A reagent for detecting ApoE gene and its application

Abstract

The present invention discloses a reagent for detecting ApoE gene and its application; the reagent comprises a primer set, the primer set comprises a forward common primer Frh for detecting ApoE gene site 112 and ApoE gene site 158, a reverse primer Rrh1 and a reaction primer Rrh2 for detecting ApoE gene site 158. The present invention obtains a reagent for ApoE gene detection and typing through creative design and screening, and by introducing rhPCR amplification, amplification forms fewer primer dimers, has high sensitivity, good specificity, significantly reduces amplification false positives, has high detection resolution, high throughput, is simple and easy to operate, is easy to automate, and has professional software to interpret the results.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a reagent for detecting ApoE gene and application thereof. Background Art

[0002] Apolipoprotein E (ApoE) is a polymorphic protein that participates in the transformation and metabolism of lipoproteins. Its gene can regulate many biological functions and plays an important role in blood lipid metabolism. ApoE gene polymorphism plays an important role in the early stage and development of cardiovascular and cerebrovascular diseases such as hyperlipidemia, atherosclerosis, and coronary heart disease. In addition, studies have shown that the ApoE gene is the closest genetic marker for Alzheimer's disease known to date, and its polymorphism is significantly correlated with susceptibility to Alzheimer's disease.

[0003] The genetic test for ApoE is mainly the base at the 112th amino acid (C.388T>C, Cys130Arg) and the 158th amino acid (C.526C>T, Arg176Cys). The exchange of two amino acid residues, arginine (Arg) and cysteine ​​(Cys), at the 112th and 158th positions of the amino acid sequence of ApoE determines the type of isomer. According to the different combinations of amino acids at positions 112 and 158, three types of ApoE are formed, namely E2, E3 and E4. There are 6 different ApoE phenotypes in the population: three homozygous (E2 / 2, E3 / 3, E4 / 4) and three heterozygous (E3 / 2, E4 / 2, E4 / 3). ApoE3 is the most common form in the population, often referred to as the "wild type", with cysteine ​​Cys at position 112 and arginine Arg at position 158 of its polypeptide chain. In the study of hyperlipidemia to assess the risk factors of cardiovascular and cerebrovascular diseases, the research using ApoE polymorphism as a means has important clinical significance.

[0004] At present, the main methods for detecting ApoE include direct sequencing, gene chip hybridization, PCR-Taqman probe typing, ARMS-PCR and PCR-melting curve, as follows:

[0005] (1) Direct sequencing method: It is the "gold standard" for SNP typing. This method designs sequencing primers, extracts nucleic acids for PCR amplification and product sequencing. The main disadvantages of this method are: 1) Before the PCR reaction, it is necessary to extract DNA from the sample, test the quality and concentration, which is complicated and there is a risk of confusing the sample; 2) When testing multiple sites, one sample needs to undergo multiple reactions, which has high testing costs, high template requirements, high operation intensity, and is prone to operational errors and contamination; 3) After the PCR reaction, the product needs to be purified and sequenced through chemical reactions. In general, the steps are cumbersome, the test cycle is long, the cost is high, and it is not conducive to large-scale clinical promotion;

[0006] (2) Gene chip hybridization method: a large number of nucleic acid molecules are arranged in a large-scale array on a very small carrier, and the technology is used to hybridize with labeled samples to detect the strength of the hybridization signal and then determine the number of molecules in the sample. The gene chip method requires gene amplification in the early stage, and the amplified product is hybridized with the chip probe. It can perform high-throughput detection, but this method has many detection steps, requires a large amount of templates, and is complex and time-consuming to operate and analyze data. It also requires opening the cover and is prone to contamination;

[0007] (3) The basic principle of PCR-Taqman probe method is to add a specific fluorescent probe at the same time as adding a pair of primers during PCR amplification. The two ends of the probe are labeled with a reporter fluorescent group and a quencher fluorescent group respectively. This method realizes SNP site typing by setting a pair of specific primers and two specific probes for one site. Differentiating one SNP requires designing two probes for one site, which is costly.

[0008] (4) ARMS-PCR allele-specific amplification method, also known as amplification-blocked mutation system, is used to detect known mutant genes. Allele-specific extension is controlled by designing the 3' end primer, and the fluorescence signal value is detected by combining a universal Taqman probe method to determine the wild-type allele and mutant wild gene. This method detects the same SNP site through different primer pairs in 2 tubes of reagents;

[0009] (5) PCR-melting curve method: a new type of DNA saturated fluorescent dye is added to the reaction system before the PCR reaction, and then the PCR product is directly introduced into a high-resolution melting analyzer. The PCR amplification product is heated and denatured within a certain temperature range. The optical detection system of the instrument collects and analyzes the fluorescent signal and draws a temperature melting curve. The (single / multiple) base differences are distinguished according to the different melting curve shapes.

