 5min, followed by 45 cycles of 95℃ for 15s and 60℃ for 45s.
[0095] Fig.10 This is the ddPCR test result diagram and Sanger sequencing result diagram for the ApoE E2 / 3 cell line genome sample involved in Example 4 of the present application, Fig.10 Part a is a diagram showing the ddPCR detection results of the ApoE 388 site of the ApoE E2 / 3 cell line genomic sample involved in Example 4 of the present application. Fig.10 Part b is a diagram showing the ddPCR detection results of ApoE 526 wild type for ApoE E2 / 3 type cell line genomic samples involved in Example 4 of the present application, Fig.10 Part c is a diagram showing the ddPCR detection results of the ApoE 526 mutant in the ApoE E2 / 3 cell line genome sample involved in Example 4 of the present application. Fig.10 Part d is a diagram of the Sanger sequencing results of the ApoE388 site and the 526 site of the ApoE E2 / 3 type cell line genomic sample involved in Example 4 of the present application.
[0096] Test results such as Fig.10 The results show that the detection results of samples 10, 11, and 12 are wild-type at 388T>C and heterozygous mutant at 526C>T, which are consistent with the results of Sanger sequencing.
[0097]
[0098] Comparative Example: Dual Probe-ddPCR Method
[0099] 1) According to the upstream and downstream gene sequences of the ApoE gene 388T>C and 526C>T mutation sites, a wild type and mutant probe (i.e., 388T>C, SEQ ID No: 22 and SEQID No: 23; 526C>T, SEQ ID No: 26 and SEQ ID No: 27) and specific primers were designed for the wild type and mutant types of ApoE 388 and ApoE 526, respectively, as shown in the following table:
[0100]
[0101] Note: The bold underlined bases in the above table correspond to the bases at the mutation site.
[0102] 2) Prepare ddPCR reaction system, including: 2×ddPCR Mix buffer 10μL, Primer (10μm) 1μL, Probe (10μm) 0.5μL, plasmid DNA (ApoE 388T>C mutant plasmid, ApoE 388T>C wild plasmid, ApoE 526C>T mutant plasmid or ApoE 526C>T mutant plasmid, the concentration of each is 5×10 3 copies / μL), add ddH2O to make up the final volume to 20μL. See the table below for details:
[0103] Components volume 2×MasterMix 10μL Upstream primer (10 μM) 1μL Downstream primer (10 μM) 1μL Probe (10 μM) 0.5μL Template DNA 2μL <![CDATA[ddH2O]]> up to 20 μL
[0104] 3) The ddPCR reaction conditions were 95°C for 5 min, followed by 45 cycles of 95°C for 15 s and 60°C for 45 s.
[0105] 4) The ddPCR method was used to detect ApoE mutations and wild plasmids. The results showed that the ddPCR detection results of the 388T>C site had many "raindrops" with unclear partitions, which may affect the final quantitative accuracy, such as Fig.11 . Fig.11 This is a ddPCR test result diagram of the comparative example of the present application using 388 mutation probes, 388 wild probes, 526 mutation probes, and 526 wild probes for detection. Fig.11 Part a of the present application is a comparative example of the ddPCR detection results using 388 mutation probes. Fig.11 Part b is a diagram of the ddPCR test results using 388 wild-type probes in the comparative example of the present application. Fig.11 Part c is a diagram of the ddPCR test results using 526 mutation probes in the comparative example of the present application. Fig.11 Part d is a graph of the ddPCR detection results using 526 wild-type probes involved in the comparative example of the present application.
[0106] 5) Further, the mutant heterozygous sample of 388T>C site (mixed sample of mutant plasmid and wild plasmid, each accounting for 50%, with a total concentration of 1×10 4 copies / μL) as mutant samples, wild-type samples at 388T>C, and heterozygous mutant samples at 526C>T (mixed samples of mutant plasmid and wild-type plasmid, 50% each, with a total concentration of 1×10 4 copies / μL) as mutant samples and wild-type samples at the 526C>T site were used to test the specificity of the Taqman probe.
