CN112375813A - Method for improving specificity of ARMS-TaqMan Blocker system - Google Patents
Method for improving specificity of ARMS-TaqMan Blocker system Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 15
- 239000003112 inhibitor Substances 0.000 claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 58
- 239000011780 sodium chloride Substances 0.000 claims description 29
- 230000035772 mutation Effects 0.000 claims description 9
- 206010028980 Neoplasm Diseases 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical group [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims 1
- 239000004202 carbamide Substances 0.000 claims 1
- 235000017281 sodium acetate Nutrition 0.000 claims 1
- 239000001632 sodium acetate Substances 0.000 claims 1
- 230000003321 amplification Effects 0.000 abstract description 10
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 6
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 18
- 238000006243 chemical reaction Methods 0.000 description 15
- 239000000243 solution Substances 0.000 description 11
- 230000035945 sensitivity Effects 0.000 description 9
- 239000000126 substance Substances 0.000 description 7
- 239000012295 chemical reaction liquid Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 230000002860 competitive effect Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
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- 238000012163 sequencing technique Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
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Abstract
The invention discloses a method for improving the specificity of an ARMS-TaqMan Blocker system, which adds a PCR inhibitor into a PCR fluorescent quantitative detection system. The PCR inhibitor is added into a PCR fluorescent quantitative detection system for the first time, and the inhibitor has obvious inhibition effect on non-specific amplification and does not inhibit specific amplification, so that the aim of reducing non-specificity is fulfilled.
Description
Technical Field
The invention relates to the technical field of tumor mutation detection, in particular to a method for improving the specificity of an ARMS-TaqMan packer system.
Background
In recent years, the non-radioactive labeling of probes has been rapidly developed while receiving a great deal of attention, and has been widely used for nucleic acid sequencing, gene detection, disease diagnosis, and the like. The ARMS technique is a commonly used fluorescent probe method, and the basic principle is that if the 3' end base of a primer is not complementary to the template base, extension cannot be performed with a general thermostable DNA polymerase. Therefore, 3 primers are designed according to the known point mutation, and the 3' end base of the primers is respectively complementary with the mutant and normal template base, so that the template with a certain point mutation is distinguished from the normal template. Sometimes, in order to improve the specificity, a wild-type template amplification Blocker (packer) is added into the system to suppress the amplification of the wild-type template, although the method can suppress the wild type to a certain extent, due to the lack of accurate packer design software and effective screening rules, the suppression effect is not ideal; alternatively, mismatched bases can be introduced at the penultimate or third base at the 3' end of the ARMS primer, but introduction of mismatched bases generally sacrifices sensitivity. Therefore, the conventional ARMS-TaqMan Blocker system is difficult to simultaneously achieve sensitivity and specificity, and complex and tedious optimization work is often required.
Disclosure of Invention
Aiming at the problem that the conventional ARMS-TaqMan packer system is difficult to simultaneously realize sensitivity and specificity, the invention provides a method for improving the specificity of the ARMS-TaqMan packer system.
The invention provides a method for improving the specificity of an ARMS-TaqMan Blocker system. The amount of PCR inhibitor added is in the range of 24-40mM, preferably 32 mM.
Conventional PCR inhibitors will in principle inhibit amplification, but for ARMS the amount of non-specifically amplified product is minimal, while the specific product is the majority. After a certain amount of PCR inhibitor is added into the system, specific amplification is not inhibited, but nonspecific self is strongly suppressed by Blocker, the content of the nonspecific self is low, and the specific PCR inhibitor can be further inhibited after the inhibitor is added.
The invention also protects the application of the method for improving the specificity of the ARMS-TaqMan Blocker system in tumor mutation detection.
The PCR inhibitor is added into a PCR fluorescent quantitative detection system for the first time, and the inhibitor has obvious inhibition effect on non-specific amplification and does not inhibit specific amplification, so that the aim of reducing non-specificity is fulfilled.
Drawings
FIG. 1 is a graph comparing the effect of the PCR inhibitor at E545K site;
FIG. 2 is a graph comparing the effect of braf site PCR inhibitors.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention have been presented for purposes of illustration and description, and are not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.
Description of the drawings: the primers, probes and Blocker used in the invention are subjected to melting point prediction by using a software TM Utility v1.3, and the sequence information of each primer, probe and Blocker is shown in Table 1, wherein F/R represents the primer, P represents the probe, B represents a competitive inhibitor Blocker, FAM represents a fluorescent group, MGB represents a quenching group, a square box represents a base modified by LNA, and PHO represents phosphorylation modification.
