CN116904562A - Method for detecting multi-type nucleic acid mutation based on double fluorescent probes - Google Patents

Method for detecting multi-type nucleic acid mutation based on double fluorescent probes Download PDF

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CN116904562A
CN116904562A CN202311177154.3A CN202311177154A CN116904562A CN 116904562 A CN116904562 A CN 116904562A CN 202311177154 A CN202311177154 A CN 202311177154A CN 116904562 A CN116904562 A CN 116904562A
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fluorescent
fluorescent probe
mutation
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赵飞
刘立雍
何利华
孟凡亮
龚杰
张建中
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National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
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Abstract

The invention relates to the technical field of biology, in particular to a method for detecting multiple types of nucleic acid mutation based on double fluorescent probes. The method comprises the following steps: designing an upstream and downstream amplification primer covering the mutation position, and designing a fluorescent probe A aiming at the mutation position, wherein the sequence of the fluorescent probe A is matched with a wild type target sequence of a sequence to be detected; and designing a fluorescent probe B for detecting a species-specific conserved target sequence of the sequence to be detected, wherein the design positions of the fluorescent probe B and the fluorescent probe A are positioned on the same template chain, the design positions of the fluorescent probes A and B are positioned between the upstream amplification primer and the downstream amplification primer and are not overlapped completely, and judging the mutation condition of the nucleic acid according to the difference value of the fluorescent signal CT values of the fluorescent probe A and the fluorescent probe B. The method can detect all types of single point mutation or multiple point mutation types in the coverage area of the fluorescent probe A, and has high sensitivity, strong specificity and wide detection linear range.

Description

Method for detecting multi-type nucleic acid mutation based on double fluorescent probes
Technical Field
The invention relates to the technical field of biology, in particular to a method for detecting multiple types of nucleic acid mutation based on double fluorescent probes.
Background
Gene mutation is a heritable mutation phenomenon of a biological genome nucleic acid molecule, and the structure of the gene is changed in base pair composition or arrangement sequence. Researches show that the gene mutation is closely related to the occurrence of diseases, the drug resistance of pathogenic bacteria and the toxicity change, and the types of the mutation mainly comprise base deletion, insertion and point mutation. Wherein, the base deletion/insertion can cause the base sequence shift after the mutation site, thereby causing the change of all nucleic acid sequences after the mutation site, and the mutation detection difficulty is less. Currently, the most difficult detection of nucleic acid mutations is the detection of single base mutations. Conventional detection techniques are often difficult to distinguish due to mutations in only 1 base in the sequence.
The current technology for detecting single base mutations has the following methods: 1. PCR amplification sequencing method: the method can detect various mutations on the target fragment by using common PCR to carry out sequencing analysis on the amplified product of the target fragment, but the amplification is dependent on PCR, the detection sensitivity is low and the time consumption is long. 2. PCR-RFLP method: and selecting specific restriction enzyme according to the point mutation position sequence to cut the PCR product so as to achieve the detection purpose. If a mutation is made at this position, the specific restriction enzyme selected will not recognize the site and will not cleave, thereby discriminating whether the site is mutated or not based on the cleavage product. The method has low detection sensitivity, is time-consuming and labor-consuming, can only detect mutation of specific bases, and is rarely used at present. 3. High resolution melting curve method: by using the fluorescent PCR technique, a specific Tm value of a target sequence is detected, and if a mutation is present in the target sequence, a minute change in the Tm value is caused. Compared with the sequencing method, the detection sensitivity is improved, but SNPs at other positions on the target sequence except for the target mutation also change the Tm value of the target sequence, so that the Tm value is not fixedly changed, and the result interpretation is wrong. 4. Hydrolysis probe method: the method is the most commonly used method for detecting single base mutation at present, and is generally detected by using a minor groove binding protein (MGB) or a short probe modified by locked nucleic acid, wherein a wild type binding probe and a mutant type binding probe are respectively designed aiming at mutation sites, and two types of fluorescence are marked by the two probes. Because of the difference of binding capacities, when the detection target site is a wild type gene sequence, the binding capacity with a wild type probe is far stronger than that of a mutant type, the detection target site is competitively bound on the target site, and the system is detected as a wild type probe fluorescent signal; similarly, the mutation is detected as a mutant fluorescent probe signal. The method has the highest detection sensitivity in the current single-base mutation, but still has the defect that the detection is difficult to overcome, namely, each type of single-base mutation is required to be designed with a corresponding mutation probe, otherwise, the detection is missed. This makes the method generally only detect one type of point mutation, at most 2-3 point mutations, and too many mutation types can reduce the sensitivity of probe detection, affecting discrimination. 5. Mass spectrometry: the method is a newer detection technology developed in recent years, and by designing a specific mass spectrum probe in front of a single base mutation site and utilizing a single base extension technology, all mutation types of the mutation site can be detected, and simultaneously a plurality of mutation sites can be detected.
