Primer probe combination for distinguishing and relatively quantifying wild type and secondary drug-resistant mutation of EGFR (Epidermal Growth Factor Receptor) gene

Abstract

The invention relates to the technical field of biomedical treatment, in particular to a primer probe combination for distinguishing and relatively quantifying wild type and secondary drug-resistant mutation of EGFR (Epidermal Growth Factor Receptor) genes. The ARMS PCR primer probe combination, the detection reagent, the kit and the detection method which take the EGFR gene as the target are obtained by optimizing the mutation site position and concentration of the primer probe and screening the annealing temperature and enzyme. The primer probe combination, the detection reagent, the kit and the detection method disclosed by the invention are used for ARMS PCR detection of EGFR genes, the amplification inhibition capability on a wild type template is improved, non-specific amplification is reduced to improve specificity, and meanwhile, a good linear relationship can be kept; the primer design process is simplified, and introduction of extra mismatched bases is avoided; a reliable relative quantitative system is established, experimental interference factors are eliminated, and abundance of mutant genes is accurately reflected; the detection period is shortened, the cost is reduced, clinical rapid detection requirements are met, and a more reliable technical means is provided for detection of mutant genes.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medical technology, and particularly relates to a primer probe combination for distinguishing and relatively quantifying wild type and secondary drug resistance mutations of an EGFR gene. BACKGROUND

[0002] The T790M mutation of an epidermal growth factor receptor (EGFR) gene is the main cause (about 50% to 60%) of resistance to first-generation and second-generation TKIs (Tyrosine kinase inhibitors). If T790M is positive, the third-generation TKI drug such as osimertinib can be used. The C797S mutation is the main drug resistance mutation (about 14% to 24%) after treatment with the third-generation TKI, and there is no suitable TKI drug for treating patients with C797S positive according to the guidelines. At this time, the patient can only use chemotherapy plus bevacizumab for treatment. Therefore, timely monitoring of secondary drug resistance mutations of non-small cell lung cancer patients after TKI treatment has important guiding significance for clinical drug decision-making.

[0003] The amplification refractory mutation system (ARMS) PCR method is a commonly used method for detecting gene mutations. The existing ARMS PCR method adds an additional mismatch at the 3' end of the primer according to the base strength mismatch rule, which can further inhibit the amplification of the wild type. Different combinations of mismatches are usually used, and the inhibition effect produced by different combinations is also different. Among them, strong mismatches include C-T, G-A and T-T; medium mismatches include A-A, G-G and C-C; and weak mismatches include T-G and A-C. The specificity can be improved according to strong+weak, medium+medium and weak+strong mismatch combinations. At this time, the primer has two mismatches with the wild type template, which significantly inhibits the amplification of the wild type. However, this method may need to design multiple mismatch combination primers, and the position of the additional mismatch is confirmed by the PCR reaction result. Finally, the best specific primer is selected from a large number of designed primers to improve the amplification specificity, which increases the difficulty and cost of primer design, and causes waste of remaining primers. At the same time, after adding an additional mismatch, the binding efficiency of the primer and the template may be reduced, which also affects the amplification of the primer and the mutant. Therefore, although this method improves the specificity, it sacrifices the sensitivity.

[0004] In addition, clinical diagnosis needs to achieve treatment decision according to the abundance of gene mutation, ARMS PCR method usually adopts absolute quantitative method to calculate the absolute copy number of mutant gene in the sample, in each determination, it needs to rely on standard product (such as recombinant plasmid) with known concentration to establish standard curve, the operation is complicated and is easily interfered by sample extraction efficiency, experimental operation difference and other factors, so that simple and rapid detection cannot be realized, and the real proportion of mutant gene in the sample cannot be accurately reflected.

[0005] Therefore, based on the problems of complex primer design, high cost, low sensitivity, difficult quantification and further improved specificity of the existing ARMS method, it is particularly necessary to develop and design an ARMS method with simple design, low cost, high sensitivity, quantification and further improved specificity. SUMMARY

[0006] Therefore, based on the problems of complex primer design, high cost, low sensitivity, difficult quantification and further improved specificity of the existing ARMS method, it is particularly necessary to develop and design an ARMS method with simple design, low cost, high sensitivity, quantification and further improved specificity.

[0007] The application provides a primer probe combination for distinguishing and relatively quantifying wild type and secondary drug resistance mutation of EGFR gene, which comprises a primer probe combination for target gene amplification.

[0008] The primer probe combination for target gene amplification comprises an upstream primer, a downstream primer and a probe.

[0009] The last nucleotide at the 3' end of the upstream primer is different from the wild type nucleotide and is the same as the nucleotide of the secondary drug resistance mutation.

[0010] The downstream primer contains the secondary drug resistance mutation in the amplification interval of the upstream primer.

[0011] Further, in the specific embodiments of the application, the target gene is EGFR gene.

[0012] The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, 2 and / or 3.

[0013] The nucleotide sequence of the downstream primer is shown in SEQ ID NO: 4.

