CN108165629B - DNA point mutation quantitative detection method - Google Patents
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Abstract
The invention provides a DNA point mutation quantitative detection method, which greatly inhibits the amplification of a wild type template by inhibiting the wild type template twice by a PNA probe and solves the problem of incomplete PNA clamping. Meanwhile, nested PCR can be carried out in a single tube by utilizing the Tm value difference of the amplification primers and the annealing temperature control during amplification, so that the amplification intensity and the amplification specificity are enhanced. Makes possible the amplification of clinical samples with extremely low copy numbers. Finally, by the closed tube detection of real-time fluorescence, the real-time detection is realized, the cross contamination of samples is avoided, and the mutation quantification is also realized. In addition, the method only needs a common real-time fluorescent PCR instrument, can obtain a detection result within 2h, has low reagent cost, and can realize rapid and low-cost detection on the sample. In a word, the successful establishment of the method can provide a novel detection method with high sensitivity, high specificity, simplicity, convenience, rapidness, no pollution and low cost for the detection of clinical DNA point mutation.
Description
Technical Field
The invention relates to the technical field of biology, in particular to the technical field of biological gene detection.
Background
Methods commonly used for clinical detection of gene mutation mainly include Sanger sequencing method, pyrosequencing method, Restriction Fragment Length Polymorphism (RFLP) analysis and the like, and the methods have low sensitivity, can only detect more than 5-20% of mutation samples, and are difficult to meet the detection requirements of clinical samples. Compared with Sanger sequencing methods, the sensitivity of methods such as Real-time fluorescent quantitative PCR (RT-PCR), Amplification Retardation Mutation System (ARMS), High-resolution Melting (HRM) method, Mutation Enrichment PCR (memPCR), and complex PCR Amplification method (Cold PCR) at low denaturation temperature is greatly improved, and more than 1% of mutations can be detected. However, these methods are difficult to detect low abundance plasma ctDNA samples with a content of less than 1%, and all of these methods can only perform qualitative detection on mutations, and are difficult to perform quantitative detection. At present, the sensitivity is more than 1%, and methods for realizing the mutation content mainly comprise Digital-PCR (polymerase chain reaction) and Next-generation Sequencing (NGs). Digital PCR is an absolute nucleic acid quantitative method based on a single-molecule PCR method for counting, and at present, the method mainly comprises BEAMing technology, droplet-type (ddPCR) and chip-type (chip dPCR, cdPCR). The method can detect gene mutation as low as 0.01 percent and can realize absolute quantification of mutation, but the flux of a digital PCR analysis sample is very low, ten thousand reaction units on each chip are used for analyzing a single sample, and the limitation of a fluorescence detection technology limits simultaneous detection of a plurality of chips. In addition, the expensive precision instrument and expensive reagent cost of the digital PCR are also an important reason for restricting the wide application thereof; the next generation sequencing technology is a sequencing technology for sequencing hundreds of thousands to millions of DNA molecules at a time, can detect the sequences of all gene fragments in a sample, and then judges whether gene mutation exists or not through the sequences. However, high-throughput sequencing requires expensive sequencers and expensive sequencing reagents, and the cost of one-time sequencing is about $ 1000, which severely limits the clinical popularization and application of the method. In addition, how to interpret the massive sequence information obtained by the second-generation sequencing is a bottleneck factor for restricting the application of the next-generation sequencing technology to clinic. In a word, the method has the problems of low sensitivity, no quantification, complex operation, high cost and the like. Therefore, it is highly desirable to develop a new method for detecting gene mutation with high sensitivity, quantitative, simplicity, rapidity and low cost.
PNA-LNA clamp PCR (PNA-LNA PCR clamp) is a novel mutation detection method based on PNA clamp reaction and LNA probe, the sensitivity of the method can reach about 1-0.1%, the operation is very simple and convenient, and no special requirements are required on instruments. However, this method also has two problems: first, the clamping reaction of PNA is incomplete. Amplification of wild type DNA cannot be completely inhibited, so that false positive results are easy to appear when low-abundance mutation is detected, and the method can only detect mutation of more than 1 per thousand; secondly, the amplification efficiency of the conventional clamp PCR is low, and the sample with extremely low template concentration is easy to fail in amplification to cause false negative results. For this reason, a nested PCR method is often used to improve the amplification strength and specificity of the clamp PCR, i.e., two pairs of PCR primers are used to amplify the fragment to be detected. Before the clamp PCR, a pair of external primers is used for pre-amplification, and the pre-amplified product is diluted and then used as a clamp PCR amplification template for clamp PCR amplification. Nested PCR has the advantage that even if the first amplification generates the wrong fragment, the probability of primer pairing and amplification on the wrong fragment is extremely low, so that the amplification specificity of nested PCR is extremely high. In addition, through the first pre-amplification, the template concentration of the second amplification is high, the template structure becomes simple, the efficiency of the second amplification is improved, and the amplification strength is enhanced. However, conventional nested amplification in two steps tends to cause cross-contamination of samples, resulting in false positive results.