In summary, the single-base mutation detection method is very numerous, and each method has advantages and disadvantages. However, in general, the method with high detection sensitivity is difficult to cover the whole area for the type of point mutation detection; methods capable of detecting multiple types of point mutations are also lacking in detection sensitivity.
Thus, there is a lack of a highly sensitive method in the art that can detect multiple types of point mutations.
Disclosure of Invention
In order to solve the technical problems, the invention provides a double-probe-based full-type point mutation detection method with high sensitivity and specificity based on a hydrolysis probe technology of point mutation detection.
First, the present invention provides a method for detecting a nucleic acid mutation, comprising:
designing an upstream and downstream amplification primer covering a mutation position, and designing a fluorescent probe A aiming at the mutation position, wherein the sequence of the fluorescent probe A is matched with a wild type target sequence of a sequence to be detected;
designing a fluorescent probe B for detecting a species-specific conserved target sequence of a sequence to be detected, wherein the design positions of the fluorescent probe B and the fluorescent probe A are positioned on the same template strand, the design positions of the fluorescent probes A and B are positioned between the upstream amplification primer and the downstream amplification primer, and the design position of the fluorescent probe B and the design position of the fluorescent probe A are completely non-overlapped;
detecting a sequence to be detected by using the upstream and downstream amplification primers, the fluorescent probe A and the fluorescent probe B;
and judging the mutation condition of the nucleic acid according to the difference value of the CT value of the fluorescent signals of the fluorescent probe A and the fluorescent probe B.
The wild-type target sequence in the present invention is a non-mutated sequence.
In the specific implementation process, a fluorescent PCR technology is adopted to detect the CT value of a fluorescent signal; the amplification conditions of the fluorescent PCR technique include: denaturation at 94-98 ℃,10-30s, annealing at 60-68 ℃,10-30s, and circulation 40-45 times.
In the specific implementation process, a detection system containing the upstream and downstream amplification primers, a fluorescent probe A and a fluorescent probe B is adopted to detect the sequence to be detected.
Also included in the detection system are, but not limited to, buffer substances.
Preferably, the detection method further comprises:
and setting an unmutated wild type sequence of the sequence to be detected as a result judgment control, and detecting the result judgment control by adopting a detection system which is the same as that for detecting the sequence to be detected.
Preferably, controlling the absolute value of the difference between the CT values of the two fluorescent signals of the result judgment control to be within 0.2 units; and then judging the mutation condition of the nucleic acid according to the difference value of the CT values of the two fluorescent signals of the sequence to be detected.
When detecting the wild template, the fluorescent probe A and the fluorescent probe B can be completely matched with the template, the binding capacity is equivalent, and fluorescent signals generated by amplification of the two fluorescent probes in the detection process are synchronous, so that the fluorescent signals are reflected on an amplification curve, and CT values of the two fluorescent signals are almost consistent (figure 1A). When detecting mutant type, the mutation position can be positioned in any area covered by the fluorescent probe A theoretically, but is positioned in the middle of the probe as much as possible in actual operation, so that the detection capability is better improved. The type of point mutation may be any type of transition or transversion (e.g., the mutation may be an A.fwdarw.G/C/T mutation at the A base position of the mutant strain (FIG. 1B), a G.fwdarw.A/C/T mutation at the G base position of the mutant strain (FIG. 1C), or a point mutation at other positions and a mutation superposition). Because the template nucleic acid sequence corresponding to the fluorescent probe A has point mutation, the binding capacity of the template and the fluorescent probe A is greatly reduced, and the fluorescent probe B is completely matched with the template sequence, so that the detection CT value of B fluorescence is not affected in the amplification process, and the detection CT value and the fluorescence signal intensity of the probe A are greatly delayed due to the great reduction of the binding capacity (FIG. 1B, C).
