CN113308519A - Primer and probe for detecting single base mutation site and detection method - Google Patents
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Abstract
The invention provides a primer, a probe and a detection method for detecting single base mutation sites. According to the single-base mutation site detection method provided by the invention, when the template is a wild type, the blocking primer has more advantages when competing with the forward primer to bind the template, along with the progress of the reaction, more and more amplification products are generated by the blocking primer and the reverse primer, and simultaneously, fewer and fewer reverse primers are in a reaction system, so that the work of the forward primer is further blocked. Compared with the existing method, the primer probe design method has higher specificity in detecting single base mutation sites, namely, the false positive condition is smaller in detecting wild type target nucleic acid sequences, and the method is more favorable for detecting mutant types.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a primer and a probe for detecting single base mutation sites and a detection method.
Background
Polymerase Chain Reaction (PCR) is a molecular biology technique that generally performs enzymatic replication of DNA without using living organisms. PCR is commonly used in medical and biological research laboratories to undertake a variety of tasks, such as gene cloning, phenotypic identification of laboratory animals, transcriptome studies, detection of genetic diseases, identification of gene fingerprints, diagnosis of infectious diseases, paternity testing, and the like. Due to its incomparable replication and precision capabilities, PCR is considered by molecular biologists to be the first method of nucleic acid detection. In the later 90 s of the last century, Real Time Quantitative PCR (qPCR) technology and related products, which were introduced by ABI of America, developed PCR into a nucleic acid sequence analysis technology with high sensitivity, high specificity and accurate quantification.
At present, the existing single base Mutation site detection reagent mostly adopts a TaqMan competitive probe method or an Amplification retardation System (ARMS). The basic principle of the TaqMan probe is that an oligonucleotide probe combined with a target sequence is cut by utilizing the 5 ' exonuclease activity of Taq enzyme in the amplification process, the 5 ' end of the probe is marked with a fluorescent reporter group, the 3 ' end of the probe is marked with a fluorescent quencher group and is phosphorylated to prevent the probe from extending, and when the primer extends to the combination position of the oligonucleotide probe, the Taq enzyme can cut the probe into small fragments, so that the fluorescent reporter group is separated from the quencher group, and fluorescence is emitted. When the probe is applied to the detection of single-base mutation sites, two competitive probes are usually adopted, wherein one competitive probe is used for a mutant target nucleic acid sequence, and the other competitive probe is used for a wild target nucleic acid sequence. However, the conventional Taqman probe is often used for detection, and has a cross reaction problem, in the point mutation detection, the wild type and the mutant type only have a difference of one base, and the cross reaction of the probe easily influences the specificity of a detection reagent, so that a false negative or false positive result is caused.
On the other hand, ARMS utilizes the feature that DNA polymerase lacks 3 'exonuclease activity, and cannot amplify a target nucleic acid sequence efficiently if the 3' terminal base of a primer cannot be paired with the target nucleic acid sequence in a correct complementary manner. However, when a single-base mutation site is detected, a common ARMS primer cannot block nonspecific amplification in many cases, and a false positive result is likely to be generated during detection, thereby resulting in insufficient detection specificity.
Disclosure of Invention
The invention aims to provide a primer, a probe and a detection method for detecting a single-base mutation site.
The basic technical principle of the invention is the ARMS technology, and the common ARMS technology is improved. The primers designed by the common ARMS technology have high probability of mismatching and are easy to generate non-specific amplification. The existing method for improving the specificity of ARMS technology is characterized in that a closed primer which is complementary with a wild template is added on the basis of artificially introducing a mismatched base at the 3 'end to improve the mismatch with the wild template, and the method is characterized in that the 3' end is specially modified to ensure that the primer cannot be extended (because the probe is arranged between an upstream primer and a downstream primer, if the closed primer is extended, the probe can be hydrolyzed to generate a signal), and the closed primer and a mutant primer compete to be combined with the wild template to improve the specificity when the wild template is detected. However, the disadvantages of this method are: when the mutant primer is mismatched with the wild-type template to generate non-specific amplification, the sequence of the amplification product is completely matched with that of the mutant primer, and the amplification product is taken as a template to be preferentially combined with the mutant primer instead of the blocked primer in the next PCR cycle. As the PCR reaction progresses, more and more amplification products complementary to the mutant type primer are obtained, and the total number of the wild type template is unchanged, the mutant type primer gradually takes advantage, and finally an amplification signal is generated, so that false positive is generated.
