CN112501281A - Human APOE genotyping detection primer set, detection method and detection kit - Google Patents
Human APOE genotyping detection primer set, detection method and detection kit Download PDFInfo
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Abstract
The invention discloses a human APOE gene typing detection primer group, a detection method and a detection kit, wherein the invention utilizes base mutation types of rs429358(T > C) site and rs7412(C > T) site of APOE gene to design amplification primers in a differentiation manner, and realizes direct interpretation of epsilon 2, epsilon 3, epsilon 4 and typing combination thereof. The detection kit comprises a specific primer group for detecting the APOE genotyping epsilon 2, epsilon 3 and epsilon 4, can be used for typing and judging by using a common fluorescent PCR instrument, has accurate detection result and high sensitivity, and can accurately type genomic DNA samples as low as 1 ng. The problems of high price of detection instruments, complex operation, easy pollution, high detection cost and the like of the traditional detection technology and the finished product detection kit are solved.
Description
Technical Field
The invention relates to gene locus detection, in particular to a detection primer group, a detection method and a detection kit for detecting human APOE genotyping.
Background
Apolipoprotein E (APOE) is a glycoprotein that plays an important role in lipoprotein metabolism, and APOE participates in lipid metabolism of the liver as a ligand of lipoprotein. The apolipoprotein E4 isomer has high affinity with LDL (low-density lipoprotein) receptors, VLDL (very low-density lipoprotein) containing apolipoprotein E4 and residues thereof are combined with LDL receptors to increase VLDL (very low-density lipoprotein), liver uptake is increased, and LDL and total cholesterol are increased. The APOE gene is closely related to various cardiovascular and cerebrovascular diseases and neurodegenerative diseases (such as Alzheimer disease).
APOE genotype is mainly determined by base changes at 2 SNP sites, rs429358 (chromosome position 44,908,684) and rs7412 (chromosome position 44,908,822). Different genotypes are formed according to the difference of the bases of the two sites. The APOE gene has 3 alleles, i.e., epsilon 2, epsilon 3, and epsilon 4, forming 6 different genotypes: 3 homozygotes (. epsilon.2/. epsilon.2,. epsilon.3/. epsilon.3,. epsilon.4/. epsilon.4) and 3 heterozygotes (. epsilon.2/. epsilon.3,. epsilon.2/. epsilon.4,. epsilon.3/. epsilon.4).
APOE has been reported to be involved in lipid metabolism regulation and is widely expressed in astrocytes, microglia and vascular wall cells as well as choroid plexus cells in the central nervous system. There are three allelic variants of APOE, APOE ∈ 2, APOE ∈ 3 and APOE ∈ 4, respectively. The research shows that APOE is the main risk factor of delayed Alzheimer's Disease (AD) after 65 years old, wherein APOE epsilon 2 has a protective effect on AD, and APOE epsilon 4 can enhance the development risk of AD. The proportion of APOE genotypes carried also affects the probability of AD, for example the risk of AD carrying one APOE 4 is increased by a factor of 3-4, while the risk of AD carrying two APOE 4 is increased by a factor of 9-15. Numerous studies have found that APOE not only regulates the expression levels of brain β -starch plaques, tau protein and TDP43 protein in AD patients, but also affects normal brain function.
In addition, epsilon 2 is a long-life genotype and is sensitive to statins, so the lipid regulation of patients can be preferably performed by statins as lipid-lowering drugs. The epsilon 3 type population accounts for most of the population, has normal effect on lipid reduction of statins, and has no obvious tendency on the attack of cardiovascular and cerebrovascular diseases. The risk of the epsilon 4 type population for generating early cardiovascular disease and senile dementia is higher, the curative effect of the statin drugs is poorer than that of epsilon 2 type and epsilon 3 type populations when the blood lipid is abnormal, probucol can be selected for treatment, and the effect of reducing the blood lipid by adopting low-fat diet is obvious. People who take statins are mostly patients with cardiovascular diseases such as coronary heart disease, myocardial infarction and the like, and accurate medication can be guided to doctors within an effective treatment period through APOE genotype detection, so that the using effectiveness of the medicine is improved, and the toxic and side effects are reduced.
At present, common methods for detecting gene SNP sites include a direct sequencing method, a gene chip hybridization method, a PCR-Taqman MGB probe method, a PCR high-resolution melting curve method, an ARMS-PCR method and the like. The methods have the defects of high reagent detection cost, complex operation, easy pollution generation, certain false negative and false positive and the like in popularization.
The direct sequencing method is a gold standard for SNP locus analysis, but the detection method is limited by a sequencing instrument, and the sequencing instrument is expensive, difficult to popularize and long in period.
