Primer probe design method for detecting mutant genes, reaction system and application

Abstract

The invention discloses a primer probe design method for detecting mutant genes, a reaction system and application. The method comprises the following steps: designing a conserved region fluorescent probe and a mutation region fluorescent probe in a single amplicon, and carrying out locked nucleic acid modification on the conserved region fluorescent probe and the mutation region fluorescent probe. The mutation frequency can be accurately interpreted through one-time PCR detection, the cross interference reaction between sites is small, and the method is not only suitable for detecting genome DNA mutation, but also suitable for mutation detection of paraffin embedded tissue sample DNA and plasma free DNA samples.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a primer probe design method for detecting mutant genes, a reaction system and an application thereof. Background Art

[0002] At present, the main detection methods for gene mutations are: Sanger sequencing, ARMS-PCR, Real-time PCR, second-generation sequencing NGS and droplet digital PCR (ddPCR). Among them, Sanger sequencing can directly detect sequence information, but the operation is complicated, the detection time is long, the data interpretation is time-consuming, and the sensitivity is not high during batch detection. The mutation frequency is required to reach 20% to 30%. These problems limit its application in clinical detection. ARMS-PCR and Real-time PCR detection accuracy can reach 1%, but there are certain limitations when detecting samples with mutation abundance less than 1%. The detection sensitivity of NGS is greatly improved, and multiple tumor markers can be detected at one time, but the detection cost is high, the detection cycle is long, and the experimental operation requirements are high. Digital droplet PCR is overall cost-effective in terms of sensitivity, cost and detection time.

[0003] The droplet digital PCR quantitative analysis technology distributes single DNA molecules to independent reaction chambers so that only a single DNA molecule exists in each unit. After the PCR amplification reaction, the positive fluorescent signal is detected to achieve single molecule quantification. There are generally two probe design methods for mutation detection using digital PCR. One is to design wild-type and mutant probes for sequences prone to mutation at the same time. The disadvantage of this method is that when there are multiple mutation forms in the system, quantitative deviations will occur. The other is to design a probe for the wild-type sequence and design another probe for the nearby conservative sequence. This method can only determine the presence of mutations in the target area but cannot determine the specific mutation form and type, and the calculation of mutation frequency is relatively complicated.

[0004] Therefore, there is an urgent need to provide a digital droplet PCR primer design method for the presence of different mutation forms at the same genome coordinate to ensure accurate interpretation of mutation frequency and minimal cross-interference reactions between sites. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a primer probe design method, reaction system and application for detecting mutant genes, which has great advantages in detecting gene mutations with multiple mutation forms at target positions in the genome (such as KRAS amino acids 12 and 13, NRAS amino acids 61, EGFR exons 19 and 20, etc.). The present invention designs a probe for a conservative region in a single amplicon to quantify the total copy number (wild-type copy number + mutant copy number), and designs several mutation region probes to quantify the copy number of each mutant type. At the same time, the mutation probe and the conservative sequence are modified to reduce the interference between the mutation sites and balance the TM value, thereby achieving rapid and accurate quantification of each site under the condition of multiple mutations.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a primer probe design method for detecting mutant genes, the method comprising: designing a conserved region fluorescent probe and a mutant region fluorescent probe in a single amplicon and performing locked nucleic acid modification on the conserved region fluorescent probe and the mutant region fluorescent probe.

[0008] Locked nucleic acid (LNA) is a nucleotide derivative. It differs from ordinary nucleotide molecules in that a methylene bridge is introduced at the 2' oxygen atom and 4' carbon atom of the carbon ring to form a lock-like structure, so it is called locked nucleic acid. When LNA binds to DNA / RNA, it follows the Watson-Crick base complementary pairing principle, and the hybridization affinity of LNA is much higher than that of the corresponding DNA / DNA or RNA / RNA. Because the binding of LNA and DNA causes a certain degree of conformational change, oligonucleotides containing LNA bases are more resistant to the action of nucleases and are not easily degraded. LNA not only has a high affinity for DNA, but also has a high sensitivity to base mismatches. It can significantly increase the difference in thermal stability between a fully matched double-stranded chain and a base mismatched double-stranded chain, making it suitable for point mutation detection.