In order to overcome the defects, the invention skillfully adds a detection area complementary with the probe at the 5 'end of the mutant primer to back position the probe, so that a repression primer complementary with a wild template can be added, and the 3' end of the primer can be extended normally without modification. When the set of primers is used for detecting the wild-type template, the blocking primer and the reverse primer form a primer pair for amplifying the wild-type template, and the reaction forms multiple inhibition on the mutant primer, so that the generation of false positive is greatly reduced: on one hand, as the reaction proceeds, more and more wild templates are available, and the competitive advantage of the blocking primer and the mutation primer is not reduced; on the other hand, since the position (penultimate base) where the repressor primer artificially introduces the mismatched base is different from that of the mutant primer (penultimate base), the amplification product sequence of the repressor primer and the reverse primer has a 3-base mismatch with the 3' end of the mutant primer, and therefore the advantage of the repressor primer and the mutant primer is greater when the product is used as a template; furthermore, the primer pair consisting of the blocking primer and the reverse primer consumes dNTP and the reverse primer for amplification, and competes Taq DNA polymerase to further block the work of the mutation primer.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a primer and a probe for detecting a single base mutation site, comprising a forward primer (F1), a repressor primer (F2), a reverse primer (R) and a TaqMan probe (P).
The forward primer sequentially comprises a detection area and a target sequence binding area along the 5 '-3' direction; wherein, the 1 st base at the 3 'end of the target sequence binding region is an amplification determining site which is complementary with the mutant template, and a mismatched base is introduced at the 2 nd or 3 rd base at the 3' end (so that multiple mismatches are formed with the wild-type template to prevent mis-extension); the detection zone comprises a region of bases complementary to the probe.
The 1 st base at the 3 ' end of the repressor primer is an amplification determining site which is complementary with the wild-type template, and a mismatched base is introduced at the 2 nd or 3 rd base at the 3 ' end (so that the mismatched base and the mutant template form multiple mismatch to prevent mis-extension), and 3-5 bases at the 5 ' end are mismatched with the template and are different from the forward primer.
The region of complementarity of the reverse primer to the target sequence is located within a region 0-200bp downstream of the region of complementarity of the forward primer to the target sequence, and may form a primer pair with either the forward primer or the blocking primer.
The TaqMan probe is complementary to the detection region of the forward primer.
In the present invention, the Tm values and lengths of the primers and probes follow the general principle of primer probe design, and the probes may be MGB probes as required.
Preferably, the target sequence binding region in the forward primer introduces a mismatch base at the 3 rd base of the 3' end.
Preferably, the repressor primer introduces a mismatched base at the 2 nd base at the 3' end.
Preferably, the 3 bases at the 5' end of the repressor primer are mismatched with the template and are different from the forward primer.
Preferably, one end of the TaqMan probe is a fluorescent group, and the other end of the TaqMan probe is a fluorescence quenching group.
In a second aspect, the present invention provides a method for detecting a single-nucleotide mutation site, which comprises performing a PCR amplification reaction using the primer and the probe, and analyzing the amplification result (analysis of the result can be performed by an amplification curve).
The method may be for non-disease diagnostic purposes.
Preferably, the PCR amplification reaction system (total volume 25. mu.l) is: mu.l of primer solution, 16. mu.l of reaction solution Mix, and 5. mu.l of DNA template.
Preferably, the concentration of the forward primer, the blocking primer and the reverse primer in the primer solution is 80-300nM respectively, and the concentration of the probe is 100-400 nM.
The PCR amplification conditions were: pre-denaturation at 92-98 deg.C for 1-10 min; denaturation at 92-98 deg.C for 10-30s, annealing at 55-60 deg.C and extension for 30-60s, and 38-45 cycles.
Preferably, the PCR amplification conditions are: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing and extension at 55 ℃ for 40s, 45 cycles.
