Primer group and method for rapidly detecting solid tumor related gene mutation

Abstract

The invention relates to a primer group and a method for rapidly detecting solid tumor related gene mutation. The primer group for mutation detection of 13 related genes of a solid tumor is obtained through a large amount of research and screening, and the primers can be used for independent amplification and can also be used for efficient amplification of a DNA template to be detected under the same PCR reaction system and reaction procedure; and specific nucleic acid sequences of 40 regions of 13 related genes of the solid tumor can be rapidly and accurately amplified by one-time multiple PCR amplification. And connecting an amplification product to a linker to construct a library, and then sequencing to realize the detection of mutations in 40 regions of 13 related genes of the solid tumor. The detection method is rapid, simple and convenient, automatic detection can be achieved, and only three days are needed from DNA extraction to result report issuing. And the kit is high in flux, low in cost, suitable for screening related genes of solid tumors and wide in application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a primer set and a method for rapidly detecting solid tumor-related gene mutations. Background Art

[0002] With the continuous deepening of tumor research and the increasing "genetization" of tumor drug development, tumor treatment has entered the era of personalized treatment. Before personalized tumor treatment, tumor patients need to undergo a series of genetic tests, and different drugs and treatment plans are used according to the patient's genetic characteristics to implement personalized treatment. Tumor surgical tissue is the first type of tumor sample we can obtain. Gene target detection of surgical samples is helpful in determining the postoperative treatment plan. During the growth process of tumors, after multiple divisions and proliferations, their daughter cells show molecular biological or genetic changes, which makes the tumor growth rate, invasive ability, drug sensitivity, prognosis and other aspects different. Due to the widespread existence of tumor heterogeneity, it is possible to identify tumor heterogeneity caused by mutations by testing multiple sites in the tumor at multiple time points, thereby providing important clues for personalized treatment. Compared with traditional tissue biopsy, liquid biopsy has many advantages such as fast, convenient, and non-invasive. Liquid biopsy technology is an effective supplement to conventional tissue biopsy, and it is used to detect targets, monitor tumor treatment response, and predict tumor recurrence.

[0003] Studies have confirmed that more and more gene mutations are associated with various forms of cancer, and more and more targeted drugs have been shown to have therapeutic effects on certain types of cancer. Taking advanced NSCLC as an example, the currently commonly used targeted therapeutic drugs in clinical practice, such as Iressa, Tarceva, and Conmena, are all epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). Studies have shown that when NSCLC patients have exon 19 deletion, exon 21 L858R, L861Q mutations, and exon 18 G719X mutations in the EGFR gene, they are sensitive to TKIs targeted therapy, but when insertion mutations occur at positions 770-775 of exon 20, TKIs resistance occurs. KRAS is a target molecule downstream of EGFR, and the EGFR growth signal needs to be transmitted through the KRAS protein. TKIs treatment is ineffective for patients carrying KRAS gene mutations. Therefore, the KRAS gene must be tested before TKIs treatment. Therefore, clinical testing of the amplification / mutation / expression of specific genes in these pathways can tailor a most suitable treatment plan for each patient, thereby maximizing the effectiveness of treatment, reducing the toxic side effects of drugs, and avoiding improper use of drugs that delay treatment. By detecting the information of tumor-related gene hotspots, the occurrence and development of tumors can be reflected. At the same time, the mutation characteristics of these genes can be analyzed. According to the individual differences of patients and combined with relevant information of pharmacogenomics, clinicians can be assisted in selecting appropriate therapeutic drugs for cancer patients and formulating a complete treatment plan, which is conducive to the realization of personalized tumor treatment "according to the person and the time".

[0004] Mutations in tumor patients mainly include single nucleotide variations (SNV), small fragment insertion-deletion (1NDEL), copy number variations (CNV), DNA fusion (FUSION), etc. Traditional detection methods such as real-time fluorescence quantitative PCR, first-generation sequencing, and gene chip methods can only detect individual and partial sites at a time, and cannot detect unknown mutations. Therefore, providing a product and method that can quickly and accurately detect mutations at multiple sites of multiple tumor-related genes is very important for realizing personalized tumor treatment "according to the person and the time". Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a primer set for rapid detection of solid tumor-related gene mutations, which can quickly and accurately amplify the specific nucleic acid sequence of the solid tumor-related gene, and then through library construction and sequencing, realize the detection of related gene mutations.