TABLE 1
Example 1 Effect of E545K site PCR inhibitors
First, primer, probe, Blocker design
The primers are designed according to the ARMS primer design principle, the length of the primers is 15-38nt, the GC content is 40-60%, the 3' end of the primers is free from hairpin structures, matching of 4 bases is avoided between the primers, the bases are uniformly distributed, and continuous GC or AT is avoided. The 3' end of the upstream ARMS primer is consistent with the mutation site, and the downstream primer is a universal primer and can amplify the wild template and the mutant template simultaneously. The sequence of the upstream primer E545K-F4 is shown as SEQ NO.01 in Table 1, and the sequence of the downstream primer E545K-R1 is shown as SEQ NO.02 in Table 1. The probe is designed according to the design principle of a TaqMan probe, the length of the probe is 14-35nt, the GC content is 60-70%, the first base at the 5' end is prevented from being G, and the sequence of the probe E545K-P is shown as SEQ NO.03 in a table 1; the competitive Blocker is E545K-B-1, the sequence is shown as SEQ NO.04 in Table 1, and 1M sodium chloride is used as a PCR inhibitor.
Second, PCR reaction system preparation
The PCR reaction solution was prepared according to the following Table 2, and deionized water was added to the reaction solution to a volume of 25. mu.l. Only pure water is added into the first group of reaction solution, and sodium chloride is not added; adding 16mM of sodium chloride into the second group of reaction liquid; adding 24mM of sodium chloride into the third group of reaction liquid; adding 32mM of sodium chloride into the fourth group of reaction liquid; the fifth group of reaction solution was added with 40mM sodium chloride.
| PCR Components | Dosage of |
| 10 × Buffer (containing Mg)2+) | 1× |
| dNTPs | 0.16mM |
| E545K-F1 | 0.8μM |
| E545K-R1 | 0.8μM |
| E545K-P | 0.4μM |
| E545-B-1 | 2μM |
| Sodium chloride | 0-40mM |
| Taq enzyme | 1U |
TABLE 2
Third, sample preparation
Fourthly, adding sample and operating the machine
In each group of reaction solution, 5. mu.l of 1%, 0.1% of standard substance and 10 ng/. mu.l of wild-type genome were added, 0.1% of the wild-type genome was used as 4 parallel channels, and the others were used as 2 parallel channels. The computer program is as follows: 2min at 95 ℃ for one cycle; 5s at 95 ℃, 30s at 56 ℃ (no fluorescence collected), 15s at 72 ℃ and 10 cycles; 3s at 93 ℃, 30s at 56 ℃ (fluorescence collected), 30s at 60 ℃, and 35 cycles. The instrument used a SLAN96 fluorescent PCR instrument.
Fifth, analysis of experimental results
Referring to FIG. 1, the first set of reaction solutions was represented by black circles with only pure water and no sodium chloride added; the fourth set of reactions was supplemented with 32mM NaCl and indicated by black squares. For ease of observation, no other set of results were put into the results of FIG. 1.
The experimental result shows that the CT value of a group (black circle) without sodium chloride is smaller than that of a group without sodium chloride; when 16-24mM sodium chloride is added, the sensitivity and specificity of the system are not influenced (results are not shown); when the amount of sodium chloride was increased to 32mM (black squares), the detection of 0.1% of the standard was not affected, and the specificity was better than that without the addition of sodium chloride, with a CT value of 10 ng/. mu.l of the wild-type genome being further posterior; when the sodium chloride dosage was further increased to 40mM, sensitivity was affected, with 0.1% standard being relatively late (results not shown).
The experimental result shows that the addition of a proper amount of PCR inhibitor does not influence the sensitivity of the system, and is more beneficial to suppressing the amplification of a trace wild type non-specific template, thereby reducing non-specific signals.
Example 2braf site PCR inhibitor Effect
First, primer, probe, Blocker design
The primers are designed according to the ARMS primer design principle, the length of the primers is 15-38nt, the GC content is 40-60%, the 3' end of the primers is free from hairpin structures, the matching of 4 bases is avoided between the primers, the base distribution is uniform, and the continuous GC or AT is avoided. The 3' end of the upstream ARMS primer is consistent with the mutation site, and the downstream primer is a universal primer and can amplify the wild template and the mutant template simultaneously. The sequence of the upstream primer braf-F is shown as SEQ NO.05 in the table 1, and the sequence of the downstream primer braf-R is shown as SEQ NO.06 in the table 1; the probe is designed according to the design principle of a TaqMan probe, the length of the probe is 14-35nt, the GC content is 60-70%, and the first basic group at the 5' end is prevented from being G. The sequence of the probe braf-P is shown as SEQ NO.07 in the table 1; the competitive Blocker is braf-B2, the sequence is shown in SEQ NO.08 of Table 1, and 1M sodium chloride is used as a PCR inhibitor.