[0009] The conservative region probe and the mutation region probe designed in the present invention are on the same amplicon, which can avoid the quantitative deviation introduced by the wild type and the mutant type due to the different amplification efficiencies. The wild type quantification uses the conservative region sequence instead of the wild type sequence, and the VAF can be accurately determined without considering whether there are other interfering mutations in the system. The VIC signal (the second fluorescent group signal) is the total template number, which simplifies the VAF calculation method. The conservative sequence probe and the mutation site probe are both modified with locked nucleic acid to ensure uniform TM values, avoid different binding efficiencies between different probes, and also avoid mutual interference between different mutation sites, so that the detection accuracy, specificity and sensitivity are better.

[0010] It can be understood that the primer probe design method for detecting mutant genes described in the present invention is applicable to all fluorescent quantitative PCR.

[0011] Preferably, the length of the single amplicon is less than 120 bp.

[0012] Preferably, the mutant gene includes the KRAS gene.

[0013] Preferably, the mutation site of the KRAS gene includes any one of KRAS p.G12D, KRAS p.G13D, KRAS p.G12V, KRAS p.G12C, KRAS p.G12S, KRAS p.G12A or KRAS p.G12R, or a combination of at least two thereof.

[0014] Preferably, the 5' end of the conserved region fluorescent probe and the 5' end of the mutation region fluorescent probe each independently contain a fluorescent group, and the 3' end each independently contain a quenching group.

[0015] Preferably, the fluorescent group includes a first fluorescent group and a second fluorescent group.

[0016] Preferably, the first fluorescent group includes VIC or FAM.

[0017] Preferably, the second fluorescent group includes VIC or FAM, and the second fluorescent group is different from the first fluorescent group.

[0018] Preferably, the quencher group comprises MGB.

[0019] Preferably, the 3' end of the conserved region fluorescent probe and the 3' end of the mutation region fluorescent probe are independently subjected to any one of dideoxy modification, amino modification or phosphorylation modification.

[0020] In a second aspect, the present invention provides a mutation region fluorescent probe for detecting a mutant gene, wherein the mutant fluorescent probe is designed by the primer probe design method described in the first aspect.

[0021] Preferably, the nucleic acid sequence of the mutation region fluorescent probe includes the sequences shown in SEQ ID NO.1-SEQ ID NO.7.

[0022] SEQ ID NO.1: CCTACGCCA / LNA_T / CAGC.

[0023] SEQ ID NO.2: CCTACG / LNA_T / CACCAGC.

[0024] SEQ ID NO.3: CCTACGCCA / LNA_A / CAGC.

[0025] SEQ ID NO.4: CCTACGCCAC / LNA_A / AGC.

[0026] SEQ ID NO.5: CCTACGCCAC / LNA_T / AGC.

[0027] SEQ ID NO.6: CCTACGCCA / LNA_G / CAGC.

[0028] SEQ ID NO.7: CCTACGCCAC / LNA_G / AGC.

[0029] In a third aspect, the present invention provides a primer and a conserved region fluorescent probe for detecting a mutant gene, wherein the primer and the conserved region fluorescent probe are designed by the primer and probe design method described in the first aspect.

[0030] Preferably, the nucleic acid sequence of the conserved region fluorescent probe includes the sequence shown in SEQ ID NO.8;

[0031] SEQ ID NO. 8: AGTGCCTTG / LNA_A / CGATACA.

[0032] Preferably, the nucleic acid sequence of the primer includes the sequences shown in SEQ ID NO.9 and SEQ ID NO.10.

[0033] SEQ ID NO.9: CTCTATTGTTGGATCATATTCGTC.

[0034] SEQ ID NO. 10: ATGACTGAATATAAACTTGTGGTAG.

[0035] In a fourth aspect, the present invention provides a ddPCR reaction system, which includes a ddPCR premix, the mutation region fluorescent probe for detecting mutant genes described in the second aspect and / or the conserved region fluorescent probe for detecting mutant genes described in the third aspect, nuclease-free water and a sample to be tested.

