CN116970687A - Fluorescent quantitative PCR method apolipoprotein E genotyping kit - Google Patents
Fluorescent quantitative PCR method apolipoprotein E genotyping kit Download PDFInfo
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- 238000003205 genotyping method Methods 0.000 title claims abstract description 15
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Abstract
The invention relates to the technical field of biological products, and discloses a fluorescent quantitative PCR method apolipoprotein E genotyping kit, which comprises the following steps: step one: the main components of the kit comprise a primer, a probe curing reaction tube and a 2 XPCR mixed solution (dNTPs, reaction enzymes, additives and the like are prepared into the 2 XPCR reaction solution together for facilitating future use, and when the kit is used, the kit can be put on a machine for operation by only mixing the same amount of purified water containing template DNA with the 2 XPCR reaction solution). The invention has simple operation and cost saving; the quality of the products of the small-scale production three batches of samples (batch number: 20120301, 20120302, 20120303) and the large-scale production three batches of samples (batch number: 20120601, 20120602, 20120603) manufactured by the production process can meet the performance requirements of the kit, the sensitivity, the specificity, the precision and the linear range meet the requirements, and the result shows that the product quality is stable and the production process is feasible.
Description
Technical Field
The invention relates to the technical field of biological products, in particular to a fluorescent quantitative PCR method apolipoprotein E genotyping kit.
Background
Human apolipoprotein E (ApoE) genes are located at 19q13.2, are about 3.6Kb in length, comprise 4 exons and 3 introns, share 1833 Single Nucleotide Polymorphism (SNP) sites in the ApoE gene region, and are 438 in the coding region, wherein rs429358 and rs7412 are the most frequent in the population, are widely and deeply studied, and have the greatest clinical value and significance.
The competitive allele-specific PCR (KompetitiveAlleleSpecificPCR, KASP) typing method is a method based on a double fluorescent marker and allele-specific primers, wherein the specific primers are designed for SNP sites and double fluorescent markers are added to type mutants and wild types.
The kit is used for establishing a fluorescent quantitative PCR method apolipoprotein E genotyping method. The method is simple and convenient to operate, low in detection cost and price, high in detection sensitivity, suitable for high-flux sample testing, and high in accuracy.
Disclosure of Invention
The invention aims to provide a fluorescent quantitative PCR method apolipoprotein E genotyping kit, which solves the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions: a fluorescent quantitative PCR method apolipoprotein E genotyping kit comprises the following steps:
step one: the main components of the kit comprise a primer, a probe curing reaction tube and a 2 XPCR mixed solution (dNTPs, reaction enzymes, additives and the like are prepared into the 2 XPCR reaction solution together for facilitating the use of future users, and when the kit is used, the kit can be put on a machine for operation by only mixing the same amount of purified water containing template DNA with the 2 XPCR reaction solution);
step two: the raw materials used are all qualified products which are subjected to self-inspection, and working solutions comprising primers, probes, dNTPs, taq enzyme, UNG enzyme, tri-HCl, (NH 4) 2SO4, mgCl2, tween-20 and DMSO solution are prepared respectively;
step three: after being checked to be qualified, the product is put into the next production; solidifying the primer and the probe in a PCR reaction tube, and directly completing the split charging of the primer and the probe; mixing dNTPs, taq enzyme, UNG enzyme, tri-HCl, (NH 4) 2SO4, mgCl2, tween-20 and DMSO solution to obtain 2×PCR reaction solution;
step four: and after the semi-finished product is inspected to be qualified, subpackaging the 2 XPCR reaction liquid, packaging the 2 XPCR reaction liquid and the primer probe curing reaction tube according to the composition of the kit and 1 person/box, and warehousing for storage after the inspection is qualified.
As a preferred embodiment of the invention, the kit combines the Taqman probe technology and the allele specific primer amplification technology, adopts a fluorescent quantitative PCR method to detect two single nucleotide polymorphism sites (388T > C,526C > T) on the apolipoprotein E gene, and identifies the apolipoprotein E genotype according to the detection result.