[0006] The first aspect of the present invention is to provide a primer set for detecting solid tumor-related gene mutations, the primer set comprising primer pairs for at least two genes among mutations in 40 regions of 13 related genes, the primer pairs being selected from the following:

[0007] A primer pair for detecting BRAF gene mutation, the primer pair being selected from: an upstream primer having a sequence as shown in SEQ ID NO.1 and a downstream primer having a sequence as shown in SEQ ID NO.2; an upstream primer having a sequence as shown in SEQ ID NO.3 and a downstream primer having a sequence as shown in SEQ ID NO.4;

[0008] A primer pair for detecting EGFR gene mutation, the primer pair being selected from: an upstream primer having a sequence as shown in SEQ ID NO.5 and a downstream primer having a sequence as shown in SEQ ID NO.6; an upstream primer having a sequence as shown in SEQ ID NO.7 and a downstream primer having a sequence as shown in SEQ ID NO.8; an upstream primer having a sequence as shown in SEQ ID NO.9 and a downstream primer having a sequence as shown in SEQ ID NO.10; an upstream primer having a sequence as shown in SEQ ID NO.11 and a downstream primer having a sequence as shown in SEQ ID NO.12; an upstream primer having a sequence as shown in SEQ ID NO.13 and a downstream primer having a sequence as shown in SEQ ID NO.14; an upstream primer having a sequence as shown in SEQ ID NO.15 and a downstream primer having a sequence as shown in SEQ ID NO.16; an upstream primer having a sequence as shown in SEQ ID NO.17 and a downstream primer having a sequence as shown in SEQ ID NO.18;

[0009] A primer pair for detecting a GNA11 gene mutation, the primer pair comprising: an upstream primer having a sequence as shown in SEQ ID NO.19 and a downstream primer having a sequence as shown in SEQ ID NO.20;

[0010] A primer pair for detecting a GNAQ gene mutation, the primer pair comprising: an upstream primer having a sequence as shown in SEQ ID NO.21 and a downstream primer having a sequence as shown in SEQ ID NO.22:

[0011] A primer pair for detecting a GNAS gene mutation, the primer pair being selected from: an upstream primer having a sequence as shown in SEQ ID NO.23 and a downstream primer having a sequence as shown in SEQ ID NO.24; an upstream primer having a sequence as shown in SEQ ID NO.25 and a downstream primer having a sequence as shown in SEQ ID NO.26;

[0012] A primer pair for detecting HRAS gene mutation, the primer pair being selected from: an upstream primer having a sequence as shown in SEQ ID NO.27 and a downstream primer having a sequence as shown in SEQ ID NO.28; an upstream primer having a sequence as shown in SEQ ID NO.29 and a downstream primer having a sequence as shown in SEQ ID NO.30;

[0013] A primer pair for detecting IDH1 gene mutation, the primer pair comprising: an upstream primer having a sequence as shown in SEQ ID NO.31 and a downstream primer having a sequence as shown in SEQ ID NO.32;

[0014] A primer pair for detecting IDH2 gene mutation, the primer pair comprising: an upstream primer having a sequence as shown in SEQ ID NO.33 and a downstream primer having a sequence as shown in SEQ ID NO.34;

[0015] A primer pair for detecting KRAS gene mutation, wherein the primer pair is selected from: an upstream primer having a sequence as shown in SEQ ID NO.35 and a downstream primer having a sequence as shown in SEQ ID NO.36; an upstream primer having a sequence as shown in SEQ ID NO.37 and a downstream primer having a sequence as shown in SEQ ID NO.38; an upstream primer having a sequence as shown in SEQ ID NO.39 and a downstream primer having a sequence as shown in SEQ ID NO.40;