[0036] Preferably, the single reaction dosage of the ddPCR premix is ​​8-12 μL; the single reaction dosage of the mutation region fluorescent probe is 0.25-1 μL, and the concentration is 125-500 nM; the single reaction dosage of the conserved region fluorescent probe is 0.25-1 μL, and the concentration is 125-500 nM; the single reaction dosage of the sample to be tested is 1-5 μL, and the concentration is 0-300 ng; the single reaction dosage of the nuclease-free water is 0-9 μL.

[0037] The point values ​​in the above 8-12 μL may specifically be 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, etc.

[0038] The point values ​​in the above 0.25-1 μL can specifically be selected as 0.25 μL, 0.26 μL, 0.3 μL, 0.4 μL, 0.5 μL, 0.6 μL, 0.7 μL, 0.8 μL, 1 μL, etc.

[0039] The point values ​​in the above 125-500nM can specifically be selected from 125nM, 128nM, 130nM, 200nM, 300nM, 400nM, 500nM and the like.

[0040] The point values ​​in the above 1-5 μL can specifically be selected as 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, etc.

[0041] The point values ​​in the above 0-300ng can specifically be selected as 0ng, 10ng, 20ng, 60ng, 100ng, 200ng, 300ng, etc.

[0042] The point values ​​in the above 0-9 μL can specifically be selected as 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, etc.

[0043] In a fifth aspect, the present invention provides a method for detecting mutant genes, the method comprising: using the genomic DNA or fragmented DNA of the sample to be tested as a template, performing a digital PCR reaction with a fluorescent probe designed using the primer probe design method described in the first aspect, obtaining the droplet copy number of the first fluorescent group and the second fluorescent group signal after the reaction is completed, and obtaining the mutation frequency of the sample to be tested based on the droplet copy number analysis and calculation.

[0044] Preferably, the mutation frequency of the sample to be tested=(the number of copies of the first fluorescent group channel / the number of copies of the second fluorescent group channel)*100%.

[0045] Preferably, the sample to be tested includes genomic DNA, paraffin-embedded tissue sample DNA or plasma free DNA.

[0046] In a sixth aspect, the present invention provides the use of the mutant region fluorescent probe for detecting mutant genes described in the second aspect and / or the conserved region fluorescent probe for detecting mutant genes described in the third aspect in the preparation of a product for detecting mutant genes.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) The conserved region probe and the mutation region probe designed by the present invention are on the same amplicon, which can avoid the quantitative deviation introduced by the different amplification efficiencies between the wild type and the mutant type;

[0049] (2) The wild-type quantification of the present invention uses the conservative region sequence instead of the wild-type sequence, and the VAF can be accurately determined without considering whether there are other interfering mutations in the system. The VIC signal is the total template number, which simplifies the VAF calculation method;

[0050] (3) In the present invention, both the conserved region probe and the mutation region probe are modified with locked nucleic acid to ensure uniform TM value, avoid different binding efficiencies between different probes, and also avoid mutual interference between different mutation sites, so as to improve detection accuracy, specificity and sensitivity;

[0051] (4) The present invention is universally applicable to samples and can be used to detect low-abundance tissue samples, puncture samples, and plasma free DNA samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is a one-dimensional graph of the ddPCR results in Example 2;

[0053] Figure 2 It is a two-dimensional graph of the ddPCR results in Example 3;

[0054] Figure 3 This is a two-dimensional graph of the ddPCR results in Example 4. DETAILED DESCRIPTION

[0055] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0056] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0057] Example 1

[0058] This embodiment provides a digital PCR method for detecting mutant genes.

[0059] (1) Prepare the reaction system shown in Table 1.