Disclosure of Invention
The invention aims to solve the problems of low sensitivity, high detection cost, difficult quantification realization and the like of a common gene mutation detection method in clinic and also aims to solve the problems of incomplete clamp of PNA-LNA clamp PCR and easy pollution of two-step nested PCR. The invention creatively combines single-tube nested PCR with PNA-LNA clamp PCR, and establishes a novel DNA mutation detection method based on single-tube nested PCR and double PNA clamp reaction.
In order to achieve the purpose, the technical scheme provided by the invention is as follows: a DNA point mutation quantitative detection method comprises the following steps:
(1) extracting DNA of a sample to be detected;
(2) determining the point mutation site of the gene to be detected, selecting two sequences in the upstream and downstream regions of the selected site,
designing 2 outer primers and 2 inner primers; the amplification product of the outer primer contains a mutation site, and the Tm value of the outer primer is higher; the inner primers are positioned inside the upstream and downstream outer primers, and the Tm value of the inner primers is lower;
designing 1PNA probe, wherein the PNA probe is wild type site specific, namely is completely complementary with wild type DNA (W-PNA) and has a base mismatch with mutant type DNA;
1 LAN probe was designed to be site-specific for the mutant, i.e., completely complementary to the mutant only
(M-LNA) with a one base mismatch to wild-type DNA;
designing 1 internal standard fluorescent probe which is complementary with the non-mutation site sequence of the amplification product of the inner primer and has no overlapping sequence with the LNA probe;
(3) putting the synthesized 2 outer primers, 2 inner primers, 1PNA probe, 1 LAN probe, 1 internal standard fluorescent probe, high fidelity Taq enzyme, high purity water and the DNA prepared in the step (1) into a reaction system;
(4) in the above reaction system, the first round of PNA clamping reaction is carried out mainly with the outer primer. During amplification, the annealing temperature is set to be 68-72 ℃, and the outer primer can be complementary with the DNA template due to the high Tm value of the outer primer; in the case of the mutant DNA, a base mismatch exists between the W-PNA and the mutant DNA, and the W-PNA probe cannot be complementary to the mutant DNA, so that the mutant DNA can be normally amplified by the outer primer. For wild-type DNA, due to the effect of inhibiting extension of the W-PNA probe, normal amplification cannot be carried out, so that the first PNA clamping reaction and enrichment of mutant DNA are carried out;
due to the fact that the concentration of the outer primers is low, after 10-20 cycles of pre-amplification, the outer primers are consumed, and interference on the amplification of the inner primers is avoided; the inner primer has a lower Tm value, so that the amplification of the outer primer is not influenced at a high annealing temperature.
(5) In the reaction system, the second round of PNA clamping reaction is carried out by using the inner primer as the main material, and the product obtained by amplification in the step (4) is used as a template. During amplification, the annealing temperature is set to be 56-62 ℃, and then the inner primer can be complementary with the template; in the case of the mutant DNA, since W-PNA has a mismatch with the mutant DNA and cannot bind to the mutant DNA at the corresponding annealing temperature, the mutant DNA can be normally amplified by the inner primer. For wild type DNA, due to the effect of inhibiting extension of the W-PNA probe, normal amplification cannot be carried out, so that the wild type DNA is inhibited for the second time, and mutant type DNA is enriched;
(6) and finally, combining an LNA fluorescent probe in the reaction system with the mutant DNA amplification product, generating fluorescence, and monitoring the content of mutant DNA in the PCR amplification system through the strength of a fluorescence signal and a standard curve. Preferably, the outer primer has a Tm of 68 ℃ to 72 ℃ and the amplification product contains the mutation site.
Preferably, the inner primers are located inside the upstream and downstream outer primers, and have Tm values of 56 ℃ to 62 ℃.