[0014] The nucleotide sequence of the probe is shown in SEQ ID NO: 5.

[0015] Further, the primer probe combination provided by the application further comprises a primer probe combination for detecting an internal reference gene.

[0016] The primer probe combination for detecting the internal reference comprises: an internal reference upstream primer with a nucleotide sequence as shown in SEQ ID NO: 6, an internal reference downstream primer with a nucleotide sequence as shown in SEQ ID NO: 7, and an internal reference probe with a nucleotide sequence as shown in SEQ ID NO: 8.

[0017] Further, in the primer probe combination, the probe for detecting the target gene is labeled with a FAM fluorescent group at the 5' end and a BHQ1 quenching group at the 3' end; and the probe for detecting the internal reference gene is labeled with a VIC fluorescent group at the 5' end and a BHQ3 quenching group at the 3' end.

[0018] The present application provides a primer probe combination for EGFR gene as a target based on an amplification refractory mutation system (ARMS) PCR method; in the traditional ARMS PCR method, an additional mismatch needs to be added in the primer design, and the additional mismatch needs to be arranged in a strong-weak manner; the present application optimizes the position of the mutation site in the primer, and obtains an ARMS PCR method primer probe combination which can distinguish the mutant type and the wild type of the EGFR gene. The primer probe combination is used for amplification of the EGFR gene, and has high specificity and good sensitivity.

[0019] The present application provides a detection reagent, which comprises the primer probe combination and SNUPP enzyme.

[0020] Further, the amplification reagent also comprises: an amplification buffer and / or a reference fluorescent dye.

[0021] In the amplification reagent,

[0022] The concentration of the upstream primer is 0.4 μM to 1.0 μM; preferably 0.4 μM;

[0023] The concentration of the probe is 0.8 μM to 1.2 μM; preferably 0.8 μM.

[0024] The enzyme in the detection reagent is screened, and after screening of various existing DNA polymerases, the results show that the specificity of the SNUPP enzyme of Shengshi Kail is significantly improved.

[0025] And the present application optimizes the addition amount of the upstream primer and the probe for amplification of the target gene in the amplification reagent, and the results show that the optimal concentration of the upstream primer is 0.4 μM; and the optimal concentration of the probe is 0.8 μM.

[0026] The present application provides a kit, which comprises: the detection reagent, the secondary drug resistance mutation positive reference product and the internal reference positive reference product.

[0027] The present application provides a method for distinguishing and relatively quantifying gene wild type and secondary drug resistance mutations, which comprises detecting a target gene by using at least one of I)~III) as follows:

[0028] I), the primer probe combination of the present application;

[0029] II), the detection reagent of the present application;

[0030] III), the kit of the present application.

[0031] Further, the method of the present application comprises the following steps: mixing a nucleic acid template with the detection reagent of the present application and then performing amplification.

[0032] In the amplification, the annealing temperature is 60℃.

[0033] In a specific embodiment of the present application, the amplification procedure is 95℃ pre-denaturation for 3min; then 95℃ denaturation for 5s, 60℃ annealing for 30s, 68℃ extension for 30s, and the cycle is repeated for 40 times; 68℃ final extension for 5min.

[0034] In the method of the present application,

[0035] The present application provides an ARMS PCR detection method for EGFR gene, which further improves the mutation detection specificity and sensitivity by optimizing the primer probe combination in the detection method; then effectively avoids the occurrence of non-specific amplification by screening DNA polymerase, further improves the mutation detection specificity; then further improves the detection sensitivity and mutation detection specificity by optimizing the reagent concentration and ratio; therefore, each parameter in the method of the present application influences each other, and jointly influences the sensitivity and mutation detection specificity of the detection result; and the method of the present application can realize relative quantification of the target gene by standard curve method.

[0036] The present application obtains ARMS PCR primer probe combination, detection reagent, kit and detection method for EGFR gene as a target by optimizing the primer probe mutation site position and concentration, annealing temperature and enzyme screening; the primer probe combination, detection reagent, kit and detection method of the present application are used for ARMS PCR detection of EGFR gene, which improves the amplification inhibition ability for wild type template, reduces non-specific amplification to improve specificity, simplifies the primer design process, avoids the introduction of additional mismatched bases, establishes a reliable relative quantification system, eliminates experimental interference factors, accurately reflects the abundance of mutant genes, shortens the detection period, reduces the cost, adapts to the clinical rapid detection demand, and provides a more reliable technical means for the detection of mutant genes. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The detection principle and relative quantitative formula of the improved ARMS PCR method are shown in the following table:

[0038] Figure 2 The wild type sequence corresponding to the mutation site is shown in the following table; Box 1 is the wild type codon corresponding to the T790M mutation site; Box 2 is the wild type codon corresponding to C797S; double underlined is the downstream primer EGFR-R; single underlined is the probe EGFR-F-UP;