[0010] In summary, the existing ApoE genotyping detection methods all have more or less defects such as complex operation process, high detection cost, long cycle, easy contamination and certain false positives. Therefore, it is urgent to establish a method for rapid detection of these two gene mutations at the same time, and provide hospitals and other medical institutions with a detection solution that is simple to operate, highly specific, highly sensitive, high throughput, highly reliable and low cost. Summary of the invention

[0011] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a reagent for detecting the ApoE gene.

[0012] The present invention also provides a kit having the above reagent.

[0013] The invention also provides the application of the above reagent and kit.

[0014] According to one aspect of the present invention, a reagent for detecting the ApoE gene is provided, the reagent comprising a primer set, the primer set comprising a forward common primer Frh for detecting the ApoE gene site 112 and the ApoE gene site 158, a reverse primer Rrh1 for detecting the ApoE gene site 158, and a reaction primer Rrh2; the nucleotide sequence of the primer set is as follows:

[0015] Frh: C3 Spacer–GGGCACGGCTGTCCAAgGAGC;

[0016] Rrh1:C3 Spacer–CTGGTACACTGCCAGGCgCTTC;

[0017] Rrh2:C3 Spacer–CTGGTACACTGCCAGGCaCTTC;

[0018] Among them, "g" and "a" are RNA bases.

[0019] The present invention realizes specific PCR amplification by designing primers Rrh1 and Rrh2 for wild type and mutant type. According to the principle of allele-specific PCR, each specific primer can only bind to the DNA template of the corresponding genotype and amplify, with high specificity.

[0020] In some embodiments of the present invention, the reagent also includes a probe sequence for detecting the ApoE gene site 112. The present invention designs two pairs of primers to amplify the wild-type and specific DNA containing the 158 site, respectively, and the product has good specificity. By adding the wild-type and specific Taqman probes of the 112 site, the wild-type or mutant analysis of the 112 site and the 158 site is realized. The whole system realizes the SNP detection of the two mutation sites through a pair of Taqman probes; the reagent components are simpler, the operation is easier, and the cost is lower.

[0021] In some embodiments of the present invention, the probe sequence includes a first fluorescent probe sequence and a second fluorescent probe sequence; the first fluorescent probe sequence is shown in SEQ ID NO:17, and the second fluorescent probe sequence is shown in SEQ ID NO:18.

[0022] In some embodiments of the present invention, the 5' end of the first fluorescent probe sequence is labeled with a first fluorescent group, and the 5' end of the second probe sequence is labeled with a second fluorescent group, and the first fluorescent group and the second fluorescent group are different.

[0023] In some embodiments of the present invention, the fluorescent group includes one of FAM, VIC, CY5 and JOE.

[0024] In some embodiments of the present invention, the first fluorescent group is FAM.

[0025] In some embodiments of the present invention, the second fluorescent group is VIC.

[0026] In some embodiments of the present invention, the 3' end of the probe sequence is also labeled with a fluorescence quenching group.

[0027] In some embodiments of the present invention, the quenching group includes one of TAMRA, Dabcyl, BHQ1 and MGB.

[0028] According to a second aspect of the present invention, a kit is provided, wherein the kit contains the above reagent.

[0029] In some embodiments of the present invention, the kit further comprises a buffer solution, a Hot Taq enzyme, a UNG enzyme and a ribonuclease. The present invention can avoid the risk of contamination and improve the accuracy of detection by adding the UNG enzyme.

[0030] In some embodiments of the present invention, the kit further comprises purified water.

[0031] According to the third aspect of the present invention, an application of the above reagents and kits is proposed, wherein the application is an application in preparing a product for detecting the ApoE gene.

[0032] In some embodiments of the present invention, the application is application in the preparation of ApoE genotyping products.

[0033] In some embodiments of the present invention, the application is application in the preparation of a product for detecting Alzheimer's disease.

[0034] In some embodiments of the present invention, the application is application in the preparation of products for detecting cardiovascular and cerebrovascular diseases.

[0035] According to a fourth aspect of the present invention, a method for detecting the ApoE gene is proposed, the method comprising the following steps: using the above reagent or kit to detect the DNA of the sample to be tested; the method is not intended for the diagnosis or treatment of the disease.

[0036] In some embodiments of the present invention, the detection is performed using fluorescent quantitative PCR.