Preferably, the 5 'end of the LNA probe is labeled with FAM fluorescent group, and the 3' end is labeled with TAMA quenching group; the 5 'end of the internal standard probe is marked with a HEX fluorescent group, and the 3' end is marked with a BHQ1 quenching group.
Preferably, in the step (4), the first round of PNA clamping reaction is carried out, and the thermal cycling reaction is set to be firstly kept at 95 ℃ for 30 sec; then, the cycle was repeated 15 times at 95 ℃ for 5sec and 68 ℃ for 34 sec.
Preferably, in the step (5), the second round of PNA clamping reaction is carried out, and the thermal cycling reaction is set to firstly maintain at 95 ℃ for 5 sec; the temperature was then reduced to 56 ℃ for 34sec for a total of 40 cycles.
Preferably, in step (1), the sample to be tested includes, but is not limited to, DNA such as tissue, whole blood or plasma.
In the step (4), the first round of PNA clamping reaction is mainly completed by an outer primer; during amplification, a higher annealing temperature (68 ℃) is set, and the inner primers IP-F and IP-R cannot be complemented with a DNA template due to a lower Tm value (60 ℃) and cannot influence the amplification of the outer primers. The higher Tm (. about.69 ℃) of the 2 outer primers (OP-F, OP-R) and the W-PNA probe was complementary to the DNA template. The PNA probe has three characteristics of (1) being incapable of being recognized by DNA polymerase as primer extension, (2) having stronger affinity and thermal stability than DNA/DNA, and (3) being sensitive to base mismatch, wherein the Tm value can be reduced by 8-20 ℃ due to mismatch of one base (average 15 ℃). For wild-type templates, normal amplification was not possible due to the effect of the PNA probe to inhibit extension (Elongation array). However, in the mutant template, since the W-PNA probe has a mismatch of one base with the W-PNA probe, the stability of PNA/DNA is reduced, and the PNA probe cannot be complementary to the mutant DNA at the annealing temperature, so that OP-F, OP-R can normally amplify the mutant DNA template. As a result, the mutant DNA can be normally amplified, and amplification of the wild type DNA is suppressed, so that the first PNA clamping reaction and enrichment of the mutant DNA are performed. Due to the fact that the concentration of the outer primers is low, after 10-20 cycles of pre-amplification, the outer primers are consumed, and interference on amplification of the inner primers is avoided.
In the step (5), carrying out a second round of PNA clamping reaction by taking the inner primers as main materials; and (3) carrying out inner primer amplification by using the product of the outer primer pre-amplification as a template. During amplification, a lower annealing temperature (60 ℃) is set, and at this time, the inner primers IP-F and IP-R can be complementary with the template. For the small amount of wild-type DNA template generated in the first round of amplification, it could not be amplified normally due to the effect of inhibition extension (Elongation array) of PNA probe. Since PNA is not recognized by DNA polymerase as primer extension, amplification of wild type DNA is again prevented. Because the PNA probe has mismatch with the mutant DNA, the PNA probe can not be combined with the mutant DNA at the corresponding annealing temperature (60 ℃) and cannot block the amplification of the mutant DNA, thereby secondarily inhibiting the enrichment of the mutant DNA by the wild type DNA.
In step (6), the LNA fluorescent probe in the reaction system binds to the mutant DNA amplification product and generates fluorescence. Because the LNA probe is mutant-specific and is sensitive to mutation, the Tm value can be reduced by 3-8 ℃ due to the mismatch of one base. Furthermore, the sequence overlap between the mutant LAN probe and the wild-type PNA probe, and the high affinity of the added wild-type PNA probe for wild-type DNA prevents hybridization of the mutant LNA probe to the wild-type DNA. Therefore, the LNA probe can specifically detect the mutant DNA product, and the specificity of the method is further improved. The internal standard probe can be complementary to either the wild-type product or the mutant product. Therefore, the fluorescence signal can be used for monitoring the PCR amplification system.