[0039] Figure 3 The thermal cycle setting for detection by the high-fidelity polymerase of the application is shown in the following table:

[0040] Figure 4 The standard curve is shown in the following table; A is the T790M standard curve; B is the C797S1 standard curve; C is the C797S2 standard curve; the abscissa is Δct, and the ordinate is the log value of the mutation abundance;

[0041] Figure 5 The amplification curve after adding to the real plasma sample is shown in the following table; A is the mutation amplification curve of the first example of T790M plasma free DNA sample; B is the mutation amplification curve of the second example of T790M plasma free DNA sample; C is the amplification curve of T / A mutation (C797S1) in EGFR C797S mutation; D is the amplification curve of G / C mutation (C797S2) in EGFR C797S mutation;

[0042] Figure 6 The consistency analysis chart of the plasma sample detection result and the second generation sequencing method result is shown in the following table (in the form of a scatter plot, the abscissa is the mutation abundance detected by the method, and the ordinate is the mutation abundance detected by the second generation sequencing method, and the consistency is evaluated by calculating the correlation coefficient);

[0043] Figure 7 The high-fidelity enzyme screening result of the application is shown in the following table; A is the amplification specificity of different high-fidelity enzymes for EGFR T790M; B is the amplification specificity of different high-fidelity enzymes for EGFR C797S1; C is the amplification specificity of different high-fidelity enzymes for EGFR C797S2;

[0044] Figure 8 The EGFR T790M, EGFR C797S1 and EGFR C797S2 primer mutation position screening result is shown in the following table; A is the EGFR T790M primer mutation position screening result; B is the EGFR C797S1 primer mutation position screening result; C is the EGFR C797S2 primer mutation position screening result;

[0045] Figure 9Figure EGFR T790M, EGFR C797S1 and EGFR C797S2 amplification primer concentration optimization results; wherein, A is the primer EGFR-790-1-1 concentration optimization result; B is the primer EGFR-797-T / A-1-1 concentration optimization result; C is the primer EGFR-797-G / C-1-1 concentration optimization result;

[0046] Figure 10 Figure probe optimization results; wherein, A is the influence of different probe concentrations on EGFR T790M amplification; B is the influence of different probe concentrations on EGFR C797S1 amplification; C is the influence of different probe concentrations on EGFR C797S2 amplification;

[0047] Figure 11 Figure annealing temperature optimization results, wherein, A is the influence of different annealing temperatures on EGFR T790M amplification; B is the influence of different annealing temperatures on EGFR C797S1 amplification; C is the influence of different annealing temperatures on EGFR C797S2 amplification;

[0048] Figure 12 Figure internal reference optimization results, wherein, A is the influence of different internal reference genes on EGFR T790M amplification; B is the influence of different internal reference genes on EGFR C797S1 amplification; C is the influence of different internal reference genes on EGFR C797S2 amplification;

[0049] Figure 13 Figure performance evaluation results of control ARMS PCR method (traditional ARMS PCR method) amplification sensitivity and mutation detection sensitivity, wherein, A is the EGFR T790M amplification sensitivity detection result; B is the EGFR T790M mutation sensitivity detection result; C is the EGFR C797S1 amplification sensitivity detection result; D is the EGFR C797S1 mutation sensitivity detection result; E is the EGFR C797S2 amplification sensitivity detection result; F is the EGFR C797S2 mutation sensitivity detection result;

[0050] Figure 14 Figure ARMS PCR method (experimental group) of the application in amplification sensitivity and mutation detection sensitivity results, wherein, A is the EGFR T790M amplification sensitivity detection result; B is the EGFR T790M mutation sensitivity detection result; C is the EGFR C797S1 amplification sensitivity detection result; D is the EGFR C797S1 mutation sensitivity detection result; E is the EGFR C797S2 amplification sensitivity detection result; F is the EGFR C797S2 mutation sensitivity detection result;

[0051] Figure 15Amplification with super high specificity DNA polymerase (SNUPPase) 10 7 ~10 0 Results of wild type template, directly from 10 7 Inhibition of wild type amplification, where A is EGFR T790M result; B is EGFR C797S1 result; C is EGFR C797S2 result;

[0052] Figure 16 Amplification with ordinary ARMS PCR enzyme (TaKaRa Premix Ex Taq™ (Probe qPCR), item number RR390A) 10 7 ~10 0 Results of wild type template, where A is EGFR T790M result; B is EGFR C797S1 result; C is EGFR C797S2 result; 10 2 Corresponding 7971 mutation to 10 4 For 7972 mutation to 10 5 Amplification;

[0053] Figure 17 Results of screening of primer positions for traditional ARMS PCR method, where A is T790M primer optimization result; B is C797S1 primer optimization result; C is C797S2 primer optimization result. DETAILED DESCRIPTION