[0037] In some embodiments of the present invention, the final concentration of the primer in the reaction system of the fluorescent quantitative PCR is about 0.1-0.4 μM.

[0038] In some embodiments of the present invention, the reaction system used in the fluorescent quantitative PCR includes an amplification reaction system 1 and an amplification reaction system 2;

[0039] The amplification reaction system 1 is:

[0040]

[0041] 3-6 μL of sample to be tested;

[0042] Add ddH2O to 45 μL;

[0043] The amplification reaction system 2 is:

[0044]

[0045]

[0046] 3-6 μL of sample to be tested;

[0047] Add ddH2O to 45 μL;

[0048] In some embodiments of the present invention, the 5×Buffer comprises the following components at final concentrations: 200-300 mM KCl, 40-60 mM Tris.HCl, 0.4-0.6% TX-100, and 10-20 mM MgSO4.

[0049] In some embodiments of the present invention, the amplification program of the fluorescent quantitative PCR is: 48-52°C for 1-3 min; 94-96°C for 4-6 min; then enter the cycling stage: 93-95°C for 13-18 s, 58-62°C for 40-50 min, 38-45 cycles, and collect fluorescence data.

[0050] In some embodiments of the present invention, the amplification program of the fluorescent quantitative PCR is: 50°C for 2 min; 95°C for 5 min; then enter the cycling stage: 94°C for 15 s, 60°C for 45 min, 40 cycles, and collect fluorescence data.

[0051] In some embodiments of the present invention, after the detection, the step of statistically analyzing the Ct value of the sample and interpreting the result is also included; the result interpretation is:

[0052] Positive: If the Ct of the sample is ≤38 and a typical S-shaped fluorescence curve appears, the sample is positive;

[0053] Negative: If the sample has no fluorescence amplification or Ct>38, the sample is negative.

[0054] According to a fifth aspect of the present invention, a method for detecting ApoE genotype is proposed, the method comprising the following steps: using the above reagent or kit to detect the DNA of the sample to be tested; the method is not intended for the diagnosis or treatment of the disease.

[0055] In some embodiments of the present invention, the detection is performed using fluorescent quantitative PCR.

[0056] In some embodiments of the present invention, the final concentration of the primer in the reaction system of the fluorescent quantitative PCR is about 0.1-0.4 μM.

[0057] In some embodiments of the present invention, the fluorescent quantitative PCR comprises an amplification reaction system 1 and an amplification reaction system 2;

[0058] The amplification reaction system 1 is:

[0059]

[0060]

[0061] 3-6 μL of sample to be tested;

[0062] Add ddH2O to 45 μL;

[0063] The amplification reaction system 2 is:

[0064]

[0065] 3-6 μL of sample to be tested;

[0066] Add ddH2O to 45 μL;

[0067] In some embodiments of the present invention, the amplification program of the fluorescent quantitative PCR is: 48-52°C for 1-3 min; 94-96°C for 4-6 min; then enter the cycling stage: 93-95°C for 13-18 s, 58-62°C for 40-50 min, 38-45 cycles, and collect fluorescence data.

[0068] In some embodiments of the present invention, the amplification program of the fluorescent quantitative PCR is: 50°C for 2 min; 95°C for 5 min; then enter the cycling stage: 94°C for 15 s, 60°C for 45 min, 40 cycles, and collect fluorescence data.

[0069] In some embodiments of the present invention, after the detection, the step of statistically analyzing the Ct value of the sample and interpreting the result is also included; the result interpretation is:

[0070] E3 / E3 genotype: If the second fluorescent sequence of amplification reaction system 1 detects Ct≤38 and a typical S-shaped fluorescence curve appears, the sample is of E3 / E3 genotype;

[0071] E2 / E2 genotype: If the second fluorescent sequence of amplification reaction system 2 detects Ct≤38 and a typical S-shaped fluorescence curve appears, the sample is of E2 / E2 genotype;

[0072] E4 / E4 genotype: If the first fluorescent sequence of amplification reaction system 1 detects Ct≤38 and a typical S-shaped fluorescence curve appears, the sample is of E4 / E4 genotype;

[0073] E3 / E4 genotype: If the first fluorescent sequence of amplification reaction system 1 detects Ct≤38, and the second fluorescent sequence of amplification reaction system 1 also detects Ct≤38, and a typical S-shaped fluorescence curve appears, the sample is of E3 / E4 genotype;

[0074] E2 / E3 genotype: If the second fluorescent sequence of amplification reaction system 1 detects Ct≤38, and the second fluorescent sequence of amplification reaction system 2 also detects Ct≤38, and a typical S-shaped fluorescence curve appears, the sample is of E2 / E3 genotype;

[0075] E2 / E4 genotype: If the first fluorescent sequence of amplification reaction system 1 detects Ct≤38, the second fluorescent sequence of amplification reaction system 1 detects Ct≤38, the first fluorescent sequence of amplification reaction system 2 detects Ct≤38, and the second fluorescent sequence of amplification reaction system 2 detects Ct≤38, and a typical S-shaped fluorescence curve appears, the sample is of E2 / E4 genotype.