[0016] A primer pair for detecting NRAS gene mutation, wherein the primer pair is selected from: an upstream primer having a sequence as shown in SEQ ID NO.41 and a downstream primer having a sequence as shown in SEQ ID NO.42; an upstream primer having a sequence as shown in SEQ ID NO.43 and a downstream primer having a sequence as shown in SEQ ID NO.44; an upstream primer having a sequence as shown in SEQ ID NO.45 and a downstream primer having a sequence as shown in SEQ ID NO.46;

[0017] A primer pair for detecting a PIK3CA gene mutation, the primer pair being selected from: an upstream primer having a sequence as shown in SEQ ID NO.47 and a downstream primer having a sequence as shown in SEQ ID NO.48; an upstream primer having a sequence as shown in SEQ ID NO.49 and a downstream primer having a sequence as shown in SEQ ID NO.50; an upstream primer having a sequence as shown in SEQ ID NO.51 and a downstream primer having a sequence as shown in SEQ ID NO.52; an upstream primer having a sequence as shown in SEQ ID NO.53 and a downstream primer having a sequence as shown in SEQ ID NO.54; an upstream primer having a sequence as shown in SEQ ID NO.55 and a downstream primer having a sequence as shown in SEQ ID NO.56; an upstream primer having a sequence as shown in SEQ ID NO.57 and a downstream primer having a sequence as shown in SEQ ID NO.58; an upstream primer having a sequence as shown in SEQ ID NO.59 and a downstream primer having a sequence as shown in SEQ ID NO.60; an upstream primer having a sequence as shown in SEQ ID NO.61 and a downstream primer having a sequence as shown in SEQ ID NO.62; an upstream primer having a sequence as shown in SEQ ID NO.63 and a downstream primer having a sequence as shown in SEQ ID NO. A downstream primer as shown in SEQ ID NO.64; an upstream primer as shown in SEQ ID NO.65 and a downstream primer as shown in SEQ ID NO.66; an upstream primer as shown in SEQ ID NO.67 and a downstream primer as shown in SEQ ID NO.68;

[0018] A primer pair for detecting a TERT gene mutation, the primer pair being selected from: an upstream primer having a sequence as shown in SEQ ID NO.69 and a downstream primer having a sequence as shown in SEQ ID NO.70; an upstream primer having a sequence as shown in SEQ ID NO.71 and a downstream primer having a sequence as shown in SEQ ID NO.72; an upstream primer having a sequence as shown in SEQ ID NO.73 and a downstream primer having a sequence as shown in SEQ ID NO.74;

[0019] A primer pair for detecting ERBB2 gene mutation, wherein the primer pair is selected from: an upstream primer with a sequence as shown in SEQ ID NO.75 and a downstream primer with a sequence as shown in SEQ ID NO.76; an upstream primer with a sequence as shown in SEQ ID NO.77 and a downstream primer with a sequence as shown in SEQ ID NO.78; an upstream primer with a sequence as shown in SEQ ID NO.79 and a downstream primer with a sequence as shown in SEQ ID NO.80.

[0020] In some embodiments, the primer set includes primer pairs for NRAS and PIK3CA genes.

[0021] In some embodiments, the primer set also includes at least one of the primer pairs for BRAF, EGFR, KRAS, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, TERT, and ERBB2 genes.

[0022] In some embodiments, the primer set includes primer pairs for BRAF, EGFR, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KRAS, NRAS, PIK3CA, TERT, and ERBB2 genes.

[0023] The second aspect of the present invention is to provide the use of the primer set described above in the preparation of a product for detecting solid tumor-related gene mutations.

[0024] In some embodiments, the product is a kit.

[0025] The third aspect of the present invention is to provide a kit for detecting solid tumor-related gene mutations, wherein the kit comprises the primer set as described above.

[0026] The fourth aspect of the present invention is to provide a method for detecting solid tumor-related gene mutations, the method comprising the following steps: using the DNA of the sample to be tested as a template, using the primer set as described above or the kit as described above to perform multiplex PCR amplification, constructing a library, and sequencing.