The gene chip method, there is a formed detection kit passing NMPA certification on the market at present, the "apolipoprotein E genotype detection kit (gene chip method)" of the Zhuhai seille-Leqi biotechnology Limited company, the kit adopts PCR-gene chip hybridization method to detect, and the APOE genotype is distinguished by hybridizing the PC amplification segment and the chip specific probe. The method has the disadvantages of complex operation, complex chip manufacturing, high cost, 3-4 hours of whole detection period and low efficiency.
A PCR-Taqman MGB probe typing method has a NMPA-certified forming kit at present, such as 'human SLCO1B1 and APOE gene polymorphism detection kit' of Wuhan Yongzhi, but two Taqman-MGB probes are needed, the price and the cost are high, and the MGB probe patent belongs to ABI company.
The SNP typing detection technology of the PCR high resolution dissolution curve can detect the mutation situation of genes with high flux, the cost of the reagent is lower, but because the SNP site is the variation of only one basic group, the detection instrument is generally more precise than the common fluorescence PCR instrument, the requirement on temperature sensitivity is higher, for the variation of different basic groups, the dissolution temperature may only be 0.2 ℃ different, and the differentiation is difficult due to the imprecise instrument, so the influence of the instrument is large, and the popularization is limited.
ARMS-PCR allele-specific amplification, also known as amplification-hindered mutation system, is used to detect known mutant genes. The method is characterized in that 2 5 ' end primers are involved, one is complementary with normal DNA and the other is complementary with mutant DNA, when two primers and a 3 ' end primer are respectively added to carry out two parallel PCR, only the primer which is completely complementary with the mutant DNA can be extended and amplified, and the PCR can not be carried out because of mismatching at the 3 ' end of the primer. The method is electrophoresis after PCR reaction, increases the chance of PCR pollution, and is time-consuming and labor-consuming.
The multiplex qPCR is not limited to conventional multicolor experiments, and can also perform multiplex analysis by Tm of the hybridization product to achieve simultaneous detection of multiple targets. However, a plurality of primer probes are required, and the reagent cost is high; it is also the most likely problem whether several reactions have mutual influence and interference. If very low abundance targets and higher targets are detected simultaneously, it is likely that missed detection of low abundance targets will result, especially when their amplification is inefficient.
Therefore, how to solve the problems existing in the process of detecting APOE genotyping and implement a detection method which is simple and easy to interpret and analyze, simple in experimental operation, high in quality and low in price and has no special instrument requirements become problems to be solved urgently.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a detection primer group, a detection method and a detection kit for human APOE genotyping, which can rapidly distinguish between the typing of the APOE genes epsilon 2, epsilon 3 and epsilon 4.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a human APOE genotyping detection primer set is used for respectively and specifically amplifying and identifying APOE genotyping epsilon 2, epsilon 3 and epsilon 4, and a specific upstream primer aiming at epsilon 2 genotyping is shown as SEQ NO.1 and a specific downstream primer SEQ NO. 2; the specific upstream primer aiming at epsilon 3 typing is shown as SEQ NO.3 and the specific downstream primer SEQ NO. 4; the specific upstream primer of epsilon 4 typing is shown as SEQ NO.5 and the specific downstream primer SEQINO. 6.
Another objective of the invention is to provide a human APOE genotyping assay, which comprises the following steps:
(1) aiming at rs429358(T > C) site and rs7412(C > T) site of APOE gene, respectively designing amplification primers in differentiation mode by utilizing site base mutation types, namely respectively designing epsilon 2, epsilon 3 and epsilon 4 specific upstream and downstream primers for specific amplification such as 3 types of typing;
(2) according to the base complementary principle, the amplification specificity and the base preference, respectively amplifying epsilon 2, epsilon 3 and epsilon 4 in a PCR reaction system with 3 pairs of primers and fluorescent dye, and converting a fluorescence acquisition signal into visual data;
(3) and collecting fluorescence signals to show that the 3 types of amplified ct values are regularly different from the typing combination for accurate typing interpretation.
The invention also aims to provide a human APOE genotyping detection kit, which comprises the specific primer pair, wherein the specific primer pair is an upstream primer pair and a downstream primer pair which are respectively designed for 1 pair of epsilon 2, epsilon 3 and epsilon 4 aiming at rs429358(T > C) site and rs7412(C > T) site of the APOE gene, and the total number is 3.
Furthermore, the human APOE genotyping detection kit also comprises PCR amplification reaction liquid, epsilon 2, epsilon 3 and epsilon 4 typing specific primer pair mixed liquid, reference positive control liquid and negative control liquid.