[0076] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention obtains reagents for ApoE gene detection and typing through creative design and screening, and by introducing rhPCR amplification, amplification forms fewer primer dimers, has high sensitivity, good specificity, significantly reduces amplification false positives, has high detection resolution, high throughput, is simple and easy to operate, is easy to automate, and has professional software to interpret the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0078] Figure 1 This is a diagram showing the detection results of system 1 in primer probe set 1 in Example 1 of the present invention;

[0079] Figure 2 This is a diagram showing the detection results of system 2 in primer probe set 1 in Example 1 of the present invention;

[0080] Figure 3 This is a diagram showing the detection results of system 1 in primer probe set 2 in Example 1 of the present invention;

[0081] Figure 4 This is a diagram showing the detection results of system 2 in primer probe set 2 in Example 1 of the present invention;

[0082] Figure 5 This is a result diagram of detecting E4E4 type using system 1 of ordinary PCR in primer probe set 2 in Example 1 of the present invention;

[0083] Figure 6 This is a result diagram of detecting E2E2 type using system 1 of ordinary PCR in primer probe set 2 in Example 1 of the present invention;

[0084] Figure 7 This is a result diagram of detecting E4E4 type using rhPCR system 1 in the rhPCR primer probe set in Example 1 of the present invention;

[0085] Figure 8 This is a result diagram of detecting E2E2 type using rhPCR system 1 in the rhPCR primer probe set in Example 1 of the present invention;

[0086] Fig. 9This is a result diagram of detecting E3 / E3 type by system 1 in the rhPCR primer probe set in Example 3 of the present invention;

[0087] Fig.10 This is a result diagram of detecting E3 / E3 type by system 2 in the rhPCR primer probe set in Example 3 of the present invention;

[0088] Fig.11 This is a result diagram of detecting E2 / E2 type by system 1 in the rhPCR primer probe set in Example 3 of the present invention;

[0089] Fig.12 This is a result diagram of detecting E2 / E2 type by system 2 in the rhPCR primer probe set in Example 3 of the present invention;

[0090] Fig.13 This is a result diagram of detecting E4 / E4 type by system 1 in the rhPCR primer probe set in Example 3 of the present invention;

[0091] Fig.14 This is a result diagram of detecting E4 / E4 type in system 2 of the rhPCR primer probe set in Example 3 of the present invention;

[0092] Fig.15 This is a result diagram of detecting E3 / E4 type by system 1 in the rhPCR primer probe set in Example 3 of the present invention;

[0093] Fig.16 This is a result diagram of detecting E3 / E4 type in system 2 of the rhPCR primer probe set in Example 3 of the present invention;

[0094] Fig.17 This is a result diagram of detecting E2 / E3 type by system 1 in the rhPCR primer probe set in Example 3 of the present invention;

[0095] Fig.18 This is a result diagram of detecting E2 / E3 type by system 2 in the rhPCR primer probe set in Example 3 of the present invention;

[0096] Fig.19 This is a result diagram of detecting E2 / E4 type by system 1 in the rhPCR primer probe set in Example 3 of the present invention;

[0097] Fig. 20 This is a result diagram of detecting E2 / E4 type by system 2 in the rhPCR primer probe set in Example 3 of the present invention;

[0098] Fig.21 This is a graph showing the results of detecting E3 / E3 type by system 1 in the rhPCR primer probe set in Example 4 of the present invention, wherein the curves from left to right are 1×10 7 / mL to 1×102 / mL concentration gradient;

[0099] Fig. 22 This is a graph showing the results of detecting E2E2 type by system 2 in the rhPCR primer probe set in Example 4 of the present invention, wherein the curves from left to right are 1×10 7 / mL to 1×10 2 / mL concentration gradient;

[0100] Fig.23 This is a graph showing the results of detecting E4E4 type by system 1 in the rhPCR primer probe set in Example 4 of the present invention, wherein the curves from left to right are 1×10 7 / mL to 1×10 2 / mL concentration gradient;

[0101] Fig.24 This is a graph showing the results of detecting E3 / E4 types by system 1 in the rhPCR primer and probe set in Example 4 of the present invention, wherein the curves from left to right are 1×10 7 / mL to 1×10 2 / mL concentration gradient;