A DNA point mutation quantitative detection kit comprises the following components:
2 outer primers, the deoxynucleotide sequences of which are respectively shown as SEQ ID No.1 and 2, wherein the amplification product of the outer primer contains a mutation site, and the Tm value of the outer primer is higher;
2 inner primers, the deoxynucleotide sequences of which are respectively shown as SEQ ID No.3 and 4, wherein the 2 inner primers are positioned inside the upstream outer primer and the downstream outer primer, and the Tm values of the inner primers are lower;
1PNA probe, the deoxynucleotide sequence of which is shown as SEQ ID No.5, is wild type site-specific, namely completely complementary (W-PNA) with wild type DNA only and has one base mismatch with mutant type DNA;
1 LAN probe, the deoxynucleotide sequence of which is shown in SEQ ID No. 6; is mutant site-specific, i.e., is only completely complementary to the mutant (M-LNA), and has a one-base mismatch with the wild-type DNA;
the deoxynucleotide sequence of the 1 internal standard fluorescent probe is shown as SEQ ID No.7, the 1 internal standard fluorescent probe is complementary with the non-mutation site sequence of the amplification product of the inner primer, and the 1 internal standard fluorescent probe does not have an overlapping sequence with the LNA probe.
In a preferred technical scheme of the invention, the kit further comprises high-fidelity Taq enzyme.
The use method of the quantitative detection kit for detecting EGFR L858R mutation sites comprises the following steps:
A. extracting DNA of a sample to be detected;
B. putting the 2 outer primers, the 2 inner primers, the 1PNA probe, the 1 LAN probe, the 1 internal standard fluorescent probe, the high fidelity Taq enzyme, the high purity water and the DNA prepared in the step (1) into a reaction system;
C. in the above reaction system, the first round of PNA clamping reaction is carried out mainly with the outer primer. During amplification, the annealing temperature is set to be 68-72 ℃, and the outer primer can be complementary with the DNA template due to the high Tm value of the outer primer; in the case of the mutant DNA, a base mismatch exists between the W-PNA and the mutant DNA, and the W-PNA probe cannot be complementary to the mutant DNA, so that the mutant DNA can be normally amplified by the outer primer. For wild-type DNA, due to the effect of inhibiting extension of the W-PNA probe, normal amplification cannot be carried out, so that the first PNA clamping reaction and enrichment of mutant DNA are carried out;
due to the fact that the concentration of the outer primers is low, after 10-20 cycles of pre-amplification, the outer primers are consumed, and interference on the amplification of the inner primers is avoided; the inner primer has a lower Tm value, so that the amplification of the outer primer is not influenced at a high annealing temperature.
D. In the reaction system, the second round of PNA clamping reaction is carried out by taking the inner primer as the main material, and the product obtained by the amplification in the step C is taken as a template. During amplification, the annealing temperature is set to be 56-62 ℃, and then the inner primer can be complementary with the template; in the case of the mutant DNA, since W-PNA has a mismatch with the mutant DNA and cannot bind to the mutant DNA at the corresponding annealing temperature, the mutant DNA can be normally amplified by the inner primer. For wild type DNA, due to the effect of inhibiting extension of the W-PNA probe, normal amplification cannot be carried out, so that the wild type DNA is inhibited for the second time, and mutant type DNA is enriched;
E. and finally, combining an LNA fluorescent probe in the reaction system with the mutant DNA amplification product, generating fluorescence, and monitoring the content of mutant DNA in the PCR amplification system through the strength of a fluorescence signal and a standard curve. Preferably, the outer primer has a Tm of 68 ℃ to 72 ℃ and the amplification product contains the mutation site.
Wherein, the 5 'end of the LNA probe is marked with FAM fluorescent group, and the 3' end is marked with TAMA quenching group; the 5 'end of the internal standard probe is marked with a HEX fluorescent group, and the 3' end is marked with a BHQ1 quenching group.
In the step C, the first round of PNA clamping reaction is carried out, and the thermal cycle reaction is set to firstly keep the temperature at 95 ℃ for 30 sec; then, the cycle was repeated 15 times at 95 ℃ for 5sec and 68 ℃ for 34 sec.
In the step D, the second round of PNA clamping reaction is carried out, and the thermal cycle reaction is set to firstly keep the temperature at 95 ℃ for 5 sec; the temperature was then reduced to 56 ℃ for 34sec for a total of 40 cycles.