[0027] In some embodiments, the multiplex PCR amplification includes a first round of PCR amplification and a second round of PCR amplification;

[0028] The reaction system of the first round of PCR amplification comprises the following components: Multiplex PCR Master Mix, DMSO, the primer pair mixture and DNA template;

[0029] The reaction system of the second round of PCR amplification comprises the following components: Multiplex PCR Master Mix, the product of the first round of PCR amplification, and a linker.

[0030] In some embodiments, the concentration of the primer pairs SEQ ID NO.1-2, SEQ ID NO.25-26, SEQ ID NO.53-54, SEQ ID NO.77-78, and SEQ ID NO.79-80 in the reaction system of the first round of PCR amplification is 0.035 uM respectively;

[0031] The concentration of the primer pairs SEQ ID NO.3-4, SEQ ID NO.17-18, SEQ ID NO.35-36, SEQ ID NO.39-40, SEQ ID NO.47-48, and SEQ ID NO.63-64 in the reaction system of the first round of PCR amplification is 0.06uM respectively;

[0032] The concentration of the primer pairs SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.15-16, SEQ ID NO.19-20, SEQ ID NO.41-42, SEQ ID NO.55-56, and SEQ ID NO.57-58 in the reaction system of the first round of PCR amplification is 0.05 uM respectively;

[0033] The concentration of the primer pairs SEQ ID NO.11-12, SEQ ID NO.29-30, SEQ ID NO.45-46, SEQ ID NO.49-50, SEQ ID NO.51-52, and SEQ ID NO.59-60 in the reaction system of the first round of PCR amplification is 0.045 uM respectively;

[0034] The concentration of the primer pairs SEQ ID NO.13-14, SEQ ID NO.21-22, SEQ ID NO.33-34, SEQ ID NO.37-38, and SEQ ID NO.67-68 in the reaction system of the first round of PCR amplification is 0.04 uM respectively;

[0035] The concentration of the primer pair SEQ ID NO. 23-24 in the reaction system of the first round of PCR amplification is 0.055 uM respectively;

[0036] The concentration of the primer pair SEQ ID NO. 27-28 in the reaction system of the first round of PCR amplification is 0.075uM respectively;

[0037] The concentrations of the primer pairs SEQ ID NO.31-32, SEQ ID NO.61-62, and SEQ ID NO.65-66 in the reaction system of the first round of PCR amplification are 0.03uM respectively:

[0038] The concentration of the primer pairs SEQ ID NO.43-44, SEQ ID NO.69-70, SEQ ID NO.71-72, and SEQ ID NO.73-74 in the reaction system of the first round of PCR amplification is 0.065 uM respectively;

[0039] The concentration of the primer pair SEQ ID NO. 75-76 in the reaction system of the first round of PCR amplification is 0.02 uM respectively.

[0040] In some embodiments, the reaction procedure of the first round of PCR amplification is: 1) 95°C, 5 min; 2) 95°C, 10 s; 60°C, 30 s; 72°C, 15 s; 20 cycles; 3) 72°C, 1 min;

[0041] The reaction procedure of the second round of PCR amplification was: 1) 95° C., 10 min; 2) 95° C., 30 s; 65° C., 90 s; 10 cycles.

[0042] In some of these embodiments, the DNA includes gDNA and / or ctDNA.

[0043] In some embodiments, the method for constructing the library is an amplicon capture method.

[0044] In some of these embodiments, the sequencing is based on the Illumina sequencing platform.

[0045] After extensive research and screening, the present invention has obtained a primer set for detecting mutations in 13 genes related to solid tumors. These primers can be amplified individually, and can also be used to efficiently amplify the DNA template to be tested under the same PCR reaction system and reaction procedure. One multiplex PCR amplification can quickly and accurately amplify the specific nucleic acid sequences of 40 regions of the 13 genes related to the solid tumor. The amplified products are connected to the linker to construct a library, and then sequenced to achieve the detection of mutations in the 40 regions of the 13 genes related to the solid tumor, assisting clinicians in selecting appropriate therapeutic drugs for cancer patients, formulating a complete treatment plan, and realizing "person-based and time-based" tumor individualized treatment.