Preferably, the final concentration of the specific primer is 0.5 uM.
Preferably, the components of the PCR amplification reaction solution comprise Taq enzyme, buffer, dNTP, 20 SYBR Green I, ROX and sterile water.
The invention has the beneficial effects that:
aiming at the 464bp distance between the rs429358 (chromosome position 44,908,684) and the rs7412 (chromosome position 44,908,822) of the APOE gene, the invention designs amplification primers in a differentiation manner by utilizing the base mutation types of the rs429358(T > C) site and the rs7412(C > T) site, and realizes the direct interpretation of epsilon 2(rs429358-T and rs7412-T), epsilon 3(rs429358-T and rs7412-C), epsilon 4(rs429358-C and rs7412-C) and the typing combination thereof. The common fluorescent PCR instrument can be used for typing and interpretation, the detection result is accurate, the sensitivity is high, and the typing of a genome DNA sample can be reduced to 1 ng. The method solves the problems that the traditional detection technology cannot be directly typed and interpreted, and the finished product detection kit has high price of detection instruments, complex operation, easy pollution, high detection cost and the like.
Drawings
Fig. 1 is a schematic diagram of the design of the present invention.
FIG. 2 is a diagram showing the results of detection of the homozygous type ε 2/ε 2 in the APOE genotyping of the present invention.
FIG. 3 is a diagram showing the results of detection of the homozygous type ε 3/ε 3 of APOE genotyping in accordance with the present invention.
FIG. 4 is a diagram showing the results of detection of the homozygous type ε 4/ε 4 of APOE genotyping in accordance with the present invention.
FIG. 5 is a diagram showing the results of detection of APOE genotyping ε 2/ε 3 heterozygotes according to an embodiment of the present invention.
FIG. 6 is a diagram showing the results of detection of APOE genotyping ε 2/ε 4 heterozygotes according to the present invention.
FIG. 7 is a diagram showing the results of detection of APOE genotyping ε 3/ε 4 heterozygotes according to the present invention.
Detailed Description
The present invention is described in detail below with reference to examples, which are intended to be illustrative only and are not to be construed as limiting the scope of the invention, and any methods and materials similar or equivalent to those described herein can be used in the present invention.
Example 1:
first, primer design
Human APOE genotype is mainly determined by base changes at 2 SNP sites, rs429358 (chromosomal position 44,908,684) and rs7412 (chromosomal position 44,908,822). Different genotypes are formed according to the difference of the bases of the two sites. There are 3 allelic genotypes of the APOE gene, i.e., ε 2(rs429358-T and rs7412-T), ε 3(rs429358-T and rs7412-C), and ε 4(rs429358-C and rs 7412-C). The inventor designs amplification primers differentially by using site base mutation types aiming at rs429358(T > C) site and rs7412(C > T) site of human APOE gene, namely designing epsilon 2, epsilon 3 and epsilon 4 specific upstream and downstream primers respectively for specific amplification, such as 3 types of typing. The primers were analyzed using Primer Premier5 software and custom synthesized by Biotechnology engineering (Shanghai) Inc.
The sequence of the specific upstream primer aiming at epsilon 2 typing is shown as SEQ NO.1 and the sequence of the specific downstream primer is shown as SEQ NO. 2; the sequence of the specific upstream primer aiming at epsilon 3 typing is shown as SEQ NO.3 and the sequence of the specific downstream primer is shown as SEQ NO. 4; the sequence of the specific upstream primer aiming at epsilon 4 typing is shown as SEQ NO.5 and the sequence of the specific downstream primer is shown as SEQ NO. 6.
The sequences of the primer group are shown in the following table I:
II, composition of kit
The kit for detecting human APOE genotyping comprises 8 tubes of PCR amplification reaction liquid, epsilon 2 primer pair mixed liquid, epsilon 3 primer pair mixed liquid, epsilon 4 primer pair mixed liquid, epsilon 2 reference positive plasmid control liquid, epsilon 3 reference positive plasmid control liquid, epsilon 4 reference positive plasmid control liquid and negative control liquid.
The PCR amplification reaction solution comprises Taq enzyme, buffer, dNTP, 20 SYBR Green I, ROX and sterile water.
Third, using method of kit
The specific detection steps of the kit for detecting human APOE genotyping in the embodiment are as follows:
DNA extraction
The specific operation of extracting DNA by using sample types such as blood or oral swabs and the like is referred to the specification of a DNA kit extraction product.
DNA quality detection
After DNA extraction, DNA quality was controlled by determining the concentration of nucleic acid DNA and the ratio of OD260/280, preferably between 1.8 and 2.0, and optimal reaction results were obtained. The gel electrophoresis imaging can be further carried out to check whether the genome DNA has degradation or not, and the main band should be not less than 1 Kb.