Second, PCR reaction system preparation
PCR reaction solution preparation was performed according to the following Table 3, and deionized water was added to 25. mu.l. Only pure water is added into the first group of reaction solution, and sodium chloride is not added; adding 12mM of sodium chloride into the second group of reaction liquid; adding 24mM of sodium chloride into the third group of reaction liquid; adding 32mM of sodium chloride into the fourth group of reaction liquid; the fifth group of reaction solution was added with 40mM sodium chloride.
TABLE 3
Third, sample preparation
Mixing braf plasmid 10^3 copies/mul with 10 ng/mul wild type genome to obtain standard substance with mutation rate of 50%, diluting 50% of standard substance with 10 ng/mul wild type genome to obtain 1% and 0.1% of standard substance, and preparing 20 ng/mul wild type genome for later use.
Fourthly, adding sample and operating the machine
In each group of reaction solutions, 5. mu.l of 1% and 0.1% of the standard and 20 ng/. mu.l of the wild-type genome were added, 0.1% of the samples were used as 4 parallel channels, and the others were used as 2 parallel channels. The computer program is as follows: 2min at 95 ℃ for one cycle; 5s at 95 ℃, 30s at 56 ℃ (no fluorescence collected), 15s at 72 ℃ and 10 cycles; 3s at 93 ℃, 30s at 56 ℃ (fluorescence collected), 30s at 60 ℃, and 35 cycles. The instrument used a SLAN96 fluorescent PCR instrument.
Fifth, analysis of experimental results
Referring to FIG. 2, the first set of reaction solutions was represented by black circles with only pure water and no sodium chloride added; the fourth set of reactions was supplemented with 32mM NaCl and indicated by black squares. For ease of observation, no other set of results were put into the results of FIG. 2.
The experimental results show that the group without sodium chloride (black circles) is most non-specific and the CT value is the smallest; when 12-24mM sodium chloride is added, the sensitivity and specificity of the system are not influenced (results are not shown); when the amount of sodium chloride was increased to 32mM (black squares), 0.1% of the standard was not affected and the specificity was better than that without the addition of sodium chloride, the CT value of 20 ng/. mu.l of wild-type genome was more posterior; while further increases in sodium chloride dosage to 40mM affected sensitivity, 0.1% standard was relatively late (results not shown).
The experimental result shows that the addition of a proper amount of PCR inhibitor does not influence the sensitivity of the system, is more favorable for suppressing the amplification of a wild non-specific template, reduces non-specific signals and obviously improves the specificity of the system.
It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by one of ordinary skill in the art and related arts based on the embodiments of the present invention without any creative effort, shall fall within the protection scope of the present invention.
Claims (5)
1. A method for improving the specificity of an ARMS-TaqMan Blocker system is characterized in that a PCR inhibitor is added into a PCR fluorescent quantitative detection system.
2. The method for improving the specificity of the ARMS-TaqMan Blocker system according to claim 1, wherein the PCR inhibitor is SDS, phenol, ethanol, isopropanol, sodium acetate, sodium chloride, EDTA or urea.
3. The method for improving the specificity of the ARMS-TaqMan Blocker system according to claim 1 or 2, wherein the amount of the PCR inhibitor added is in the range of 24-40 mM.
4. The method for improving the specificity of the ARMS-TaqMan Blocker system according to claim 3, wherein the amount of the PCR inhibitor added is 32 mM.
5. Use of the method of any one of claims 1 to 3 for increasing the specificity of the ARMS-TaqMan Blocker system in the detection of tumor mutations.
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| CA2483507A1 (en) * | 2001-04-24 | 2002-10-31 | Isis Innovation Limited | Enzyme and snp marker for disease |
| CN106498060A (en) * | 2016-11-03 | 2017-03-15 | 江苏然科生物技术有限公司 | A kind of fluorescence quantitative PCR reaction solution and method |
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| US20200010846A1 (en) * | 2018-07-03 | 2020-01-09 | Rice Research Institute, Anhui Academy Of Agricultural Sciences | Specific expression promoters in rice and the use thereof |
| CN112522369A (en) * | 2020-12-08 | 2021-03-19 | 合肥欧创基因生物科技有限公司 | Method for designing Blocker double chain of ARMS-TaqMan Blocker system |
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