As a preferred implementation mode of the invention, the kit is added with a plurality of Taqman fluorescent probes in a reaction system, thereby realizing the purpose of real-time monitoring of the synchronous accumulation of fluorescent signals and the formation of PCR products; the DNA polymerase is Taq enzyme; to prevent contamination of the PCR products, dTTP in dNTPs was replaced with dUTP and UNG enzyme was added; in addition to the pH value-adjusted Tri-HCl, proper metal ions (NH 4) 2SO4 and MgCl2, tween-20 and DMSO are added into the reaction buffer solution to increase the reaction efficiency of PCR.
As a preferred embodiment of the present invention, the reaction conditions of the kit are: the possibility of contamination of the PCR product was removed at 50℃for 5 minutes, pre-denaturation at 95℃for 2 minutes, 20 cycles at 95℃for 5 seconds, 60℃for 1 second, and 72℃for 30 seconds, followed by 30 cycles under the following conditions: the analysis results were concluded at 95℃for 5 seconds, 60℃for 30 seconds. In the first 20-cycle reaction, after the conditions are repeatedly optimized, the annealing temperature is set to 60 ℃ and the time is shortened to 1 second, so that the reaction conditions are strictly controlled, under the conditions, the extension of the 3 '-end of the primer and the unmatched template base is more difficult than the extension of the annealing time, and the extension of the 3' -end of the primer and the matched template base is less influenced, thereby increasing the specificity of the reaction.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention has simple operation and cost saving; the quality of the products of the small-scale production three batches of samples (batch number: 20120301, 20120302, 20120303) and the large-scale production three batches of samples (batch number: 20120601, 20120602, 20120603) manufactured by the production process can meet the performance requirements of the kit, the sensitivity, the specificity, the precision and the linear range meet the requirements, and the result shows that the product quality is stable and the production process is feasible.
2. In the invention, dUTP is used for replacing dTTP in the PCR reaction, and T in amplified product fragments is completely replaced by U, so that PCR amplified products containing dU base are formed. The UNG enzyme can selectively break the glycosidic bond of U base in single-stranded and double-stranded DNA, degrade the DNA containing U in the reaction system, effectively eliminate the residual pollution of PCR products, greatly reduce the false positive caused by the pollution of amplified products, and further ensure the specificity and accuracy of amplification.
3. In the invention, after the first 20-cycle reaction is repeatedly optimized, the annealing temperature is set to 60 ℃ and the time is shortened to 1 second, so that the aim is to strictly control the reaction condition, under the condition, the extension of the 3 '-end of the primer and the unmatched template base is much more difficult than that of the annealing time, and the extension of the 3' -end of the primer and the matched template base is less influenced, thereby increasing the specificity of the reaction, and under the conditions of a specific primer probe reaction system and the reaction, the ARMS-PCR can realize full or no amplification and has ultrahigh accuracy.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1 is a schematic diagram of a process flow of a fluorescent quantitative PCR method apolipoprotein E genotyping kit of the present invention;
FIG. 2 is a schematic diagram showing the sequence of placing the reaction tube of the apolipoprotein E genotyping kit according to the fluorescent quantitative PCR method in a fluorescent quantitative PCR instrument for recording samples.
Detailed Description
Referring to fig. 1, the present invention provides a technical solution: a fluorescent quantitative PCR method apolipoprotein E genotyping kit;
the production process of the kit is provided with three quality control points, and products in each step can be continuously produced after being inspected to be qualified, wherein raw material working solution is prepared into key production procedures, primer probe working solution preparation, curing and split charging, other raw material working solution preparation, mixing and split charging are all completed in a hundred thousand-level clean area, and a main production process flow chart is shown in figure 1.