[0054] The present application provides a primer probe combination for distinguishing and relatively quantifying wild type and secondary drug resistance mutations of EGFR gene, and those skilled in the art can improve the process parameters according to the content herein. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0055] The traditional ARMS-PCR is to realize high-specificity detection of DNA mutation by using the principle that the 3' end base of the primer is not complementary to the template base, and the DNA polymerase cannot be extended. The technology has the advantages of simple operation, short reaction time, low detection cost without using expensive special reagents and instruments. However, the technology also has problems of low specificity (≥1%) and difficulty in quantification. With the aid of the ultra-high-specificity DNA polymerase (Shengshi Kail SNUPP enzyme), the enzyme has 3'-5' exonuclease activity, i.e. correction activity, when the mismatch between the template and the primer is recognized, the incorrectly paired base is immediately removed, effectively avoiding the occurrence of non-specific amplification, so even if there is no additional mismatch at the 3' end of the primer, the mutant and the wild type template can be accurately distinguished, and the amplification specificity is significantly improved. At the same time, without adding additional base mismatches, the same effect as the traditional ARMS PCR method can be achieved, greatly simplifying the primer design process; in addition, by using a relative quantification formula, the mutation abundance of the mutant gene is calculated according to the difference between the Ct values of the mutant gene and the selected internal reference gene, which can eliminate the influence of non-specific factors such as sample size and extraction efficiency, save the steps of standard preparation, calibration and verification, reduce the experimental period and operation complexity, and make the quantitative results of the mutant gene closer to the real initial template amount, thereby ensuring the accuracy of the calculation of the mutation abundance; at the same time, the standard curve of the application only needs to be constructed once and can be used multiple times.

[0056] In the specific detection, the DNA of the sample to be detected needs to be extracted; the DNA of the sample to be detected is extracted and the quality of the DNA is evaluated, the content and the OD260 / 280 ratio of the DNA are determined by a ultraviolet spectrophotometer, and the DNA purity is ensured to be between 1.8 and 2.0;

[0057] In the specific detection of the application, the internal reference gene is a stable and high-abundance gene in tissues or plasma, which can be stably expressed without being affected by experiments, and the fluorescent probe group is different from the fluorescent group of the mutant amplification product, so that there are two amplification curves; the CT difference between the target gene and the internal reference gene can realize the relative quantification of the target gene.

[0058] The relative quantification method is that the actin gene (β-actin) is used as the internal reference gene, the target mutant gene and the internal reference gene are amplified by PCR respectively, the probes of the target gene and the internal reference gene are modified and designed by different fluorescent groups respectively, different fluorescent signals are generated after amplification, the threshold cycle number Ct values corresponding to the target gene and the internal reference gene are obtained, the Ct values of the target gene and the internal reference gene are subtracted to obtain △ct, and the linear regression equation of the △ct of the standard and the mutation abundance is: Log (mutation abundance) = k·△Ct + b, wherein k and b are constants, R 2=0.99, then detect the clinical sample, use the obtained delta ct to calculate the mutation abundance, and compared with the second generation sequencing method, good consistency is obtained.

[0059] Compared with the prior art, the present application has the following advantages: first, compared with the second generation sequencing method, the present application does not require complex instruments and cumbersome experimental procedures, only needs to design a conventional allele-specific primer and combine with real-time fluorescent PCR amplification detection, the requirement for laboratory hardware conditions is low, and the present application is very suitable for primary medical institutions or clinical rapid detection scenes, and is convenient for large-scale popularization and application; second, the entire experimental process, including detection and result analysis, can be completed within several hours, which is much faster than the several days required by the second generation sequencing method, and can quickly provide a strong basis for clinical treatment decision, and is especially suitable for patients who need to adjust the treatment scheme in time. In addition, the present application does not require high sequencing instrument investment and reagent cost, the primer design and fluorescent probe preparation cost are much lower than those of the second generation sequencing method, and the present application is suitable for large-scale screening or routine detection requirements which are sensitive to detection cost, and effectively reduces the economic burden of clinical diagnosis.

[0060] Compared with the traditional ARMS PCR method, the present application avoids the step of adding an additional base mismatch when designing a primer, and simplifies the primer screening process, under the action of ultrahigh specificity DNA polymerase, the amplification of wild-type genes is significantly inhibited according to the base mismatch strength rule; the mutation abundance of the mutant gene can be accurately determined through the relative quantitative formula, as long as the internal reference genes are consistent, the change of the mutation abundance can be directly shown after correction by the same internal reference, the result misjudgment caused by the fluctuation of experimental conditions is reduced, the change of the mutation gene abundance in the patient's body is dynamically monitored, the treatment effect can be understood in time, which provides a basis for adjusting the treatment scheme, and helps to improve the treatment success rate and life quality of the patient. Especially suitable for scenes with complex samples, low abundance and large demand, and provides a reliable quantitative basis for disease diagnosis, treatment evaluation and prognosis.