[0046] The present invention establishes suitable multiplex PCR amplification conditions for the primer set through repeated exploration and optimization of multiplex PCR amplification conditions, including the concentration of each primer in the primer set in the amplification system and the amplification procedure, which can efficiently and accurately achieve the amplification of each relevant gene detection region, thereby ensuring the depth uniformity, accuracy and sensitivity of subsequent sequencing.

[0047] The detection method of the present invention is fast and simple, and can realize automated detection. It only takes 3 days from DNA extraction to the issuance of result reports. It also has high throughput and low cost, and is suitable for related gene screening of solid tumors, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of library construction using the amplicon capture method for detecting solid tumor-related gene mutations.

[0049] Figure 2 Experimental workflow for detecting solid tumor-related gene mutations.

[0050] Figure 3 Schematic diagram of the size determination of library fragments using the 2100 Bioanalyzer.

[0051] Figure 4 Bioinformatics analysis pipeline for detecting solid tumor-related gene mutations.

[0052] Figure 5 Statistics of recall and precision of each algorithm software.

[0053] Figure 6 This is a comparison chart of the IGV results of the primer sequence test for the BRAF gene chr7:140453103-140453221 region.

[0054] Figure 7 This is a comparison chart of the IGV results of the primer sequence test for the chr7:55248975-55249100 region of the EGFR gene.

[0055] Figure 8 This is a comparison chart of the IGV results of the primer sequence test for the chr12:25378550-25378658 region of the KRAS gene.

[0056] Fig. 9 This is a comparison chart of the IGV results of the primer sequence test for the chr1:115256505-115256584 region of the NRAS gene.

[0057] Fig.10 This is a comparison chart of the IGV results of the primer sequence test for the chr3:178951997-178952097 region of the PIK3CA gene.

[0058] Fig.11 Comparison of sequencing depths optimized for primer concentrations.

[0059] Fig.12 is the cumulative frequency of false matching rate.

[0060] Fig.13 is the frequency of mismatched bases at each site.

[0061] Fig.14 Statistics for sequencing data with different DNA input amounts.

[0062] Fig.15 This is the SNV distribution diagram of the detected samples.

[0063] Fig.16 This is the indel distribution diagram of the detected samples.

[0064] Fig.17 It is the linear distribution diagram of precision. DETAILED DESCRIPTION

[0065] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0066] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0067] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0068] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

[0069] The present invention is further described in detail below with reference to specific embodiments.

[0070] Example 1 Specific primers for detecting solid tumor-related gene mutations

[0071] In this embodiment, 13 genes including solid tumor-related genes BRAF, EGFR, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KRAS, NRAS, PIK3CA, TERT, and ERBB2 were selected as target amplification regions, and a set of primer sets suitable for solid tumor-related gene mutation detection were designed and screened. The specific primer sequences and the corresponding detection genes and detection region information are shown in Table 1.

[0072] Table 1 Primer sequences, detected genes and detection range for detecting solid tumor-related gene mutations

[0073]

[0074]

[0075]

[0076] Example 2 Method for detecting gene mutations associated with solid tumors

[0077] Please refer to Figure 1-Figure 2 ,in, Figure 1 This is a schematic diagram of the library construction principle of the amplicon capture method for detecting solid tumor-related gene mutations in the present invention. Figure 2 The flowchart of the method for detecting solid tumor-related gene mutations of the present invention is as follows. The primer set in Table 1 of Example 1 is used to detect 13 solid tumor-related genes of 96 samples, which specifically includes the following steps:

[0078] 1. DNA extraction

[0079] A commercial nucleic acid extraction and purification kit (Qiagen) was used to extract gDNA or ctDNA from peripheral blood samples.

[0080] 2. First round of PCR amplification and purification

[0081] (1) PCR amplification

[0082] The SKa Multiplex PCR Master Mix of Qikai Biotechnology was used as the amplification enzyme reagent. The reagent was thawed on an ice plate, mixed and microcentrifuged, and then PCR amplification was performed (the template was gDNA or ctDNA extracted from peripheral blood samples) to obtain the amplification products of solid tumor-related genes. The reaction system for PCR amplification is shown in Table 2; the primer concentrations in the PCR amplification component Primer Mix are shown in Table 3; the PCR amplification reaction program: [95°C, 5min]; [95°C, 10s; 60°C, 30s; 72°C, 15s;] 20times; [72°C, 1min, 15°C, ∝]. "∝" means keeping at 15°C, the same below.