1. Preparation of three samples for pilot production
1.1 preparation and inspection of primer probe working solution
1.2 curing and sub-packaging the primer probe
1.3 preparation of working solution of dNTPs, reaction enzyme and additive and inspection
1.4 inspection of semifinished products
Preparation and split charging of 1.52 XPCR reaction solution
1.6 kit packaging
1.7 inspection of finished products
1.8 warehouse entry and preservation
2. Preparation of three batches of samples for mass production
2.1 preparation and inspection of primer probe working solution
2.2 curing and sub-packaging the primer probe
2.3 preparation and inspection of working solution of dNTPs, reactive enzymes and additives
2.4 inspection of semi-finished products
Preparation and split charging of 2.52 XPCR reaction solution
2.6 kit packaging
2.7 inspection of finished products
2.8 warehouse entry and preservation
The production process is simple to operate, and the cost is saved; the quality of the products of the small-scale production three batches of samples (batch number: 20120301, 20120302, 20120303) and the large-scale production three batches of samples (batch number: 20120601, 20120602, 20120603) manufactured by the production process can meet the performance requirements of the kit, the sensitivity, the specificity, the precision and the linear range meet the requirements, and the result shows that the product quality is stable and the production process is feasible.
TABLE 1 test results for finished products of pilot and Large production samples
2. Composition of reaction system and dosage of reagents
Five essential elements of the PCR (polymerase chain reaction ) reaction are: template DNA, primers, dNTPs (4 deoxynucleotides, oxygen-ribonucleoside triphosphate), DNA polymerase and suitable buffers (suitable pH, mg2+, etc.).
The kit combines the Taqman probe technology and the allele specific primer amplification technology, adopts a fluorescent quantitative PCR method to detect two single nucleotide polymorphism sites (388T > C,526C > T) on the apolipoprotein E gene, and identifies the apolipoprotein E genotype according to the detection result. The kit is added with a Taqman fluorescent probe in a reaction system, so that the aim of synchronous real-time monitoring of fluorescent signal accumulation and PCR product formation is fulfilled; the DNA polymerase is Taq enzyme; to prevent contamination of the PCR products, dTTP in dNTPs was replaced with dUTP and UNG enzyme was added; in addition to the pH value-adjusted Tri-HCl, proper metal ions (NH 4) 2SO4 and MgCl2, tween-20 and DMSO are added into the reaction buffer solution to increase the reaction efficiency of PCR. Finally, the composition of the reaction system of the kit is as follows:
TABLE 2 composition of reaction System of apolipoprotein E genotyping kit (fluorescent quantitative PCR method)
1. Primer(s)
2. Probe with a probe tip
3、dNTPs
4. Taq enzyme
5. UNG enzyme
dUTP is used for replacing dTTP in the PCR reaction, and T in the amplified product fragment is completely replaced by U, so that a PCR amplified product containing dU base is formed. The UNG enzyme can selectively break the glycosidic bond of U base in single-stranded and double-stranded DNA, degrade the DNA containing U in the reaction system, effectively eliminate the residual pollution of PCR products, greatly reduce the false positive caused by the pollution of amplified products, and further ensure the specificity and accuracy of amplification.
6、Tri-HCl
7、(NH 4 ) 2 SO 4
8、MgCl 2
9、Tween-20
10、DMSO
3. Sample requirement
4. Reaction conditions of the System
1. Test method
1.1 sample DNA extraction (sample treatment zone)
For specific steps, reference is made to the instructions for the DNA extraction kit used.
1.2 preparation and sample addition of amplification reagents (preparation area of PCR reaction solution)
The reaction components were removed from the kit, slowly thawed in an ice bath and mixed upside down. And taking a proper amount of 2 XPCR mixed solution, adding the same amount of water and the extracted DNA sample, enabling the concentration of the final PCR mixed solution to be 1×, fully and uniformly mixing, split charging into 4 connecting tubes containing the immobilized primer probes, enabling the final volume of the reaction of each tube to be 25 μl, covering a tube cover, and transferring to a PCR amplification region after instantaneous centrifugation.
2. Reaction conditions of the system
2.1 cycle condition set-up
2.2 instrument detection channel selection
The primer probe curing reaction tubes provided by the kit all contain a mixture of two fluorescent probes, namely an apolipoprotein E gene probe and an internal reference gene probe, so that the detection channels are set as follows:
the reference fluorescence is ROX, the collection temperature of the fluorescence signal is set to 62 ℃, the reaction system is 25 μl, and the specific setting method is referred to the corresponding instrument instruction.