In some embodiments, one or more fluorescent probes a are designed in the same detection system, depending on the number of mutation positions.
Preferably, the design site of the upstream and downstream amplification primers is located within 300bp upstream and downstream of the mutation position.
Preferably, the Tm values of the fluorescent probe a and the fluorescent probe B are 5 to 10 ℃ higher than the Tm values of the upstream amplification primer or the downstream amplification primer.
In some embodiments, the fluorescent groups of fluorescent probe a and fluorescent probe B are different.
Such fluorescence includes, but is not limited to, FAM and VIC fluorescence.
In some embodiments, the fluorescent probe a or fluorescent probe B is independently selected from: MGB probe, LNA probe, molecular beacon probe, taqMAN probe.
In some embodiments, fluorescent probe a is a hydrolysis probe with single base discrimination capability.
In some embodiments, fluorescent probe B is a probe with high sensitivity and high specificity detection capability.
In some embodiments, the fluorescent probe is labeled with a fluorescent group at the 5 'end, a BHQ1 group or minor groove binding protein (MGB) modified at the 3' end, or a locked nucleic acid modification is performed on a portion of the bases.
Preferably, the absolute value of the difference between the two fluorescence signal CT values is Δct (Δct= |ct) A -CT B |) is provided; when the absolute value of the difference between the CT values of the two fluorescent signals of the result judgment control is controlled to be within 0.2 units,
when delta CT is less than 0.5, judging that the sequence to be detected is wild type;
when DeltaCT is more than 2.0, the sequence to be detected is judged to be mutant.
Preferably, when detecting single base mutations, the Δct value is delayed by at least 2; when any two base mutations are detected, the Δct value is deferred by at least 4; when the mutation is larger than two bases, the DeltaCT value is delayed by at least 6 or more.
In some embodiments, after fluorescent PCR amplification is complete, the fluorescence thresholds of fluorescent probes a and B are manually dragged so that the Δct value of both fluorescent signals of the result judgment control (unmutated plasmid) is controlled within 0.2.
In some embodiments, the detection method is used for non-diagnostic therapeutic purposes.
Those skilled in the art can further combine the above preferred embodiments to arrive at other preferred embodiments.
The invention mainly solves the problem of detection of single base mutation which is most difficult to distinguish in mutation detection, and the invention not only can be applied to single base mutation detection, but also is more suitable for mutation of gene insertion or deletion, and the distinguishing effect is more obvious, so that the detection of mutation of gene insertion or deletion by using the detection method of the invention belongs to the protection scope of the invention.
Compared with the prior art, the invention has the beneficial effects that:
the detection method of the invention has high sensitivity (about 5-10 copies) which is equivalent to that of a fluorescent PCR method, is superior to a single base mutation technology which depends on a common PCR amplification technology, and is the most sensitive point mutation detection technology at present. The detection method can detect any single point mutation and multipoint mutation within the coverage area of the fluorescent probe A, and solves the problem that the point mutation technology of the current probe method can only detect 2-3 short plates with point mutation types at most. In addition, the detection method has strong specificity, wide detection linear range (at least covering 7 orders of magnitude concentration gradient range) and can perform quantitative detection, which is not possessed by all the current point mutation detection technologies.
Drawings
FIG. 1 is a schematic diagram of a method for detecting a nucleic acid mutation according to the present invention.
FIG. 2 is a graph showing the results of fluorescent PCR detection of sample A of example 1.
FIG. 3 is a graph showing the result of fluorescent PCR detection of sample B of example 1.