[0102] Fig.25 This is a graph showing the results of detecting E3E4 types by system 2 in the rhPCR primer probe set in Example 4 of the present invention, wherein the curves from left to right are 1×10 7 / mL to 1×10 2 / mL concentration gradient;

[0103] Fig.26 This is a graph showing the results of detecting E2 / E3 types by system 1 in the rhPCR primer and probe set in Example 4 of the present invention, wherein the curves from left to right are 1×10 7 / mL to 1×10 2 / mL concentration gradient;

[0104] Fig. 27 This is a graph showing the results of detecting E2 / E3 types by system 2 in the rhPCR primer and probe set in Example 4 of the present invention, wherein the curves from left to right are 1×10 7 / mL to 1×10 2 / mL concentration gradient;

[0105] Fig.28 This is a graph showing the results of detecting E2 / E4 types by system 1 in the rhPCR primer and probe set in Example 4 of the present invention, wherein the curves from left to right are 1×10 7 / mL to 1×10 2 / mL concentration gradient;

[0106] Fig.29 This is a graph showing the results of detecting E2 / E4 types by system 2 in the rhPCR primer and probe set in Example 4 of the present invention, wherein the curves from left to right are 1×10 7 / mL to 1×10 2 / mL concentration gradient;

[0107] Fig.30 This is a graph showing the results of detecting E2 / E4 types by system 2 in the rhPCR primer and probe set in Example 4 of the present invention, wherein the curves from left to right are 1×10 7 / mL to 1×10 2 / mL concentration gradient;

[0108] Fig.31 This is a graph showing the results of detecting E2 / E4 types by system 2 in the rhPCR primer and probe set in Example 4 of the present invention, wherein the curves from left to right are 1×10 7 / mL to 1×10 2 The concentration gradient was 1000 μg / mL. DETAILED DESCRIPTION

[0109] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0110] Example 1 Design and determination of primer probes for detecting ApoE gene

[0111] 1. Primer design and optimization

[0112] (1) Primer design and screening

[0113] According to the mutation sites of ApoE112 (C.388T>C, Cys130Arg) and 158 (C.526C>T, Arg176Cys), specific primers and probes were designed using the software DNAMAN 6.0, and two pairs of primer-probe sets with the best performance such as amplification efficiency were screened, among which the leading primer F was a universal primer for ApoE112 and 158 sites, the reverse primer (R1 or R3) was a specific primer for 158 site (C.526C), and the reverse primer (R2 or R4) was a specific primer for 158 site (C.526T); the P1, P2, P3, and P4 probes were wild-type and mutant probes for ApoE112 site (C.388T>C, Cys130Arg). The specific sequences are shown in Table 1. Primers and probes were synthesized by Liuhe BGI Biotechnology Co., Ltd.

[0114] Table 1

[0115]

[0116]

[0117] (2) Optimization of primer probe sets

[0118] The different sets of primers and probes designed in step (1) (primer probe set 1 and primer probe set 2, respectively) were used to conduct experiments to screen out the optimal detection primers and probes (three parallels were set for each set).

[0119] The reaction systems are shown in Tables 2 and 3.

[0120] Table 2

[0121] Material Name concentration Addition amount 5×Buffer 5× 10μL dUTP mix 25μmol / L 0.4μL F-Primer 10μmol / L 1μL R1-Primer 10μmol / L 1μL P1 10μmol / L 0.5μL P2 10μmol / L 0.5μL Hot taq 5U / μL 0.3μL UNG enzyme 1U / μL 0.2μL Template DNA - 5μL <![CDATA[ddH2O]]> - Add water to make up to 45 μL

[0122] Table 3

[0123]

[0124]

[0125] The reaction cycle parameters of fluorescence PCR were: 50°C for 2 min; 95°C for 5 min; then entering the cycling stage: 95°C for 15 sec, 60°C for 45 sec (collecting fluorescence), and 40 cycles.

[0126] The detection results are shown in Figures 1-4. It can be seen from the figure that both primer probe set 1 and primer probe set 2 have good detection effects, but primer probe set 2 is better than primer probe set 1. Therefore, primer probe set 2 is selected for the subsequent rhPCR primer-probe design.

[0127] (3) Design of rhPCR primer probe set

[0128] Based on the traditional PCR principle, rhPCR uses a closed and cleavable rhPCR primer. The rhPCR primer does not have the ability to extend at the beginning of the reaction. It can only be digested by ribonuclease (RNase) H when it is complementary to the target DNA base sequence, thereby gaining the ability to extend and start the amplification reaction. rhPCR can effectively solve the problems of primer dimer formation and off-target amplification in PCR and improve the specificity of the reaction.