In a third aspect, the present invention provides a detection reagent or kit comprising the above primer and probe.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
according to the single-base mutation site detection method provided by the invention, when the template is a wild type, the blocking primer has more advantages when competing with the forward primer to bind the template, along with the progress of the reaction, more and more amplification products are generated by the blocking primer and the reverse primer, and simultaneously, fewer and fewer reverse primers are in a reaction system, so that the work of the forward primer is further blocked. Compared with the existing method, the primer probe design method has higher specificity in detecting single base mutation sites, namely, the false positive condition is smaller in detecting wild type target nucleic acid sequences, and the method is more favorable for detecting mutant types.
Drawings
FIG. 1 is a preferred embodiment of the present invention.
FIG. 2 shows the result of detecting a wild-type template using the primer probe of the present invention.
FIG. 3 shows the results of wild-type template detection using a common ARMS primer probe.
FIG. 4 shows the result of detecting mutant plasmids in the preferred embodiment of the present invention.
FIG. 5 is a diagram showing the results of the blank control test according to the preferred embodiment of the present invention.
Detailed Description
The invention provides a design method of a fluorescent quantitative PCR primer and a probe for improving the discrimination between a mutant type and a wild type in single base mutation site detection. On the basis, the invention also provides a single base mutation site detection method.
The invention adopts the following technical scheme:
the invention provides a fluorescent quantitative PCR primer and a probe for detecting single-base mutation sites, which comprise a forward primer (F1), a repressor primer (F2), a reverse primer (R) and a TaqMan probe (P). The specific design method is as follows:
the forward primer F1 comprises an upstream detection region and a target sequence binding region from 5 'end to 3' end, wherein:
the 1 st base at the 3 'end of the target sequence binding region is an amplification determining site which is complementary with the mutant template, and a mismatch base is introduced into the 3 rd base at the 3' end, so that the mismatch base and the wild template form multiple mismatches to prevent the incorrect extension;
the upstream detection zone is a portion having a sequence complementary to probe P from the 5 'to 3' end.
The 1 st base at the 3 ' end of the repression primer F2 is an amplification determining point which is complementary with a wild template and a mismatch base is introduced into the 2 nd base at the 3 ' end, so that the amplification determining point and the mutant template form multiple mismatch to prevent erroneous extension, a middle target sequence binding region is the same as a forward primer target sequence binding region, and the 3 bases at the 5 ' end are mismatched with the template and are different from the forward primer.
③ the complementary region of the reverse primer R and the target sequence is located in the region of 0-200bp downstream of the complementary region of the forward primer and the target sequence, and can form a primer pair with the forward primer F1 or the repressor primer F2.
And fourthly, the TaqMan probe P is complementary with the upstream detection area of the forward primer F1.
The primer probe design scheme is shown in FIG. 1. Wherein, the forward primer F1 comprises an upstream detection region and a target sequence binding region in sequence from 5 'end to 3' end, wherein the 3 'end of the target sequence binding region has an amplification decision site which is complementary with a variation detection site on the target sequence, and the 3 rd base of the 3' end introduces a mismatched base; the upstream detection zone is a portion having a sequence complementary to probe P from the 5 'to 3' end. The 1 st base at the 3 ' end of the repressor primer F2 is an amplification determining site which is complementary to a wild type template and introduces a mismatched base at the 2 nd base at the 3 ' end, the middle target sequence binding region is the same as the forward primer target sequence binding region, and the 3 th base at the 5 ' end is mismatched with the template and is different from the forward primer.
When the template is a mutant type, only the 3 rd base at the 3 ' end of the forward primer F1 is mismatched with the template, while the 2 nd base at the 3 ' end of the blocking primer F2 and the 1 st base are both mismatched with the template, so that F1 preferentially binds to the template for amplification, simultaneously, the amplification product has a binding sequence with the probe P, then forms a primer pair with the reverse primer R and performs template amplification, a fluorescent signal is released by the TaqMan hydrolysis probe principle, and the 1 st, 2 nd and 3 rd bases at the 3 ' end of the blocking primer F2 are all mismatched with the amplification product sequence, further reducing the competitive power with F1.