FIG. 4 is a graph showing the result of fluorescent PCR detection of sample C of example 1.
FIG. 5 is a graph showing the result of fluorescent PCR detection of sample D of example 1.
FIG. 6 is a graph showing the results of fluorescent PCR detection of sample A of example 2.
FIG. 7 is a graph showing the result of fluorescent PCR detection of specimen E of example 2.
FIG. 8 is a graph showing the result of fluorescent PCR detection of sample F of example 2.
FIG. 9 is a graph showing the result of fluorescent PCR detection of specimen G of example 2.
FIG. 10 is a graph showing the results of fluorescent PCR detection of sample A of example 3.
FIG. 11 is a graph showing the result of fluorescent PCR detection of sample B of example 3.
FIG. 12 is a graph showing the result of fluorescent PCR detection of sample C in example 3.
FIG. 13 is a graph showing the result of fluorescent PCR detection for sample D of example 3.
FIG. 14 is a graph showing the results of fluorescence PCR detection of all specimens of example 4.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The examples are not intended to identify the particular technology or conditions, and are either conventional or are carried out according to the technology or conditions described in the literature in this field or are carried out according to the product specifications. The reagents and instruments used, etc. are not identified to the manufacturer and are conventional products available for purchase by regular vendors.
Example 1
The embodiment provides a method for detecting nucleic acid mutation, which evaluates the effects of various single-point mutations, and comprises the following steps:
mycoplasma pneumoniae is selected as a mutation detection embodiment, and 23S rRNA gene 2063 site mutation is detected, wherein a specimen A (wild type), a specimen B (A2063G mutant type), a specimen C (A2063C mutant type) and four representative point mutation specimens of a specimen D (A2063T mutant type) are taken as detection objects, wherein the specimens A, B and D are mutant strains which can be separated from the natural world, and the specimen C is a artificially synthesized mutant fragment (the mutant type strain has not been detected in China, only a mutant sequence can be artificially synthesized, as shown in SEQ ID No. 1): the sequence shown in SEQ ID No.1 was ligated to pUC57 plasmid.
Firstly designing a mutation site (nt 2063) for amplifying an upstream primer and a downstream primer, wherein the upstream primer and the downstream primer are positioned at 118bp and 120bp of the upstream and downstream of the mutation site respectively and are respectively shown as SEQ ID No.2 and SEQ ID No.3, then designing a mutation site detection fluorescent probe A, wherein the sequence of the fluorescent probe A is matched with a wild type target sequence, the 5 'end of the fluorescent probe A is marked with a VIC fluorescent group, the 3' end of the fluorescent probe A is modified with minor groove binding protein (MGB), and the sequence of the fluorescent probe A is shown as SEQ ID No. 4; a fluorescent probe B (internal reference probe) is designed in a mutation-free region of the same template chain downstream of the fluorescent probe A, the 5 'end of the fluorescent probe B is marked with FAM fluorescent groups, the 3' end is modified with minor groove binding protein (MGB), and the sequence of the fluorescent probe B is shown as SEQ ID No. 5; both probes were located between the upstream and downstream amplification primers (FIG. 1).
By means of a general purpose deviceDetecting mutation sites by using a fluorescent PCR reagent and two-step circulation detection conditions, pre-denaturing at 95 ℃ for 5min, circulating for 1 time, and then denaturing at 95 ℃ for 15s;66 degrees of extension, 15s, 45 cycles. After the fluorescent PCR amplification is finished, the two fluorescence thresholds are manually dragged, so that the result judges the DeltaCT (DeltaCT= |CT) of the two fluorescence signals of the control (unmutated plasmid) A -CT B I) value is controlled within 0.2.
As a result, as shown in fig. 2 to 5, sample a fluorescence Δct=0.06, sample B fluorescence Δct=4.11, sample C fluorescence Δct=3.82, and sample D fluorescence Δct=2.86. And judging that A is wild type and BCD is mutant according to the fluorescent signal delta CT < 0.5 is wild type and delta CT > 2.0 is mutant, wherein the detection result accords with the specimen type.