In order to solve the problems of PNA-LNA clamp PCR and two-step nested PCR, the invention establishes a novel method for quantitatively detecting low-content DNA point mutation based on single-tube nested PCR and double Peptide Nucleic Acid (PNA) clamp reaction. The method establishes a single-tube nested PCR method by controlling the Tm difference and the thermal cycle temperature of the inner primer and the outer primer during nested amplification, and solves the problem that two-step nested PCR is easy to cause pollution. And adding a PNA probe complementary to wild type DNA and a LAN probe complementary to mutant type DNA while performing single-tube nested PCR amplification. The inhibition of the PNA probe on the wild type DNA twice greatly inhibits the amplification of the wild type DNA, and solves the problem of incomplete PNA clamping reaction in the clamp PCR technology. Finally, the amplified product of the nested PCR is specifically detected by a fluorescence-labeled LNA probe.
Thus, through the inhibition of the PNA probe on the wild type template twice, the amplification of the wild type template is greatly inhibited, and the problem of incomplete PNA clamping is solved. Meanwhile, nested PCR can be carried out in a single tube by utilizing the Tm value difference of the amplification primers and the annealing temperature control during amplification, so that the amplification intensity and the amplification specificity are enhanced. Makes possible the amplification of clinical samples with very low copy numbers, such as plasma ctDNA. Finally, by the closed tube detection of real-time fluorescence, the real-time detection is realized, the cross contamination of samples is avoided, and the mutation quantification is also realized. In addition, the method only needs a common real-time fluorescent PCR instrument, can obtain a detection result within 2h, has low reagent cost, and can realize rapid and low-cost detection on the sample. In a word, the successful establishment of the method can provide a novel detection method with high sensitivity, high specificity, simplicity, convenience, rapidness, no pollution and low cost for the detection of clinical DNA point mutation.
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FIG. 1 shows the principle of the mutation quantitative detection method based on single-tube nested PCR and double Peptide Nucleic Acid (PNA) clamp reaction according to the present invention;
FIG. 2 thermal cycling settings for the mutation quantitative detection method of the present invention;
FIG. 3 shows typical results of clinical specimen testing using the present method; FIG. 3.A is a standard curve for the present method; FIG. 3.B shows typical results of the method for detecting mutation positivity of EGFR L858R site in non-small cell lung cancer tumor tissue sample; FIG. 3.C is a typical result of the method for detecting mutation positivity of EGFR L858R site in plasma sample of non-small cell lung cancer; FIG. 3.D is a typical result of the method for detecting mutation negativity of EGFR L858R site in non-small cell lung cancer tumor tissue samples; FIG. 3.E shows typical results of the method for detecting mutation negativity of EGFR L858R site in plasma samples of non-small cell lung cancer;
FIG. 4 compares the difference between the amplification sensitivity and the mutation detection sensitivity of the method of the present invention and the PNA-LNA clamp PCR method; FIG. 4A shows the amplification sensitivity of PNA-LNA clamp PCR; FIG. 4B shows the amplification sensitivity of the method of the present invention; FIG. 4C shows the mutation detection sensitivity of PNA-LNA clamp PCR; FIG. 4D shows the mutation detection sensitivity of the method of the present invention.
Detailed Description
The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes and are not intended to limit the scope of the present invention. The conditions employed in the examples may be further adjusted according to the conditions of the investigator, and the conditions not specified are generally the conditions in routine experiments.
Introduction and summary
The present invention is illustrated by way of example and not by way of limitation. It should be noted that references to "an" or "one" embodiment in this disclosure are not necessarily to the same embodiment, but to at least one.
Various aspects of the invention are described below. It will be apparent, however, to one skilled in the art that the present invention may be practiced according to only some or all aspects of the present invention. For purposes of explanation, specific numbers, materials and configurations are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without specific details. In other instances, well-known features are omitted or simplified in order not to obscure the present invention.
Various operations will be described as multiple discrete steps in turn, and in a manner that is most helpful in understanding the present invention; however, the description in order should not be construed as to imply that these operations are necessarily order dependent.
Various embodiments will be described in terms of typical classes of reactants. It will be apparent to those skilled in the art that the present invention may be practiced using any number of different types of reactants, not just those provided herein for purposes of illustration. Furthermore, it will also be apparent that the invention is not limited to any particular hybrid example.
Example one
The invention establishes a high-sensitivity point mutation detection method for evaluating the detection condition of the method on clinical samples. The method is applied to the detection of EGFR L858R mutation site related to targeted drug administration in plasma and tissue samples of non-small cell lung cancer.