Accordingly, when the template is wild type, the repressor primer F2 preferentially binds to the template for amplification, no fluorescent signal is generated because F2 has no probe-binding sequence, and the amplification reaction consumes the raw materials of the reverse primer R, and the amplification products mismatch with the 1 st, 2 nd and 3 rd bases at the 3' end of the forward primer F1, thereby further repressing the amplification of F1.
The primer and probe design method and the use method thereof can obviously improve the specificity of single base mutation sites.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions. Example 1 detection of the N501Y mutation site in the New coronavirus 2019-nCOV-B.1.1.7 mutant
The specific sequences of the primers and probes designed in this example are as follows (experimental group):
forward primer F1: 5'-CACGGGCCTTTACAATTTTATGGTTTCCAACCCAGTT-3'
Blocking primer F2: 5'-AACTATGGTTTCCAACCCACAA-3'
Reverse primer R: 5'-CTGGTGCATGTAGAAGTTCAAAAGA-3'
And (3) probe P: 5'-ATTGTAAAGGCCCGTG-3', VIC (fluorescent group) is modified at the 5 'end of the probe, and MGB (fluorescence quenching group) is modified at the 3' end of the probe.
Primers and probes designed based on the general ARMS technology (control group):
a forward primer: 5'-CATATGGTTTCCAACCCAGTT-3'
Reverse primer: 5'-CTGGTGCATGTAGAAGTTCAAAAGA-3'
And (3) probe: 5'-CTACTACTCTGTATGGTT-3', VIC (fluorescent group) is modified at the 5 'end of the probe, and MGB (fluorescence quenching group) is modified at the 3' end of the probe.
The fluorescent quantitative PCR reaction is carried out by using the combination of the detection primer and the probe, and the method comprises the following steps: PCR amplification was performed using plasmids carrying the N501Y mutant and wild type as templates.
The following reaction system was prepared in a PCR tube: 4 ul of primer solution, 16 ul of reaction solution Mix, 5 ul of DNA template (N501Y mutant or wild type plasmid) and 25 ul of total volume; when a blank control reaction is set, double distilled water is used to replace the template.
And (3) uniformly mixing the prepared PCR tubes, centrifuging, and amplifying in a fluorescent quantitative PCR instrument. The amplification conditions were: 95 deg.C
5 min; 95 ℃ 10s, 55 ℃ 40s, 45 cycles.
The experimental primer solutions were as follows:
| primer solution | Final concentration |
| Forward primer F1 | 200nM |
| Blocking primer F2 | 200nM |
| Reverse primer R | 200nM |
| Probe P | 400nM |
The reaction solution of the experimental group is as follows:
| reaction solution Mix | Volume of | Suppliers of goods |
| Reaction buffer | 10μl | Second Sage Biotechnology (Shanghai) Ltd |
| 100mM Mg2+ | 1.5μl | TaKaRa |
| Taq DNA polymerase | 1μl | TaKaRa |
| dNTPs | 0.1μl | Feipeng biology Co.,Ltd. |
| DEPC water | 3.4μl | SANGON BIOTECH (SHANGHAI) Co.,Ltd. |
The control primer solutions were as follows:
| primer solution | Final concentration |
| Forward primer F1 | 400nM |
| Reverse primer R | 400nM |
| Probe P | 200nM |
The control reaction solution was as follows:
| reaction solution Mix | Volume of | Suppliers of goods |
| Reaction buffer | 10μl | Second Sage Biotechnology (Shanghai) Ltd |
| 100mM Mg2+ | 1.5μl | TaKaRa |
| Taq DNA polymerase | 1μl | TaKaRa |
| dNTPs | 0.1μl | Feipeng biology Co.,Ltd. |
| DEPC water | 3.4μl | SANGON BIOTECH (SHANGHAI) Co.,Ltd. |
The DNA template concentrations are shown in Table 1:
TABLE 1
| Template concentration | Wild type | Mutant plasmids | |
| 1 | 109copies/ |
106copies/ml | |
| 2 | 108copies/ |
105copies/ |
|
| 3 | 107copies/ |
104copies/ml | |
| 4 | 106copies/ |
103copies/ml |
And (4) judging a result: and analyzing the result through an amplification curve.