Example 2
The embodiment provides a method for detecting nucleic acid mutation, which evaluates complex mutation effects, and comprises the following steps:
in this example, mycoplasma pneumoniae was selected as a mutation detection example by using the primer probe and detection system of example 1, mutation at the 23S rRNA gene 2063 and 2064 was detected, single-base mutation at the specimen 2064 was detected, and simultaneous mutation of 2063 and 2064 was detected, and the mutation detection ability was tested. Sample A (wild type), sample E (A2064G), sample F (A2064C), sample G (A2063G and A2064G were used for simultaneous mutation). Wherein the samples A, E are mutant strains which can be separated from the natural world, the samples F and G are artificially synthesized mutant fragments, the sequences of which are respectively shown as SEQ ID No.6 and SEQ ID No.7, and are connected with pUC57 plasmids.
As a result, as shown in fig. 6 to 9, sample a had two kinds of fluorescence Δct=0.27, sample E had two kinds of fluorescence Δct=4.33, sample F had two kinds of fluorescence Δct=4.01, and sample G had two kinds of fluorescence Δct=5.65. And judging that A is wild type and EFG is mutant according to the fluorescent signal delta CT < 0.5 is wild type and delta CT > 2.0 is mutant, wherein the detection result accords with the specimen type.
Example 3
The sensitivity, specificity and limit of detection of the detection method were studied in this example.
The sample A (wild type), sample B (A2063G mutant type), sample C (A2063C mutant type), sample D (A2063T mutant type) four representative point mutant samples were selected as detection targets, and each sample nucleic acid was obtained by mixing 10 7 The copies were diluted 10-fold in concentration gradient to 1 copy. The above four concentration gradient samples were subjected to detection using the detection method in example 1, and repeated three times.
The results show that 10 copies can be detected for all four specimens, and whether mutation occurs or not can be accurately identified (fig. 10-13). In addition, the method was performed using 40 cases of nucleic acids of the same species as specimen A, and the method had a detection sensitivity of 100% (40/40) for 40 cases of nucleic acids, and correctly distinguished 31 cases of mutants, 9 cases of wild type, and a discrimination accuracy of 100% (40/40) in 40 cases of specimens. Method specificity verification was performed using nucleic acids of other species than the non-specimen A-G templates (8 bacterial nucleic acids, 6 fungal nucleic acids, 4 viral nucleic acids, mouse and human genomic nucleic acids), all detected as negative results, with a specificity of 100%.
Example 4
The samples of examples 1-3 were all derived from mycoplasma pneumoniae, a respiratory pathogen; in this example, the effect of the point mutation was evaluated using helicobacter pylori, a pathogenic bacterium of the gastrointestinal tract.
For the mutation site (nt 2143 site), three representative point mutation samples of H (wild type) and sample I (A2143G mutant type) and sample J (A2143C mutant type) were selected as detection subjects, and sample H, I, J was a mutant strain which was isolated from nature. Firstly designing a mutation site (nt 2143) amplification upstream and downstream primer which is positioned at the upstream and downstream positions of the mutation site and at 78bp and 56bp respectively and is shown as SEQ ID No.8 and SEQ ID No.9, and then designing a mutation site detection fluorescent probe A, wherein the sequence of the fluorescent probe A is matched with a wild-type target sequence, the 5 'end of the fluorescent probe A is marked with a VIC fluorescent group, the 3' end of the fluorescent probe A is modified with minor groove binding protein (MGB), and the sequence of the fluorescent probe A is shown as SEQ ID No. 10; a fluorescent probe B (internal reference probe) is designed in a mutation-free region of the same template chain downstream of the fluorescent probe A, the 5 'end of the fluorescent probe B is marked with FAM fluorescent groups, the 3' end is modified with minor groove binding protein (MGB), and the sequence of the fluorescent probe B is shown as SEQ ID No. 11; both probes are located between the upstream and downstream amplification primers.