[0129] Primer probe set 2 was used to design the rhPCR primer probe set, including adding C3 Spacer modification to the 3' end of the forward primer and the reverse primer, respectively, adding a "g GAGC" sequence to the 5' end of the forward primer, wherein g is an RNA base; adding a "gCTTC" sequence to the 5' end of the reverse primer R3, and adding an "aCTTC" sequence to the 5' end of the reverse primer R4, wherein "g" and "a" are RNA bases.

[0130] (4) Choice between conventional PCR and rhPCR

[0131] APOE genotypes were detected by conventional PCR (primer probe set 2) and rhPCR (rhPCR primer probe set), respectively. The samples used were E2 / E2 and E4 / E4 positive specimens (positive plasmid concentration was 1×10 3 / mL).

[0132] The system used for ordinary PCR detection is shown in Table 2-3. The reaction program used is: 50℃2min; 95℃5min; then enter the cycle stage: 95℃15sec, 60℃45sec (collecting fluorescence), 40 cycles.

[0133] The system used for rhPCR detection is shown in Table 4-5, wherein 5×Buffer includes components with the following final concentrations: 250mM KCl, Tris.HCl 50mM, TX-100 0.5%, MgSO4 15mM; the reaction procedure is shown in Table 6.

[0134] Table 4

[0135]

[0136]

[0137] Table 5

[0138] Material Name concentration Addition amount 5×Buffer 5× 10μL dNTP mix 25μmol / L 0.4μL F-Primer 10μmol / L 1μL R2-Primer 10μmol / L 1μL P5 10μmol / L 0.5μL P6 10μmol / L 0.5μL Hot taq 5U / μL 0.3μL RNase HⅡ 5U / μL 0.2μL Template DNA - 5μL <![CDATA[ddH2O]]> - Add water to make up to 45 μL

[0139] Table 6

[0140]

[0141] Test results such as Figure 5-8 As shown in the figure, it can be seen that the fluorescence curve of ordinary PCR will be slightly upward and the specificity is poor. The use of rhPCR can improve the specificity and avoid the occurrence of false positives.

[0142] Example 2 A kit for detecting the ApoE gene

[0143] In this example, a kit for detecting the ApoE gene was prepared, the components of which included the rhPCR primer probe set prepared in Example 1 (the sequences are shown in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively), 5×Buffer, dNTP mix, Hot taq, RNase HⅡ and ddH2O.

[0144] The detection reaction system is shown in Table 4 and Table 5 in Example 1.

[0145] The detection cycle parameters are as shown in Table 6 in Example 1.

[0146] The sample detection and interpretation criteria are:

[0147] Positive control: Ct value of FAM and HEX / VIC channels ≤ 38, and the amplification curve has an obvious exponential growth period;

[0148] Negative control: no amplification in each channel, Ct>38 or no Ct value;

[0149] If the above conditions are not met, the experiment will be deemed invalid and the instruments, reagents, amplification conditions, experimental operations, etc. should be checked.

[0150] Sample validity determination:

[0151] Internal standard gene: In all sample tests, the Ct value of the CY5 channel is ≤38, and the amplification curve has an obvious exponential growth period. If this condition is not met, the sample is considered an invalid sample.

[0152] The genotype determination method when the results are valid is shown in Table 7 below.

[0153] Table 7

[0154]

[0155] The ApoE genotype results are interpreted as:

[0156] E3 / E3 genotype: If the second fluorescent sequence of amplification reaction system 1 detects Ct≤38 and a typical S-shaped fluorescence curve appears, the sample is of E3 / E3 genotype;

[0157] E2 / E2 genotype: If the second fluorescent sequence of amplification reaction system 2 detects Ct≤38 and a typical S-shaped fluorescence curve appears, the sample is of E2 / E2 genotype;

[0158] E4 / E4 genotype: If the first fluorescent sequence of amplification reaction system 1 detects Ct≤38 and a typical S-shaped fluorescence curve appears, the sample is of E4 / E4 genotype;

[0159] E3 / E4 genotype: If the first fluorescent sequence of amplification reaction system 1 detects Ct≤38, and the second fluorescent sequence of amplification reaction system 1 also detects Ct≤38, and a typical S-shaped fluorescence curve appears, the sample is of E3 / E4 genotype;

[0160] E2 / E3 genotype: If the second fluorescent sequence of amplification reaction system 1 detects Ct≤38, and the second fluorescent sequence of amplification reaction system 2 also detects Ct≤38, and a typical S-shaped fluorescence curve appears, the sample is of E2 / E3 genotype;

[0161] E2 / E4 genotype: If the first fluorescent sequence of amplification reaction system 1 detects Ct≤38, the second fluorescent sequence of amplification reaction system 1 detects Ct≤38, the first fluorescent sequence of amplification reaction system 2 detects Ct≤38, and the second fluorescent sequence of amplification reaction system 2 detects Ct≤38, and a typical S-shaped fluorescence curve appears, the sample is of E2 / E4 genotype.