PCR reaction
1) Preparing a 15ul PCR reaction system:
calculate the PCR reaction tubes required for the number of samples (PCR reaction tube 3+3 reference positive +3 negative control), i.e. 9 tubes for 1 sample and 36 tubes for 10 samples. Taking an epsilon 2 typing amplification system as an example, the typing reaction system is 15 ul: wherein 7.5ul of PCR amplification reaction solution, 1.5ul of ε 2 primer pair mixture, 1ul of sample, and the rest of water were supplemented to 15ul of total system. In the same manner, an epsilon 3 typing amplification system and an epsilon 4 typing amplification system were prepared. The PCR tube was capped and labeled according to known templates.
2) Performing a PCR reaction
The PCR amplification procedure is shown in table two below:
watch two
The PCR reaction is carried out on a QuantStaudio 6 ABI Q6, ABI7500 and Agilent MX3000P fluorescent quantitative PCR instrument.
4. Analysis and interpretation of results
1) Judging the effectiveness of the kit according to 3 tubes of reference positive control and negative control; wherein 3 tubes of positive control are adopted, and the amplification curve is good in amplification and has no abnormality; the detection result of the negative control should have no specific amplification, otherwise, pollution may exist, and the detection result of the sample is unreliable.
2) Determination of genotyping of kits
Collecting fluorescence signals to show that the amplified ct values of 3 types of typing are regularly different from the typing combination, and determining a curve 1 (epsilon 2) appearing first and a curve 2 (epsilon 3) and a curve 3 (epsilon 4) appearing after the curve 1 in sequence; when the curve 2 and the curve 1 are delta CT <1, 1/2 heterozygote (epsilon 2/epsilon 3) is formed; when curve 2 and curve 3 and curve 1. DELTA. CT > 1, 1/1 homozygotes (. epsilon.2/. epsilon.2) are obtained.
The results of the sample testing are shown in detail in FIGS. 2-7.
It should be understood that the above examples are only for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. It will be apparent to those skilled in the art that other variations and modifications may be made in the invention without departing from the spirit or scope of the invention as defined in the following claims. However, obvious variations or modifications which fall within the spirit of the invention are intended to be covered by the scope of the invention.
Sequence listing
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Claims (6)
1. A human APOE genotyping detection primer set is characterized in that the primer set is used for respectively and specifically amplifying and identifying APOE genotyping epsilon 2, epsilon 3 and epsilon 4, and a specific upstream primer aiming at epsilon 2 genotyping is shown as SEQ NO.1 and a specific downstream primer SEQ NO. 2; the specific upstream primer aiming at epsilon 3 typing is shown as SEQ NO.3 and the specific downstream primer SEQ NO. 4; the specific upstream primer of epsilon 4 typing is shown as SEQ NO.5 and the specific downstream primer SEQINO. 6.
2. A human APOE genotyping detection method is characterized by comprising the following steps:
(1) aiming at rs429358(T > C) site and rs7412(C > T) site of APOE gene, respectively designing amplification primers in differentiation mode by utilizing site base mutation types, namely respectively designing epsilon 2, epsilon 3 and epsilon 4 specific upstream and downstream primers for specific amplification such as 3 types of typing;
(2) according to the base complementary principle, the amplification specificity and the base preference, respectively amplifying epsilon 2, epsilon 3 and epsilon 4 in a PCR reaction system with 3 pairs of primers and fluorescent dye, and converting a fluorescence acquisition signal into visual data;
(3) and collecting fluorescence signals to show that the 3 types of amplified ct values are regularly different from the typing combination for accurate typing interpretation.
3. The human APOE genotyping detection kit is characterized by comprising the specific primer pair as claimed in claim 1, wherein the specific primer pair is an rs429358(T > C) site and an rs7412(C > T) site aiming at the APOE gene, and 1 pair of upstream and downstream primer pairs aiming at epsilon 2, epsilon 3 and epsilon 4 are respectively designed, and 3 pairs are calculated.
4. The human APOE genotyping detection kit of claim 3, wherein the detection kit further comprises PCR amplification reaction solution, a mixture of epsilon 2, epsilon 3 and epsilon 4 typing specific primer pairs, reference positive control solution and negative control solution.
5. The human APOE genotyping detection kit of claim 4, wherein the final concentration of the specific primers is 0.5 uM.
6. The human APOE genotyping detection kit of claim 4, wherein the PCR amplification reaction comprises Taq enzyme, buffer, dNTP, 20 SYBR Green I, ROX, sterile water.
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