EFGR is a common driver of non-small cell lung cancer, and its mutation occurs mainly in the 18/19/20/21 exon. EGFR L858R (NCBI accession number: NG _007726.1) is formed by changing leucine to arginine in the 858 th amino acid encoded by exon 21 of EGFR gene, and the mutation can cause activation of EGFR downstream pathway and further cause tumorigenesis. Iressa and Tarceva are common EGFR tyrosine kinase inhibitors and are commonly used target drugs for treating non-small cell lung cancer at present. A large number of researches show that EGFR L858R is one of sensitive mutations of Iressa and Tarceva, and the effective rate of Iressa and Tarceva to EGFR L858R mutation patients is far higher than that of EGFR L858R non-mutation patients. Therefore, the National Comprehensive Cancer Network (NCCN) guidelines and the clinical application guidelines for antitumor drugs published by the ministry of health of china both stipulate that sensitive mutations of the EGFR gene (e.g., EGFR L858R) of patients are detected before tyrosine kinase inhibitors such as ressa (Iressa) and tarceva are used, and individualized targeted drug treatment regimens are formulated according to the mutation detection results. 1. Primers and sequences: the inner primers, the outer primers and the probes required by EGFR L858R are shown in Table 1, wherein 2 outer primers are OP-F and OP-R, the sequences of which are respectively shown in SEQ ID Nos. 1 and 2, 2 inner primers are IP-F and IP-R, the sequences of which are respectively shown in SEQ ID Nos. 3 and 4, 1PNA Probe is W-PNA, the sequence of which is shown in SEQ ID No.5, and 1 LAN Probe is M-LNA Probe, the sequence of which is shown in SEQ ID No. 6; and an internal standard probe, the sequence of which is shown as SEQ ID No. 7.
Table 1 primers and probes required for detecting EGFR L858R by using the method of the invention
2. Tissue and plasma sample extraction. The Simgen Circulating Nucleic Acid Kit is adopted to extract a plasma ctDNA sample. DNA was extracted from tumor Tissue using the DNeasy Blood & Tissue Kit.
3. The reagent formula comprises: the 50. mu.l reaction reagent formula is: premix ExTaq (Probe) 1X, OP-F, OP-R each 0.05. mu.M, IP-F, IP-R each 0.2. mu.M, reference fluorescence ROX-II 1X, M-LNA Probe 0.6. mu.M, W-PNA 0.8. mu.M, internal standard Probe 0.4. mu.M, ddH2O make up to 50. mu.l.
4. Thermal cycling program and setting of standard curve: the thermal cycle is as follows: 95 ℃ for 30 sec; 15 times (95 ℃ C., 5 sec; 68 ℃ C., 34sec) and a further 40 times (95 ℃ C., 5 sec; 60 ℃ C., 34 sec). At 105,104,103,102,101The known copy number of EGFR positive cell DNA of (a) is a standard.
5. Typical results of sample testing
The ctDNA of 100 lung cancer tissue samples and 100 plasma samples were tested, and typical results are shown in fig. 3. As can be seen from FIG. 3, the method of the present invention can quantitatively detect mutations of EGFR L858R in tissue and plasma samples.
Example two
The method is the development and improvement of PNA-LNA clamp PCR technology, so that the method and the PNA-LNA clamp PCR technology are compared and tested. The difference between the two methods in amplification sensitivity and mutation detection sensitivity was mainly compared.
1. The primers and sequences of the experimental group and the control group 1 are the same as those of the first embodiment;
2. preparation of standards of known content: NCI-H1975 cells containing mutant EGFR L858R and H293T cells containing wild-Type EGFR L858R were purchased from American Type Culture Collection, Rockville, USA;
the EGFR L858R wild-type and mutant DNAs in the cells were quantified by digital PCR and used as standards for known contents of wild-type and mutant DNAs, respectively. Diluting the EGFR L858R mutant cell DNA with known concentration to 10 times according to the method of 10-fold incremental dilution4，103，102，101And 100As amplification template, to evaluate amplification sensitivity. At 10510 of the DNA of the wild-type cell of EGFR L858R, respectively5，104，103，102，1015 and 10050% of EGFR L858R mutant cell DNA (M: W10)5：105)，10％(M:W＝104：105)，1％(M:W＝103：105)，1‰(M:W＝102：105)，0.1‰(M:W＝101：105)，0.05‰(M:W＝5：105) And 0.01 ‰ (M: W ═ 10)0：105) A mixture of wild type and mutant DNA to evaluate the sensitivity of mutation detection;
3. the reagent formula comprises:
the reagent formula of the experimental group is the same as that of the first embodiment;
the PCR technology formula of the control group 1PNA-LNA clamp is as follows: the 50. mu.l reaction reagent formula is: premix ExTaq (Probe) 1X, IP-F, IP-R each 0.2. mu.M, reference fluorescence ROX-II 1X, M-LNA Probe 0.6. mu.M, W-PNA 0.8. mu.M, internal standard Probe 0.4. mu.M, ddH2O complement system to 50. mu.l;
4. program settings for the thermal cycling program:
the thermal cycling procedure of the experimental group of the invention is the same as that of the first embodiment;
control group 1PNA-LNA clamp PCR technique thermocycling procedure: 95 ℃ for 30 sec; 40 times (95 ℃ C., 5 sec; 60 ℃ C., 34 sec).