[0061] In the present application, C797S1 and C797S2 are also written as C797S1 and C797S2, which are used to distinguish the two mutations of C797S;

[0062] EGFR-T790M / C797S1 / C797S2-Im-ARMS PCR refers to the method of the present application;

[0063] The reagents used in the present application are all ordinary market products, and can be purchased in the market. The present application is further described below in combination with examples:

[0064] Example 1: Primer probe combination for distinguishing and relatively quantifying wild-type and secondary drug resistance mutations of EGFR gene

[0065] The application establishes a high-sensitivity, high-specificity and quantifiable point mutation detection method, and the detection principle is as shown in the formula (I) : Figure 1 In the calculation formula of Δct, M is the ct value of the mutant; C is the ct value of the internal reference; and after obtaining Δct, a standard curve is established to obtain the log value of the mutation abundance (log(mutation abundance)).

[0066] In order to evaluate the detection of the method, the EGFR T790M and C797S mutation sites related to non-small cell lung cancer TKI secondary drug resistance are taken as examples for method display.

[0067] Four plasma samples with known T790M mutant type, C797S1 mutant type and C797S2 mutant type are taken as four plasma free DNA samples (of the four samples, two are T790M plasma free DNA samples, and the remaining mutant plasma free DNA samples are each one; the known amount of T790M, C797S1, C797S2 mutant plasmid product and internal reference plasmid product is added to the plasma sample, wherein the mutant plasmid product is 8×10 4 copies; the internal reference plasmid product is 8×10 5 copies), and a control plasma sample (8×10 5 copies of internal reference plasmid product are added to the control sample), wherein two T790M samples, one control / C797S1 / C797S2 sample, the above samples are divided into two parts; one part is detected by the method of the application, and the plasma ctDNA is extracted from the plasma by TheSimgen Circulating Nucleic Acid Kit to obtain a simulated plasma free DNA sample, which is used for subsequent method establishment and detection, and the other part is sent to a second-generation sequencing company for detection:

[0068] I. Four plasma free DNA plasma samples and control plasma samples are detected by the ARMS-PCR method of the application

[0069] 1. Primer sequence: the primers and probes required for EGFR T790M (EGFR T790M is C / T mutation), EGFR C797S1 (T\A mutation in EGFR C797S mutation) and EGFR C797S2 (G / C mutation in EGFR C797S mutation) are shown in Table 1:

[0070] Table 1. Primers and probes required for detecting EGFR T790M / C797S of the application

[0071]

[0072] EGFR-790-1-1 is an upstream primer for amplifying EGFR T790M mutation; EGFR-797-T / A-1-1 is an upstream primer for amplifying T\A mutation in EGFR C797S mutation; EGFR-797-G / C-1-1 is an upstream primer for amplifying G / C mutation in EGFR C797S mutation; EGFR-R is a downstream primer corresponding to the above three upstream primers, and after amplification, the mutation site is located in the amplification product; EGFR-F-UP is a probe for EGFR T790M and EGFR C797S. Among the primers EGFR-790-1-1, EGFR-797-T / A-1-1 and EGFR-797-G / C-1-1, the last base, i.e. the first base at the 3' end, is mismatched with the wild type and completely identical with the mutant, and the wild type sequence containing the mutation segment is shown in Figure 2 .

[0073] 2. Reagent formula (20 μL reaction system): template DNA 2 μL, SNUPP Taq Buffer 1x, SNUPP Taq enzyme 0.1 U / μL, EGFR-790-1-1 (or EGFR-797-T / A-1-1 or EGFR-797-G / C-1-1) 0.4 μM, EGFR-R 0.4 μM, β-actin-F 0.4 μM, β-actin-R 0.4 μM, reference fluorescence ROX-II 0.5x, ordinary fluorescence probe (EGFR-F-UP) 0.8 μM, internal standard fluorescence probe (β-actin-probe) 0.8 μM, ddH2O to make up the reaction system to 20 μL.

[0074] 3. Amplification program setting: pre-amplification at 95℃ for 3 min; then 95℃ for 5 sec, 60℃ for 30 sec, 68℃ for 30 sec, 40 cycles; finally, 68℃ for 5 min for the final extension step, as shown in Figure 3 .

[0075] II. Establishment of standard curve

[0076] The amount of internal standard detected in the amplification system is 10 5 copies / μL, and 10 5 , 10 4 , 10 3 , 10 2 , 10 1 copies / μL mutant plasmid product combined with 10 5 copies / μL internal standard plasmid product to make a standard curve of mutation abundance and Ct value difference, as shown in Figure 4 .

[0077] III. Detection Results of the ARMS-PCR Method of the Present Invention

[0078] like Figure 5 As shown, the method of the present invention can achieve highly sensitive, highly specific, and quantitative detection of EGFR T790M mutation, T / A mutation in EGFR C797S mutation, and G / C mutation in EGFR C797S mutation in plasma samples. According to... Figure 5 The difference between the mutant and the internal reference ct value is used to obtain Δct, which is then substituted into... Figure 4 The vertical axis log (mutation abundance) was obtained by standardizing the curve, and then converted into exponential form as shown in Table 2. The mutation abundance of the T790M mutant is 6.14% and 6.83%, respectively, the mutation abundance of the C797S1 mutant is 5.11%, and the mutation abundance of the C797S2 mutant is 7.26%.