[0162] Example 3 Accuracy Verification

[0163] In this example, the ApoE gene kit in Example 2 was used to detect the ApoE genotype. The sample used was plasma with a known genotype through sequencing (whole blood genomic DNA was obtained using a DNA extraction kit purchased from Shanghai Shenggong Biotechnology Co., Ltd.), and the reaction system and detection parameters were the same as in Example 2.

[0164] The results are as follows Figure 9-20 As shown in the figure, it can be seen that the scheme of the present invention can accurately detect different ApoE genotypes.

[0165] Example 4 Sensitivity Test

[0166] In this example, the ApoE gene kit in Example 2 was used to detect the ApoE genotype. The samples used were E3 / E3, E2 / E2, E4 / E4, E3 / E4, E2 / E3, and E2 / E4 positive specimens (1×10 7 Positive samples were diluted to 1×10 / mL with normal saline. 2 / mL as a sensitivity test reference) by sequencing plasma with known genotype (whole blood genomic DNA was obtained using a DNA extraction kit purchased from Shanghai Shenggong Biotechnology Co., Ltd.), the reaction system and detection parameters were consistent with Example 2.

[0167] The results are as follows Figure 21-31 As shown in the figure, it can be seen that the ApoE gene kit of the present invention can effectively detect 1×10 2 The sensitivity reference positive sample was 0.1 / mL, and the detection limit was low.

[0168] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A reagent for detecting ApoE gene, characterized in that: The reagent includes a primer set, which includes a forward common primer Frh for detecting ApoE gene site 112 and ApoE gene site 158, a reverse primer Rrh1 for detecting ApoE gene site 158, and a reaction primer Rrh2; the nucleotide sequence of the primer set is as follows: Frh: C3 Spacer –GGGCACGGCTGTCCAAgGAGC; Rrh1:C3 Spacer –CTGGTACACTGCCAGGCgCTTC; Rrh2:C3 Spacer –CTGGTACACTGCCAGGCaCTTC; Among them, "g" and "a" are RNA bases; The reagents also include RNase HII enzyme.

2. The reagent according to claim 1, characterized in that The reagent also includes a probe sequence for detecting site 112 of the ApoE gene.

3. The reagent according to claim 2, characterized in that The probe sequence includes a first fluorescent probe sequence and a second fluorescent probe sequence; the first fluorescent probe sequence is shown in SEQ ID NO:17, and the second fluorescent probe sequence is shown in SEQ ID NO:

18.

4. The reagent according to claim 3, characterized in that The 5' end of the first fluorescent probe sequence is labeled with a first fluorescent group, and the 5' end of the second probe sequence is labeled with a second fluorescent group, and the first fluorescent group and the second fluorescent group are different.

5. The reagent according to claim 4, characterized in that The fluorescent group includes one of FAM, VIC, CY5 and JOE.

6. A kit, characterized in that: The kit comprises the reagent according to any one of claims 1 to 5.

7. The kit according to claim 6, characterized in that The kit also includes a buffer solution, Hot taq enzyme, UNG enzyme and ribonuclease.

8. Use of the reagent according to any one of claims 1 to 5 or the kit according to claim 6 or 7 in any one of the following (1) to (4): (1) Application in the preparation of products for detecting ApoE gene; (2) Application in the preparation of ApoE genotyping products; (3) Application in the preparation of products for detecting Alzheimer's disease; (4) Application in the preparation of products for detecting cardiovascular and cerebrovascular diseases.

9. A method for detecting ApoE gene, characterized in that: The method comprises the following steps: using the reagent described in any one of claims 1 to 5 or the kit described in claim 6 or 7 to detect the DNA of the sample to be tested; the method is not intended for the diagnosis or treatment of a disease.

10. The detection method according to claim 9, characterized in that: The detection is performed using fluorescent quantitative PCR.