2.3 detection type setting
When using a Eppendorf Mastercycler Realplex fluorescent quantitative PCR instrument, the detection type can be set before or after the amplification is started or after the amplification is finished, the template-free control is set as 'NTC', and the sample to be detected is set as 'Unknown'.
3. Interpretation of test results
3.1 quality control Standard
Taking Eppendorf Mastercycler Realplex as an example, a baseline (baseline) is set using the fluorescent signal of the first 3-15 cycles, and a threshold (threshold) is set based on the instrument automatically taking value or just above the highest point of the template-free control (NTC) amplification curve; when the minimum detection of 1ng of human genome DNA of the kit is achieved, namely the Cq value of the internal reference gene-GAPDH is between standards, the reaction can be normally carried out; otherwise, the test should be repeated.
3.2 judgment of results
3.2.1 detecting whether the reference gene in each reaction tube emits a VIC signal. If the internal reference genes all obtain positive results, continuing to analyze. If both the FAM response and the internal reference gene are not effective, the data must be discarded and the experiment repeated, as inhibitors or sample degradation may be present, leading to false negative test results.
3.2.2 Apolipoprotein E genes have 3 isoforms, apoE2, apoE3 and ApoE4 respectively, wherein E3 (112 cysteine-3838T, 158 arginine-526C) is wild-type and the base mutations encoding amino acids 112 and 158 of the protein produce two mutant genes E4 (112 arginine-3838C, 158 arginine-526C) and E2 (112 cysteine-3838C, 158 cysteine-526C) respectively. Genes on the human chromosome are present in pairs, so that the six genotypes of apolipoprotein E are E2/2, E2/3, E3/3, E4/2, E4/3 and E4/4, respectively.
The primer probe curing reaction tubes of the kit 1 to 4 detect T, C (388 base site) of amino acid number 112 and C, T (526 base site) of amino acid number 158 respectively. If 1 has a curve of increase T,2 is C,3 is C,4 is T, and the apolipoprotein E genotype is identified with reference to the following table:
3.2.3 if neither of the reaction tubes 1 and 2 had reacted, then the detection of a polymorphic site that was rare and located short upstream of 112 was performed again using reaction tubes 5 and 6, and if there was a curve growth of tube 5 as T and 6 as C, and the genotype was identified according to the above table.
4. The reaction condition of the system is determined according to
PCR consists of three basic reaction steps of denaturation, annealing and extension: (1) denaturation of template DNA: after the template DNA is heated to about 94 ℃ for a certain time, the double strand of the template DNA is dissociated into single strands so as to be combined with the primer, and preparation is made for the next round of reaction; (2) annealing (renaturation) of template DNA to primer: after the template DNA is denatured into single strands by heating, the temperature is reduced to about 55 ℃, and the primer is combined with the complementary sequence of the single strands of the template DNA in a pairing way; (3) extension of the primer: under the action of Taq DNA polymerase, the DNA template-primer conjugate uses dNTPs as reaction raw material and target sequence as template, and according to base pairing and semi-reserved replication principle, a new semi-reserved replication chain complementary with template DNA chain is synthesized, and the three processes of cyclic denaturation, annealing and extension are repeated so as to obtain more 'semi-reserved replication chains', and the new chain can become the template for next cycle.
The reaction conditions of the kit are as follows: the possibility of contamination of the PCR product was removed at 50℃for 5 minutes, pre-denaturation at 95℃for 2 minutes, 20 cycles at 95℃for 5 seconds, 60℃for 1 second, and 72℃for 30 seconds, followed by 30 cycles under the following conditions: the analysis results were concluded at 95℃for 5 seconds and 60℃for 30 seconds. In the first 20-cycle reaction, after the repeated optimization of conditions, the annealing temperature is set to 60 ℃ and the time is shortened to 1 second, so that the aim is to strictly control the reaction conditions, under the conditions, the extension of the 3 '-end of the primer and the unmatched template base is much more difficult than that of the annealing time, and the extension of the 3' -end of the primer and the matched template base is less influenced, thereby increasing the specificity of the reaction, and under the conditions of our specific primer probe reaction system and the reaction, the amplification of our ARMS-PCR can be realized completely or non-completely, and the method has ultrahigh accuracy.