[0079] Furthermore, the detection results of this method were compared with those of next-generation sequencing to analyze their consistency. The results are shown in Table 2 and... Figure 6 As shown, the method of this invention exhibits a good linear relationship with next-generation sequencing in mutation abundance detection, R 2 The value of 0.9377 indicates that this method has good accuracy and reliability in the detection of real plasma samples.

[0080] Table 2. Mutation abundance detection of EGFR T790M / C797S single mutant samples

[0081] Comparison table with second-generation sequencing results

[0082]

[0083] Example 3: Optimization of the ARMS PCR method described in this invention

[0084] I. Screening of High-Fidelity Enzyme Products

[0085] We searched for high-fidelity enzyme products from both domestic and international biotechnology companies as candidates for screening, as detailed below:

[0086] Table 3. High-fidelity enzymes to be screened

[0087]

[0088] The screening criteria were as follows: EGFR T790M, C797S1, and C797S210 were screened according to the conditions specified in the instructions for each high-fidelity enzyme. 6The wild type W and mutant M amplification, the wild type ct value obtained by inserting 3' end mismatched primer and inserting additional mismatched primer and the mutant ct value difference (amplification specificity, the greater the value, the higher the specificity), while high fidelity enzyme amplification should not affect the amplification of mutant M (when the mutant amplification is inhibited, the greater the value, the more enhanced the inhibition of mutant amplification), and the SNUPP enzyme is selected according to the above (SNUPP enzyme Figure 7 ).

[0089] II. EGFR T790M, EGFR C797S1 and EGFR C797S2 primer mutation position screening results

[0090] The position of the mismatched base in the primer EGFR-790-1-1, the primer EGFR-797-T / A-1-1, and the primer EGFR-797-G / C-1-1 in Table 1 is explored; specifically, the above three primers are added with base mismatches with the wild type at the 3' end and completely complementary to the mutant, 1-X (X is any integer from 1 to 8) represents that in addition to the addition of mismatched bases at the 3' end, an additional mismatch is added at the Xth position from the end, and the primer setting is specifically shown in Table 4; the primers in Table 4 are used to replace the primers EGFR-790-1-1, the primer EGFR-797-T / A-1-1, and the primer EGFR-797-G / C-1-1 in Table 1, respectively, and the reagent formula and amplification procedure of Example 1 are used to screen the appropriate primer mutation position.

[0091] Table 4. Mutation position optimization

[0092]

[0093] The experimental results show that in the reaction system, 1-1~1-8 primers are added respectively to carry out 10 6 Wild type W and mutant M amplification, according to Figure 8 It can be concluded that the selected ultra-high specificity DNA polymerase does not need to insert additional mismatched bases, but only needs to insert 1 mismatched base at the 3' end to achieve ultra-high specificity amplification.

[0094] III. Optimization of primer concentration

[0095] In the reaction system, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 μM of the primer EGFR-790-1-1, the primer EGFR-797-T / A-1-1, and the primer EGFR-797-G / C-1-1 are added, respectively, the other components of the reagent formula are as in Example 1, the amplification procedure is as in Example 1, and the amplification intensity and specificity difference under each concentration are compared, Figure 9For the mutant amplification curve, the curve height can represent the amplification intensity, and the ct value can represent the specificity. Therefore, the primer concentration of 0.4 μM was selected as the reaction condition.

[0096] IV. Optimization of probe concentration

[0097] In the reaction system, 0.2, 0.4, 0.6, 0.8, 0.6, 1.0, and 1.2 μM of the probe (EGFR-F-UP) were added, respectively. The other components of the reagent formula were as described in Example 1, and the amplification procedure was as described in Example 1. The amplification intensity and specificity difference at each concentration were compared. Figure 10 For the mutant amplification curve, the curve height can represent the amplification intensity, and the ct value can represent the specificity. Therefore, the primer concentration of 0.4 μM was selected as the reaction condition.

[0098] V. Optimization of annealing temperature

[0099] After the optimal primer position, primer concentration, and probe concentration were determined (the specific primer and probe sequences were as described in Example 1, and the reagent formula was as described in Example 1), the annealing temperature in the amplification procedure (pre-amplification at 95°C for 3 min; then 95°C for 5 sec, 60°C (annealing temperature) for 30 sec, 68°C for 30 sec, 40 cycles, and finally 68°C for 5 min for terminal extension) was set to 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, 60°C, 62°C, 64°C, 66°C, and 68°C, respectively. The influence on amplification was observed. The blue line represents the ct value of the mutant template, and the red line represents the ct value difference between the wild type and the mutant. The selection criteria are as described above. Finally, 60°C was selected as the annealing temperature. Figure 11 ).