[0060] Table 1

[0061] Component name Single reaction dosage ddPCR master mix (2×) 10μL Upstream primer (18 μM) 1μL Downstream primer (18 μM) 1μL Conserved region probe (10 μM) 0.5μL Mutation region probe (10 μM) 0.5μL Nuclease-free water 6μL Sample (10ng / μL) 1μL Total volume 20μL

[0062] (2) Use the Automated Droplet Generator in the Bio-Rad QX200 AutoDG Droplet Digital PCR System to generate "oil-in-water" droplets for the ddPCR reaction system. After the droplet system is completed, all droplets are transferred to a 96-well plate. After sealing the plate, the next PCR reaction can be carried out.

[0063] (3) The microdroplet system was placed on a PCR instrument for amplification reaction. The amplification condition parameters were as shown in Table 2.

[0064] Table 2

[0065]

[0066] (4) The 96-well plate was placed in a QX200 Droplet Reader for fluorescence signal reading. The KRAS gene mutation type and total copy number of the sample to be tested were statistically analyzed based on the actual FAM and VIC signal values ​​read. The actual detected copy number and mutation ratio of the sample to be tested were calculated to determine the positive or negative status of the sample.

[0067] Example 2

[0068] Specific detection of KRAS p.G12D / G12V / G12C / G12S / G12A / G12R / G13D.

[0069] The experiments were carried out according to the steps of Example 1 using gDNA of 7 KRAS-positive cell lines as templates, respectively, wherein the sample input amount was 20 ng, and the mutant probes used were probes corresponding to KRAS p.G12D / G12V / G12C / G12S / G12A / G12R / G13D. The sample and corresponding site information are shown in Table 3.

[0070] Table 3

[0071] sample Site (p.dot) Site (c.dot) LS180 KRAS p.G12D c.35G>A HCT15 KRAS p.G13D c.38G>A SW620 KRAS p.G12V c.35G>T NCI-H358 KRAS p.G12C c.34G>T A549 KRAS p.G12S c.34G>A H2009 KRAS p.G12A c.35G>C MDAMB134VI KRAS p.G12R c.35G>A

[0072] The probe sequence information is shown in Table 4.

[0073] Table 4

[0074]

[0075]

[0076] The results are as follows Figure 1 As shown, there was no cross-reaction between the KRAS p.G12D / G12V / G12C / G12S / G12A / G12R / G13D probe and the 7 common KRAS gene positive mutation templates, indicating good specificity.

[0077] Example 3

[0078] Detect KRAS p.G12D positive cell line sample LS180.

[0079] Using LS180 cell line gDNA as template, the experiment was carried out according to the steps in Example 1, wherein the amount of sample to be tested was 20 ng, the primers and probes were SEQ ID NO.1, SEQ ID NO.8-10, and the test results were shown in Figure 2 The fourth quadrant signal is a droplet with only conservative sequence signals (single VIC positive signal), and the first quadrant signal group is a droplet with both mutant probes and conservative sequence signals (both FAM and VIC positive signals). The FAM channel detects the KRAS p.G12D positive signal in the template, and its copy number is 65 copies / μL according to the software calculation; the VIC channel detects the total signal of mutant + wild type in the template, and its copy number is 127 copies / μL according to the software calculation; then the mutation frequency VAF of this sample is 51.18%, and the copy number of the wild-type template is VIC channel-FAM channel, that is, 62 copies / μL.

[0080] Example 4

[0081] Detection of fragmented DNA samples.

[0082] Using LS180 cell line simulated cfDNA as a template, the experiment was carried out according to the steps in Example 1, wherein the sample input amount was 20 ng, the primers and probes were SEQ ID NO.1, SEQ ID NO.8-10, and the results are shown in the attached Figure 3 The fourth quadrant signal is a droplet with only conservative sequence signals (single VIC positive signal), and the first quadrant signal group is a droplet with both mutant probe and conservative sequence signals (both FAM and VIC positive signals). The FAM channel detects mutant signals, and its copy number is 56.1 copies / μL according to the software calculation; the VIC channel detects total template (mutant + wild type) signals, and its copy number is 109 copies / μL according to the software calculation; the mutation frequency VAF of this sample is 51.47%.