5. Quality control method of system
When the minimum detection of 1ng of human genome DNA of the kit is achieved, namely the Cq value of the internal reference gene-GAPDH is between standards, the reaction can be normally carried out; otherwise, the test should be repeated.
6. Verification of main production process and reaction system
According to the determined production process, reaction system and reaction conditions, three small-scale production samples and three large-scale production samples are manufactured and tested, and the test results are as follows:
TABLE 6 test results for finished products of pilot and Large production samples
Test results show that the sensitivity, the specificity, the precision and the linear range meet the requirements, and the production process, the reaction system and the reaction conditions are stable, so that the kit is suitable for manufacturing and application.
Claims (4)
1. A fluorescent quantitative PCR method apolipoprotein E genotyping kit comprises the following steps:
step one: the main components of the kit comprise a primer, a probe curing reaction tube and a 2 XPCR mixed solution (dNTPs, reaction enzymes, additives and the like are prepared into the 2 XPCR reaction solution together for facilitating the use of future users, and when the kit is used, the kit can be put on a machine for operation by only mixing the same amount of purified water containing template DNA with the 2 XPCR reaction solution);
step two: the raw materials used are all qualified products which are subjected to self-inspection, and working solutions comprising primers, probes, dNTPs, taq enzyme, UNG enzyme, tri-HCl, (NH 4) 2SO4, mgCl2, tween-20 and DMSO solution are prepared respectively;
step three: after being checked to be qualified, the product is put into the next production; solidifying the primer and the probe in a PCR reaction tube, and directly completing the split charging of the primer and the probe; mixing dNTPs, taq enzyme, UNG enzyme, tri-HCl, (NH 4) 2SO4, mgCl2, tween-20 and DMSO solution to obtain 2×PCR reaction solution;
step four: and after the semi-finished product is inspected to be qualified, subpackaging the 2 XPCR reaction liquid, packaging the 2 XPCR reaction liquid and the primer probe curing reaction tube according to the composition of the kit and 1 person/box, and warehousing for storage after the inspection is qualified.
2. A fluorescent quantitative PCR method apolipoprotein E genotyping kit is characterized in that the kit combines a Taqman probe technology and an allele specific primer amplification technology, and adopts a fluorescent quantitative PCR method to detect two single nucleotide polymorphism sites (388T > C,526C > T) on an apolipoprotein E gene and identify the apolipoprotein E genotype according to the detection.
3. A fluorescent quantitative PCR method apolipoprotein E genotyping kit is characterized in that a Taqman fluorescent probe is added into a reaction system of the kit, so that the aim of synchronous real-time monitoring of fluorescent signal accumulation and PCR product formation is fulfilled; the DNA polymerase is Taq enzyme; to prevent contamination of the PCR products, dTTP in dNTPs was replaced with dUTP and UNG enzyme was added; in addition to the pH value-adjusted Tri-HCl, proper metal ions (NH 4) 2SO4 and MgCl2, tween-20 and DMSO are added into the reaction buffer solution to increase the reaction efficiency of PCR.
4. A fluorescent quantitative PCR method apolipoprotein E genotyping kit is characterized in that the kit reaction conditions are as follows: the possibility of contamination of the PCR product was removed at 50℃for 5 minutes, pre-denaturation at 95℃for 2 minutes, 20 cycles at 95℃for 5 seconds, 60℃for 1 second, and 72℃for 30 seconds, and then 30 cycles again under the following conditions: the analysis results concluded at 95 ℃ for 5 seconds, 60 ℃ for 30 seconds. In the first 20-cycle reaction, after the conditions are repeatedly optimized, the annealing temperature is set to 60 ℃ and the time is shortened to 1 second, so that the reaction conditions are strictly controlled, under the conditions, the difficulty of extending the 3 '-end of the primer and the unmatched template base is much higher than that of the annealing time, and the influence of extending the 3' -end of the primer and the matched template base is small, so that the specificity of the reaction is increased.
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