[0100] VI. Results of EGFR T790M / C797S mutation combined with different internal control genes

[0101] According to the primers and probes shown in Table 5, 10 6 The EGFR exon 7 (Exon 7), 15 (Exon 15), 17 (Exon 17), 25 (Exon 25), and β-actin internal control plasmid products (amplification products) with the same mutant content were added to the reaction system as internal controls.

[0102] Table 5. Primers and probes for internal control genes

[0103]

[0104] The control was without the addition of internal control primers and probes; the control group and the different internal control groups were different from the internal control primers and probes and the template (at a concentration of 1 × 10 6The mixed concentration of wild-type and mutant positive plasmid amplification products was 1×10⁻⁶ copies / μL. 6 (The amplification product of the internal control F / R primer (copy / μL) is used as a template) differs from Example 1, but everything else is the same as in Example 1. Results are as follows: Figure 12 As shown, the difference in ct values ​​between wild-type and mutant groups compared with the control group can reflect the amplification specificity and observe whether the different internal reference genes affect amplification. Since the content is the same, the ct values ​​of mutant and internal reference should be the same. If there is a difference, it means that amplification is affected. In summary, β-actin was selected as the internal reference gene.

[0105] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0106] Comparative Example 1: Comparison between the control ARMS PCR method (traditional ARMS PCR method) and the present invention

[0107] Using simulated samples, the ARMS PCR method (experimental group) of the present invention was compared with the ARMS PCR method of the control group; the primer and probe, reagent formulation and amplification program of the ARMS PCR method (experimental group) of the present invention are as in Example 1.

[0108] The simulated samples were obtained by extracting plasmid amplification products using the Tiangen Common DNA Product Purification Kit. Known concentrations of mutant plasmid amplification products (EGFR T790M mutation, EGFR C797S T / A mutation, and EGFR C797S G / C mutation amplification products) were then diluted 10-fold serially to a final concentration. 7 10 6 10 5 10 4 10 3 10 2 10 1 and 10 0 Different concentrations of copies / μL were used as amplification templates to evaluate amplification sensitivity. At 10... 5 Add 10 to the wild-type plasmid amplification product respectively 5 10 4 10 3 10 2 10 1 and 10 0 The mutant plasmid amplification product was prepared at 50% (M:W=10) copies / μL. 5 10 5 ), 10% (M:W=10) 4:10 5 :10 3 :10 5 :10 2 :10 5 :10 1 :10 5 :10 0 :10 5 :10 5 :10 5 :10

[0109] I. Primer, reagent and detection steps of control group ARMS PCR method (traditional ARMS PCR method)

[0110] 1. The primer sequence of the control group ARMS PCR method is shown in Table 6:

[0111] Table 6. Primer and sequence required for detecting EGFR T790M C797S by control ARMS PCR method

[0112]

[0113] EGFR-790-MIP1-7 is an upstream primer for amplifying EGFR T790M mutation; EGFR-797T / A-MIP1-6 is an upstream primer for amplifying T\A mutation in EGFR C797S mutation; EGFR-797G / C-MIP1-6 is an upstream primer for amplifying G / C mutation in EGFR C797S mutation; EGFR-R is a downstream primer corresponding to the above three upstream primers; EGFR-F-UP is a probe for EGFR T790M and EGFR C797S. Among them, in addition to the 3' terminal base mismatch in primers EGFR-790-MIP1-7, EGFR-797-T / A-1-6 and EGFR-797G / C-MIP1-6, there is also a mismatch from the 3' end Xth base.

[0114] 2. The reagent formula of the ARMS PCR method of the control group (20 μL reaction system): template 2 μL, Premix Ex Taq™ (Probe qPCR, Takara, containing Taq enzyme) 1×, EGFR-790-MIP1-7 (or EGFR-797-T / A-1-6, or EGFR-797G / C-MIP1-6) 0.2 μM, EGFR-R 0.2 μM, β-actin-F 0.2 μM, β-actin-R 0.2 μM, reference fluorescence ROX-II 1×, ordinary fluorescence probe (EGFR-F-UP) 0.8 μM, internal reference fluorescence probe (β-actin-probe) 0.8 μM, ddH2O to supplement the reaction system to 20 μL.

[0115] 3. The amplification program setting of the control group: pre-amplification is 95℃ for 30 sec; then 95℃ for 5 sec, 60℃ for 34 sec for 40 cycles.

[0116] Secondly, the ARMS PCR method (experimental group) of the application is compared with the ARMS PCR method of the control group.

[0117] The comparison results of the sensitivity and mutation detection specificity of the experimental group method and the control group method are as follows: Figure 13 and 14 The results show that the amplification sensitivity of the ARMS PCR method of the control group is 10 0 copies / reaction, the linear range is 10 7 ~10 0 copies / reaction, and R 2 is 0.99; while the amplification sensitivity of the ARMS PCR method of the application (experimental group) is 10 0 ~10 1 copies / reaction, the linear range is 10 1 ~10 7 copies / reaction, and R 2 is 0.99. However, for the inhibition of wild type, the ARMS PCR method of the control group can only inhibit T790M10 2 , C797S1 10 4 and C797S2 10 5 wild type, while the ARMS PCR method of the experimental group of the application can directly inhibit 10 7 wild type amplification, which is improved by at least two orders of magnitude, and the influence of wild type on amplification is suppressed at the source. Figure 15 and Figure 16 .