Carrying out mutation site detection by using a universal fluorescent PCR reagent and two-step circulation detection conditions, wherein the mutation site detection is firstly performed at 95 ℃ for 5min, the mutation site detection is circulated for 1 time, and then performed at 95 ℃ for 15s;67 degrees of extension, 15s, 45 cycles. After the fluorescent PCR amplification is finished, the two fluorescence thresholds are manually dragged, so that the result judges the DeltaCT (DeltaCT= |CT) of the two fluorescence signals of the control (unmutated plasmid) A -CT B I) value is controlled within 0.2.
As a result, as shown in fig. 14, sample H had two kinds of fluorescence Δct=0.27, sample I had two kinds of fluorescence Δct=3.39, and sample J had two kinds of fluorescence Δct=4.59. According to the fluorescent signal DeltaCT < 0.5 is wild type, deltaCT > 2.0 is mutant, H is judged to be wild type, I, J is mutant, and the detection result accords with the specimen type.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A method for detecting a nucleic acid mutation, comprising:
designing an upstream and downstream amplification primer covering a mutation position, and designing a fluorescent probe A aiming at the mutation position, wherein the sequence of the fluorescent probe A is matched with a wild type target sequence of a sequence to be detected;
designing a fluorescent probe B for detecting a species-specific conserved target sequence of a sequence to be detected, wherein the design positions of the fluorescent probe B and the fluorescent probe A are positioned on the same template strand, the design positions of the fluorescent probes A and B are positioned between the upstream amplification primer and the downstream amplification primer, and the design position of the fluorescent probe B and the design position of the fluorescent probe A are completely non-overlapped;
detecting a sequence to be detected by using the upstream and downstream amplification primers, the fluorescent probe A and the fluorescent probe B;
and judging the mutation condition of the nucleic acid according to the difference value of the CT value of the fluorescent signals of the fluorescent probe A and the fluorescent probe B.
2. The method according to claim 1, wherein the fluorescent signal CT value is detected by a fluorescent PCR technique; the amplification conditions of the fluorescent PCR technique include: denaturation at 94-98 ℃,10-30s, annealing at 60-68 ℃,10-30s, and circulation 40-45 times.
3. The method of detecting according to claim 2, further comprising:
and setting an unmutated wild type sequence of the sequence to be detected as a result judgment control, and detecting the result judgment control by adopting a detection system which is the same as that for detecting the sequence to be detected.
4. The method according to claim 3, wherein the absolute value of the difference between the CT values of the two fluorescent signals of the result judgment control is controlled to be within 0.2 units; and then judging the mutation condition of the nucleic acid according to the difference value of the CT values of the two fluorescent signals of the sequence to be detected.
5. The method according to any one of claims 1 to 4, wherein one or more fluorescent probes A are designed in the same detection system according to the number of mutation sites.
6. The method according to claim 1, wherein the design site of the upstream and downstream amplification primers is located within 300bp upstream and downstream of the mutation site.
7. The method according to claim 1, wherein the Tm values of the fluorescent probe A and the fluorescent probe B are 5 to 10℃higher than the Tm value of the upstream amplification primer or the downstream amplification primer.
8. The method according to claim 1, wherein the fluorescent groups of fluorescent probe A and fluorescent probe B are different.
9. The method of detection according to claim 8, wherein the fluorescent probe a or fluorescent probe B is independently selected from: MGB probe, LNA probe, molecular beacon probe, taqMAN probe.
10. The method according to claim 4, wherein,
the absolute value of the difference value of the CT values of the two fluorescent signals is delta CT;
when the absolute value of the difference between the CT values of the two fluorescent signals of the result judgment control is controlled to be within 0.2 units,
when delta CT is less than 0.5, judging that the sequence to be detected is wild type;
when DeltaCT is more than 2.0, the sequence to be detected is judged to be mutant.
CN202311177154.3A 2023-09-13 2023-09-13 Method for detecting multi-type nucleic acid mutation based on double fluorescent probes Pending CN116904562A (en)

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CN115678974A (en) * 2022-11-16 2023-02-03 福州迈新生物技术开发有限公司 Double-probe method applied to fluorescent quantitative PCR

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