5. The invention relates to a method for experimental group and a comparison group 1PNA-LNA clamp PCR technology sensitivity comparison result.
As shown in FIG. 4, for the amplification sensitivity, the PNA-LNA clamp PCR technique can amplify >10 copies of the template, and less than 10 copies cannot be amplified accurately (FIG. 4A); the method established by the invention can amplify the template with 1 copy, and the amplification sensitivity is improved by 10 times (figure 4B). For mutation detection sensitivity, the PNA-LNA clamp PCR technique can detect > 1% of mutations, consistent with literature reports (fig. 4C). The mutation detection method established by the invention can detect the mutation of more than 0.1 per mill, and the mutation detection sensitivity is improved by 10 times (figure 4D). As can be seen, the method has greatly improved sensitivity compared with the existing method, and can realize the detection of point mutation samples with low copy number and low abundance.
EXAMPLE III
In order to evaluate the accuracy of the method of the invention in detecting clinical samples, the detection result of the method is compared with the digital PCR result. Digital PCR is an absolute nucleic acid quantification method based on a single-molecule PCR method that can detect as low as 0.01% of gene mutations and can achieve absolute quantification of mutations. Is a method which is widely applied in the field of clinical point mutation detection at present. To this end, we compared the method of the present invention with the result of ddPCR detection to evaluate the accuracy of the method.
1. The primers and sequences of the experimental group method and the control group 2 of the invention are the same as those of the first embodiment,
2. the tissue sample extraction method is the same as that of the first embodiment,
3. the reagent formula comprises:
the reagent formula of the experimental group is the same as that of the first embodiment;
the digital PCR of the contrast group 2 adopts a PrimePCRTM ddPCRTM Mutation Assay (EGFR, p.L858R, Human) kit, the reagent formula is according to the instruction of the kit,
4. program settings for the thermal cycling program:
the thermal cycle procedure of the method of the invention is the same as that of the first embodiment;
the control group 2 digital PCR thermal cycle program is set according to the requirements of the kit instruction;
5. the method of the present invention was compared with the results of ddPCR.
The results are shown in Table 1. As can be seen from Table 1, ddPCR detected 25 EGFR L858R mutations and 75 EGFR L858R negatives in 100 tumor tissues. For 25 samples with positive ddPCR detection, the detection result of the method is positive. For 75 samples with negative ddPCR detection results, the detection results of the method are all negative. The consistency of the two is 100%, if ddPCR is used as a gold standard method, the sensitivity of the method established in the research is 100%, and the specificity is also 100%, which shows that the method can accurately detect the EGFR L858R locus.