11. The detection method according to claim 10, characterized in that: The reaction system used in the fluorescent quantitative PCR includes an amplification reaction system 1 and an amplification reaction system 2; The amplification reaction system 1 is: 5×Buffer1×; dNTP mix 0.2-0.44 μM; Forward common primer Frh 0.1-0.4 μM; Reverse primer Rrh10.1-0.4 μM; Probe 0.1-0.4 μM; Hot taq30-40U / L; RNase HⅡ20-30U / L; 3~6µL of sample to be tested; Add ddH2O to 45 μL; The amplification reaction system 2 is: 5×Buffer1×; dNTP mix 0.2-0.44 μM; Forward common primer Frh 0.1-0.4 μM; Reverse primer Rrh20.1-0.4 μM; Probe 0.1-0.4 μM; Hot taq30-40U / L; RNase HⅡ20-30U / L; 3~6µL of sample to be tested; Add ddH2O to make up to 45 μL.

12. The detection method according to claim 10, characterized in that: The amplification program of the fluorescent quantitative PCR is: 50°C for 2 min; 95°C for 5 min; then enter the cycling stage: 94°C for 15 s, 60°C for 45 min, 40 cycles, and collect fluorescence data.

13. The detection method according to claim 9, characterized in that: After the detection, the step of statistically analyzing the Ct value of the sample and interpreting the result is also included; the result interpretation is: Positive: If the Ct of the sample is ≤38 and a typical S-shaped fluorescence curve appears, the sample is positive; Negative: If the sample has no fluorescence amplification or Ct>38, the sample is negative.

14. A method for detecting ApoE genotype, characterized in that: The method comprises the following steps: using the reagent described in any one of claims 1 to 5 or the kit described in claim 6 or 7 to detect the DNA of the sample to be tested; the method is not intended for the diagnosis or treatment of a disease.

15. The detection method according to claim 14, characterized in that: The detection is performed using fluorescent quantitative PCR.

16. The detection method according to claim 15, characterized in that: The reaction system used in the fluorescent quantitative PCR includes an amplification reaction system 1 and an amplification reaction system 2; The amplification reaction system 1 is: 5×Buffer1×; dNTP mix 0.2-0.44 μM; Forward common primer Frh 0.1-0.4 μM; Reverse primer Rrh10.1-0.4 μM; Probe 0.1-0.4 μM; Hot taq30-40U / L; RNase HⅡ20-30U / L; 3~6µL of sample to be tested; Add ddH2O to 45 μL; The amplification reaction system 2 is: 5×Buffer1×; dNTP mix 0.2-0.44 μM; Forward common primer Frh 0.1-0.4 μM; Reverse primer Rrh20.1-0.4 μM; Probe 0.1-0.4 μM; Hot taq30-40U / L; RNase HⅡ20-30U / L; 3~6µL of sample to be tested; Add ddH2O to make up to 45 μL.

17. The detection method according to claim 15, characterized in that: The amplification program of the fluorescent quantitative PCR is: 50°C for 2 min; 95°C for 5 min; then enter the cycling stage: 94°C for 15 s, 60°C for 45 min, 40 cycles, and collect fluorescence data.

18. The detection method according to claim 14, characterized in that: After the detection, the step of statistically analyzing the Ct value of the sample and interpreting the result is also included; the result interpretation is: E3 / E3 genotype: If the second fluorescent sequence of amplification reaction system 1 detects Ct≤38 and a typical S-shaped fluorescence curve appears, the sample is of E3 / E3 genotype; E2 / E2 genotype: If the second fluorescent sequence of amplification reaction system 2 detects Ct≤38 and a typical S-shaped fluorescence curve appears, the sample is of E2 / E2 genotype; E4 / E4 genotype: If the first fluorescent sequence of amplification reaction system 1 detects Ct≤38 and a typical S-shaped fluorescence curve appears, the sample is of E4 / E4 genotype; E3 / E4 genotype: If the first fluorescent sequence of amplification reaction system 1 detects Ct≤38, and the second fluorescent sequence of amplification reaction system 1 also detects Ct≤38, and a typical S-shaped fluorescence curve appears, the sample is of E3 / E4 genotype; E2 / E3 genotype: If the second fluorescent sequence of amplification reaction system 1 detects Ct≤38, and the second fluorescent sequence of amplification reaction system 2 also detects Ct≤38, and a typical S-shaped fluorescence curve appears, the sample is of E2 / E3 genotype; E2 / E4 genotype: If the first fluorescent sequence of amplification reaction system 1 detects Ct≤38, the second fluorescent sequence of amplification reaction system 1 detects Ct≤38, the first fluorescent sequence of amplification reaction system 2 detects Ct≤38, and the second fluorescent sequence of amplification reaction system 2 detects Ct≤38, and a typical S-shaped fluorescence curve appears, the sample is of E2 / E4 genotype.

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