[0118] Meanwhile, the LOD of mutation detection of the two methods in the presence of wild-type DNA was also evaluated. The LOD values of T790M, C797S1, and C797S2 in the control group ARMS PCR method were 0.1%, 0.01%, and 0.001%, respectively, and the LOD values of the three mutant types after the ARMS PCR method in the experimental group were 0.01%, 0.01%, and 0.001%, respectively, indicating that the improved ARMS PCR method significantly improved the inhibition ability of wild-type DNA and the detection ability of low-abundance mutations while maintaining high linearity, and is especially suitable for precise detection of low-concentration mutation samples such as ctDNA.

[0119] Example 2 Optimization of traditional ARMS PCR method

[0120] Replace EGFR-790-MIP1-7, EGFR-797T / A-MIP1-6, and EGFR-797G / C-MIP1-6 in Table 6 with EGFR-790-1-1 (1-1) ~ EGFR-790-1-8 (1-8), EGFR-797-T / A-1-1 (1-1) ~ EGFR-797-T / A-1-8 (1-8), and EGFR-797-G / C-1-1 (1-1) ~ EGFR-797-G / C-1-8 (1-8) in Table 4, respectively, and use 1 × 10 6 copies / μL wild-type and 1 × 10 6 copies / μL mutant template amplification product as the detection sample, and perform detection according to the reagent formula and amplification program of the control group ARMS PCR method in Example 1.

[0121] The results are shown in Table 7. Figure 17 The difference between the wild-type and mutant ct values, i.e., the amplification specificity, is greater, the specificity is better, and the screening condition requires that it does not affect the amplification of the mutant, i.e., there is no significant amplification inhibition. In summary, the primer positions represented by EGFR-790-1-7 (1-7), EGFR-797-T / A-1-6 (1-6), and EGFR-797-G / C-1-6 (1-6) arrows are selected, and the SNUPPase mentioned in the patent only needs to be inserted with a mismatch at the 3' end. Unlike the traditional method, inserting one mismatch at the 3' end cannot achieve the best amplification sensitivity and mutation detection specificity. Therefore, under the amplification of the traditional ARMS PCR method, in addition to the base mismatch at the 3' end of the primer, a mismatch needs to be inserted at other positions to achieve better amplification effect.

[0122] In addition, the detection sensitivity and specificity of the control group ARMS PCR method shown in Table 6 are not as good as the present application after optimization of the primer and probe concentrations and optimization of the annealing temperature.

Claims

1. A primer probe combination for distinguishing and relatively quantifying wild type and secondary resistance mutations of the EGFR gene, characterized in that, The primer probe combination for target gene amplification comprises an upstream primer, a downstream primer and a probe. The primer probe combination for target gene amplification comprises an upstream primer, a downstream primer and a probe. The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, 2 and / or 3. The nucleotide sequence of the downstream primer is shown in SEQ ID NO:

4. The nucleotide sequence of the probe is shown in SEQ ID NO:

5.

2. The primer probe combination according to claim 1, characterized in that, The primer probe combination for detecting an internal reference gene is also included; the internal reference gene is β-actin.

3. The primer probe combination of claim 2, wherein, The primer probe combination for detecting an internal reference comprises an internal reference upstream primer with a nucleotide sequence shown in SEQ ID NO: 6, an internal reference downstream primer with a nucleotide sequence shown in SEQ ID NO: 7 and an internal reference probe with a nucleotide sequence shown in SEQ ID NO:

8.

4. A test reagent, characterized by The primer probe combination of any one of claims 1-3 and SNUPP enzyme are included.

5. The amplification reagent of claim 4, wherein The amplification reagent further comprises an amplification buffer and / or a reference fluorescent dye.

6. The amplification reagent of claim 5, wherein In the amplification reagent, The concentration of the upstream primer is 0.4-1.0 μM; The concentration of the probe is 0.8-1.2 μM.

7. A kit characterized in that, The detection reagent of any one of claims 4-6, the secondary drug resistance mutation positive reference and the internal reference positive reference are included. The detection of the target gene is performed by using at least one of the following I)-III):

8. A method for distinguishing and relatively quantifying wild-type and secondary drug resistance mutations for non-diagnostic purposes, characterized in that, I) the primer probe combination of any one of claims 1-3; II) the detection reagent of any one of claims 4-6; III) the kit of claim 8. The amplification is performed after mixing the nucleic acid template with the detection reagent of any one of claims 4-6.

9. The method of claim 8, wherein, In the amplification, the annealing temperature is 60°C.

10. The method of claim 8, wherein, ​

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