The above-described specific embodiments are merely preferred embodiments of the present invention, and it should be noted that, for those skilled in the art, various modifications or substitutions can be made without departing from the principle of the present invention, and these modifications or substitutions should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Suzhou university affiliated first hospital
<120> DNA point mutation quantitative detection method
<160> 7
<210> 1
<211> 26
<212> outer primer OP-F
<213> Artificial Synthesis
<400> 1
ctacttggag gaccgtcgct tggtgc 26
<210> 2
<211> 27
<212> outer primer OP-R
<213> Artificial Synthesis
<400> 2
cctggtccct ggtgtcagga aaatgct 27
<210> 3
<211> 22
<212> inner primer IP-F
<213> Artificial Synthesis
<400> 3
<210> 4
<211> 20
<212> inner primer IP-R
<213> Artificial Synthesis
<400> 4
<210> 5
<211> 11
<212> PNA Probe
<213> Artificial Synthesis
<400> 5
tgggctggcc a 11
<210> 6
<211> 14
<212> LAN probe
<213> Artificial Synthesis
<400> 6
6-FAM-ttgggcgggc caaa-TAMRA 14
<210> 7
<211> 23
<212> internal standard probe
<213> Artificial Synthesis
<400> 7
HEX-tggtattctt tctcttccgc acc-BHQ1 23
Claims (6)
1. A DNA point mutation quantitative detection method comprises the following steps:
(1) extracting DNA of a sample to be detected;
(2) determining the point mutation site of the gene to be detected, selecting two sequences in the upstream and downstream regions of the selected site,
designing 2 outer primers and 2 inner primers; the amplification product of the outer primer contains a mutation site, and the Tm value of the outer primer is higher; the inner primers are positioned inside the upstream and downstream outer primers, and the Tm value of the inner primers is lower;
designing 1PNA probe W-PNA, which is wild type site specific, namely completely complementary with wild type DNA and mismatched with mutant type DNA by one base;
designing 1 LNA probe M-LNA which is mutant site specific, namely being completely complementary with mutant DNA and having one base mismatch with wild DNA;
designing 1 internal standard fluorescent probe which is complementary with the non-mutation site sequence of the amplification product of the inner primer and has no overlapping sequence with the LNA probe;
(3) putting the synthesized 2 outer primers, 2 inner primers, 1PNA probe, 1 LNA probe, 1 internal standard fluorescent probe, high fidelity Taq enzyme, high purity water and the DNA prepared in the step (1) into a reaction system;
(4) in the reaction system, a first round of PNA clamping reaction is carried out mainly by using an outer primer, and during amplification, the annealing temperature is set to 68-72 ℃, so that the outer primer can be complementary with a DNA template due to a higher Tm value; for the mutant DNA, the base mismatch exists between the W-PNA and the mutant DNA, the W-PNA probe cannot be complementary with the mutant DNA, so that the mutant DNA can be normally amplified by an outer primer, and for the wild type DNA, the W-PNA probe cannot be normally amplified due to the extension inhibition effect of the W-PNA probe, so that the first PNA clamping reaction and the enrichment of the mutant DNA are carried out;
due to the fact that the concentration of the outer primers is low, after 10-20 cycles of pre-amplification, the outer primers are consumed, and interference on the amplification of the inner primers is avoided; the inner primer has low Tm value, so that the amplification of the outer primer cannot be influenced at high annealing temperature;
(5) in the reaction system, carrying out a second round of PNA clamping reaction by taking an inner primer as a main material, carrying out amplification by taking the product obtained in the step (4) as a template, and setting the annealing temperature to be 56-62 ℃ during amplification, wherein the inner primer can be complementary with the template; for the mutant DNA, the W-PNA is mismatched with the mutant DNA and cannot be combined with the mutant DNA at the corresponding annealing temperature, so that the mutant DNA can be normally amplified by the inner primer, and for the wild type DNA, the W-PNA probe can not be normally amplified due to the inhibition of the extension effect, so that the wild type DNA is inhibited for the second time, and the mutant DNA is enriched;
(6) and finally, combining an LNA fluorescent probe in the reaction system with the mutant DNA amplification product, generating fluorescence, and monitoring the content of mutant DNA in the PCR amplification system through the strength of a fluorescence signal and a standard curve.
2. The detection method according to claim 1, wherein the Tm value of the outer primer is 68 ℃ to 72 ℃, and the amplification product contains a mutation site.
3. The detection method according to claim 1, wherein the inner primers are positioned inside the upstream and downstream outer primers, and the Tm value thereof is 56 ℃ to 62 ℃.
4. The detection method according to claim 1, wherein the LNA probe is labeled at the 5 'end with a FAM fluorophore and at the 3' end with a TAMA quencher; the 5 'end of the internal standard probe is marked with a HEX fluorescent group, and the 3' end is marked with a BHQ1 quenching group.
5. The detection method according to claim 1, wherein in the step (4), the first round of PNA clamping reaction is performed, and the thermal cycling reaction is set to be firstly maintained at 95 ℃ for 30 sec; then, the cycle was repeated 15 times at 95 ℃ for 5sec, and at 68 ℃ for 34 sec.
6. The assay of claim 1, wherein in step (5), the second round of PNA clamping reaction, the thermal cycling reaction is set to first maintain 95 ℃ for 5 sec; then the temperature is reduced to 60 ℃ for 34sec, and the circulation is carried out for 40 times.
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