[0107] Fig.12This is a ddPCR test result diagram of further testing mutant samples and wild samples using 388 mutation probes, 388 wild probes, 526 mutation probes, and 526 wild probes involved in the comparative example of the present application, Fig.12 Part a of the present application is a comparative example of the use of 388 mutation probes to further detect mutant samples and wild samples ddPCR test results, Fig.12 Part b is a diagram of ddPCR test results of further testing mutant samples and wild samples using 388 wild probes involved in the comparative example of this application, Fig.12 Part c is a diagram of ddPCR test results of further testing mutant samples and wild-type samples using 526 mutation probes in the comparative example of the present application. Fig.12 Part d is a ddPCR test result diagram of further testing mutant samples and wild samples using 526 wild probes in the comparative example of this application. The results show that the 388T>C mutant probe has positive droplets in the wild sample, the 388T>C wild probe has positive droplets in the mutant sample, and both probes at the 388T>C site have non-specific detection and cannot be further tested, such as Fig.12 The 526C>T mutant probe has positive droplets in the wild-type sample, and the 388T>C wild-type probe has positive droplets in the mutant sample. Both probes at the 526C>T site have nonspecific detection and cannot be further tested. Fig.12 Part c to d.
[0108] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.
Claims
1. A kit for ddPCR detection of ApoE genotype, characterized in that: It includes a first primer probe set for detecting ApoE 388T>C and a second primer probe set for detecting ApoE 526C>T; The first primer probe set includes: forward primer 388T-F, the sequence of which is shown in SEQ ID NO.1; forward primer 388C-F, the sequence of which is shown in SEQ ID NO.2; reverse primer 388-R, the sequence of which is shown in SEQ ID NO.3; and probe 388-P, the sequence of which is shown in SEQ ID NO.4; The second primer probe group includes: forward primer 526C-F, the sequence of which is shown in SEQ ID NO.5; reverse primer 526C-R, the sequence of which is shown in SEQ ID NO.6; forward primer 526T-F, the sequence of which is shown in SEQ ID NO.7; reverse primer 526T-R, the sequence of which is shown in SEQ ID NO.8; and probe 526-P, the sequence of which is shown in SEQ ID NO.
9.
2. The kit according to claim 1, characterized in that The fluorescent groups of the probe 388-P and the probe 526-P are independently selected from FAM, FITC, HEX, VIC, TET, JOE, ROX, CY3, CY5, CY5.5, CY7, ATTO 425 and SF670, and the quenching groups of the probe 388-P and the probe 526-P are independently selected from BHQ1, BHQ2, BHQ3, MGB and DABCYL.
3. The kit according to claim 2, characterized in that The fluorescent group of the probe 388-P is different from the fluorescent group of the probe 526-P.
4. The kit according to claim 1, characterized in that The kit further comprises at least one of the following: a ddPCR premix, a droplet generation oil, a positive control substance, and a negative control substance.
5. The kit according to claim 4, characterized in that The ddPCR premix includes at least one of the following: PCR buffer, Taq DNA polymerase, Mg 2+ and dNTPs.
6. A device for detecting ApoE genotype, characterized in that: include: Amplification module: using the kit as described in any one of claims 1 to 5 to perform ddPCR detection on the nucleic acid sample to be tested; Judgment module: judge the genotype and content of ApoE according to the results of the ddPCR test.
7. A method for detecting ApoE genotype for non-diagnostic and therapeutic purposes, characterized in that: include: Obtaining nucleic acid samples to be tested; Using the kit according to any one of claims 1 to 5 to prepare a ddPCR reaction system, and performing the reaction according to the reaction procedure; Determine the genotype and content of ApoE.
8. The detection method according to claim 7, characterized in that: In the ddPCR reaction system, the concentration of forward primer 388T-F, forward primer 388C-F, reverse primer 388-R, forward primer 526C-F, reverse primer 526C-R, forward primer 526T-F or reverse primer 526T-R is 300 nM to 500 nM; Preferably, in the ddPCR reaction system, the concentration of forward primer 388T-F, forward primer 388C-F, reverse primer 388-R, forward primer 526C-F, reverse primer 526C-R, forward primer 526T-F or reverse primer 526T-R is 400 nM.
9. The method according to claim 7 or 8, characterized in that: In the ddPCR reaction system, the concentration of probe 388-P or probe 526-P is 150 nM to 250 nM; Preferably, in the ddPCR reaction system, the concentration of probe 388-P or probe 526-P is 200 nM.
10. The method according to claim 7, characterized in that In the reaction procedure, the annealing temperature is 59°C to 62°C; Preferably, in the reaction procedure, the annealing temperature is 60°C.