[0083] In summary, the present invention designs a probe for a conserved sequence in a single amplicon to quantify the total copy number (wild-type copy number + mutant copy number) to achieve wild-type copy number quantification, and designs several mutant probes to quantify each mutant copy number. At the same time, by modifying the mutation probe and the conserved sequence, the interference between the mutation sites is reduced and the TM value is balanced, thereby achieving rapid and accurate quantification of each site under the condition of multiple mutations.

[0084] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A primer probe design method for detecting mutant genes, It is characterized in that The method comprises: designing a conserved region fluorescent probe and a mutation region fluorescent probe in a single amplicon and performing locked nucleic acid modification on the conserved region fluorescent probe and the mutation region fluorescent probe.

2. The primer probe design method according to claim 1, It is characterized in that The length of the single amplicon is less than 120 bp; Preferably, the mutant gene includes the KRAS gene; Preferably, the mutation site of the KRAS gene includes any one of KRAS p.G12D, KRAS p.G13D, KRAS p.G12V, KRAS p.G12C, KRAS p.G12S, KRAS p.G12A or KRAS p.G12R, or a combination of at least two thereof.

3. The primer probe design method according to claim 1, It is characterized in that The 5' end of the conserved region fluorescent probe and the 5' end of the mutant region fluorescent probe each independently contain a fluorescent group, and the 3' end each independently contain a quenching group; Preferably, the fluorescent group includes a first fluorescent group and a second fluorescent group; Preferably, the 3' end of the conserved region fluorescent probe and the 3' end of the mutation region fluorescent probe are independently subjected to any one of dideoxy modification, amino modification or phosphorylation modification.

4. A fluorescent probe for detecting mutation regions of mutant genes, It is characterized in that The mutation region fluorescent probe is designed by the primer probe design method according to any one of claims 1 to 3; Preferably, the nucleic acid sequence of the mutation region fluorescent probe includes the sequences shown in SEQ ID NO.1-SEQ ID NO.

7.

5. A primer and a conserved region fluorescent probe for detecting mutant genes, It is characterized in that The primers and conserved region fluorescent probes are designed by the primer-probe design method according to any one of claims 1 to 3; Preferably, the nucleic acid sequence of the conserved region fluorescent probe includes the sequence shown in SEQ ID NO.8; Preferably, the nucleic acid sequence of the primer includes the sequences shown in SEQ ID NO.9 and SEQ ID NO.

10.

6. A ddPCR reaction system, It is characterized in that The ddPCR reaction system comprises a ddPCR premix, the mutant region fluorescent probe for detecting mutant genes according to claim 4 and / or the conserved region fluorescent probe for detecting mutant genes according to claim 5, nuclease-free water and a sample to be tested.

7. According to the ddPCR reaction system of claim 6, It is characterized in that The single reaction dosage of the ddPCR premix is ​​8-12 μL; the single reaction dosage of the mutation region fluorescent probe is 0.25-1 μL, and the concentration is 125-500 nM; the single reaction dosage of the conserved region fluorescent probe is 0.25-1 μL, and the concentration is 125-500 nM; the single reaction dosage of the sample to be tested is 1-5 μL, and the concentration is 0-300 ng; the single reaction dosage of the nuclease-free water is 0-9 μL.

8. A method for detecting a mutant gene, It is characterized in that The method comprises: using genomic DNA or fragmented DNA of a sample to be tested as a template, performing a digital PCR reaction using a fluorescent probe designed by the primer probe design method according to any one of claims 1 to 3, obtaining the droplet copy number of the first fluorescent group and the second fluorescent group signal after the reaction is completed, and obtaining the mutation frequency of the sample to be tested based on the droplet copy number analysis and calculation.

9. The method for detecting a mutant gene according to claim 9, It is characterized in that The mutation frequency of the sample to be tested = (the number of copies of the first fluorescent group channel / the number of copies of the second fluorescent group channel) * 100%; Preferably, the sample to be tested includes genomic DNA, paraffin-embedded tissue sample DNA or plasma free DNA.

10. Use of the mutant region fluorescent probe for detecting mutant genes according to claim 4 and / or the conserved region fluorescent probe for detecting mutant genes according to claim 5 in preparing a product for detecting mutant genes.

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