In this example, plasmids of the N501Y mutant and N501Y wild type were subjected to fluorescent quantitative PCR amplification using the primer and probe combination described above and detected. FIGS. 2 and 3 show the results of detection of wild-type template using the primer set of the present invention and the conventional ARMS primer set. It can be seen that the primers of the present invention were usedThe concentration of each group was 109copies/ml、108copies/ml、107copies/ml、106No wild type plasmids of copies/ml were amplified non-specifically, whereas the common ARMS primer set was used even for detection 106Non-specific amplification of wild-type plasmids of copies/ml was still present. FIG. 4 shows the detection results of mutant plasmids of the primer set according to the present invention; FIG. 5 shows the detection results of the primer set blank control group of the present invention.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The primer and the probe for detecting the single-base mutation site are characterized by comprising a forward primer, a repression primer, a reverse primer and a TaqMan probe;
the forward primer sequentially comprises a detection area and a target sequence binding area along the 5 '-3' direction; wherein, the 1 st base at the 3 'end of the target sequence binding region is an amplification determining site which is complementary with the mutant template, and a mismatched base is introduced at the 2 nd or 3 rd base at the 3' end; the detection zone comprises a region of bases complementary to the probe;
the 1 st base at the 3 ' end of the repression primer is an amplification determining site which is complementary with a wild type template, a mismatched base is introduced at the 2 nd or 3 rd base at the 3 ' end, and 3-5 bases at the 5 ' end are mismatched with the template and are different from the forward primer;
the complementary region of the reverse primer and the target sequence is positioned in a region which is 0-200bp downstream of the complementary region of the forward primer and the target sequence, and can form a primer pair with the forward primer or the repression primer;
the TaqMan probe is complementary to the detection region of the forward primer.
2. The primer and probe of claim 1, wherein the target sequence binding region of the forward primer incorporates a mismatched base at the 3 rd base at the 3' end.
3. The primers and probe of claim 1, wherein the blocking primer introduces a mismatch base at the 2 nd base at the 3' end.
4. The primers and probe of claim 1, wherein the 3 bases at the 5' end of the repressor primer are mismatched to the template and are different from the forward primer.
5. The primers and probe of any one of claims 1-4, wherein one end of the TaqMan probe is a fluorophore and the other end is a fluorescence quencher.
6. A method for detecting a single base mutation site, comprising carrying out a PCR amplification reaction using the primer and the probe according to any one of claims 1 to 5, and analyzing the amplification result;
the method is for non-disease diagnostic purposes.
7. The method of claim 6, wherein the PCR amplification reaction system is: 4 mul of primer solution, 16 mul of reaction solution Mix and 5 mul of DNA template;
the concentrations of the forward primer, the repression primer and the reverse primer in the primer solution are respectively 80-300nM, and the concentration of the probe is 100-400 nM.
8. The method of claim 7, wherein the PCR amplification conditions are: pre-denaturation at 92-98 deg.C for 1-10 min; denaturation at 92-98 deg.C for 10-30s, annealing at 55-60 deg.C and extension for 30-60s, and 38-45 cycles.
9. The method according to any one of claims 6 to 7, wherein the analysis of the results is performed by means of an amplification curve.
10. A detection reagent or kit comprising the primer and the probe according to any one of claims 1 to 5.
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Cited By (2)
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| CN113846147A (en) * | 2021-09-01 | 2021-12-28 | 上海市儿童医院 | SNV epitaxial probe type qPCR detection method |
| CN117327821A (en) * | 2023-11-09 | 2024-01-02 | 上海市肺科医院（上海市职业病防治院） | Kit for quantitatively detecting ultra-low-proportion rifampicin drug-resistant mutation and detection method |
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| JP2006075126A (en) * | 2004-09-13 | 2006-03-23 | National Institute Of Advanced Industrial & Technology | Method for detecting monobasic mutation and probe for detection |
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Application publication date: 20210827 Assignee: Shanghai Fangyuan Biaoping Biotechnology Co.,Ltd. Assignor: Shanghai Berger Medical Technology Co.,Ltd. Contract record no.: X2024310000035 Denomination of invention: Primers and probes for detecting single base mutation sites and detection methods Granted publication date: 20220607 License type: Common License Record date: 20240402 |