[0083] Table 2 First round PCR reaction system

[0084]

[0085] Table 3 Primer concentrations in PCRMix

[0086]

[0087]

[0088] (2) Purification

[0089] The first round of amplification product purification was performed by magnetic bead purification method. The purification operation was performed by Agilent automated instrument, which was simple, fast and pollution-proof.

[0090] 3. Second round of PCR amplification and purification

[0091] (1) PCR amplification

[0092] Place the SKa Multiplex PCR Master Mix enzyme reagent on an ice plate, prepare the reaction system according to Table 4 (except the first round of amplification products and adapters) and dispense into a 96-well PCR plate, then add 22.5 μL of the purified first round of PCR amplification products, and then add 2.5 μL of the corresponding adapter (concentration is 2.5 μM, adapter sequence UDI-001-096, as shown in Table 5, each sample is added with the corresponding adapter to distinguish the samples), and perform the second round of PCR reaction. Reaction program: [95°C, 10 min]; [95°C, 30 s; 65°C, 90 s;] 10 times; [15°C, ∝].

[0093] Table 4 Second round PCR reaction system

[0094] Components Volume (μL) SKa Multiplex PCR Master Mix 25 The first round of PCR amplification products 22.5 Linker (2.5μM) 2.5 Total 50

[0095] Table 5 Linker sequences

[0096]

[0097]

[0098]

[0099]

[0100] (2) Purification

[0101] The second round of PCR amplification product purification was performed by magnetic bead purification. The purification operation was performed by Agilent automated instruments, which was simple, fast and pollution-proof.

[0102] 4. Library concentration determination and quality control

[0103] The library concentration was determined using Qubit, and the library concentration was required to be ≥10 ng / μL. The library fragment size was determined using Agilent 2100 bioanalyzer. Figure 3 This is a schematic diagram for determining the size of library fragments. The size of the library fragment is about 300 bp, which is consistent with the expected fragment size.

[0104] 5. Sequencing

[0105] Using high-throughput sequencer Miniseq TM DX-CN platform for sequencing. Miniseq selected by the detection method of the present invention TM The DX-CN platform uses SBS sequencing technology and amplicon capture method for detection. SBS sequencing technology can effectively reduce sequencing errors of homopolymers and repetitive sequences by continuously improving and updating sequencing reagents and detection systems, and has high base coverage and accuracy. At the same time, the simplification of the experimental process of amplicon capture technology has greatly reduced the professional threshold of operators, enabling more people to complete the experiment and completely liberating the risk of insufficient manpower. Amplicon capture sequencing is very useful as a complementary technology to whole genome sequencing, which greatly simplifies the experimental process and analysis objectives. This technology has been proven to be a fast and effective technology, and plays a unique role in the new generation of high-throughput sequencing. It has produced many exciting new discoveries and its application areas are becoming more and more extensive.

[0106] 6. Bioinformatics analysis

[0107] For bioinformatics analysis workflow, please refer to Figure 4 The Raw Data (bcl) file generated by sequencing is transferred to a dedicated computer server in the laboratory. After the fastq file is generated by dedicated software, the sequencing data is analyzed and processed, including filtering low-quality data in sequencing, removing adapters and primers, aligning to the corresponding position of the reference genome data, and statistically analyzing the sequencing mapping information, sequence number, target area coverage size, average sequencing depth, sequencing uniformity, etc., and selecting and testing the corresponding software or Selfscript for mutation analysis for various types of mutations, and then determining the gene, coordinates and other annotations of the mutation site through annotation software combined with the database, and finally outputting the visual table results.

[0108] Among them, the variation detection algorithm for detecting solid tumor-related gene mutations is as follows:

[0109] p=1-CDF Poisson (x, m)

[0110] Q=-10*Log 10 (p).

[0111] In this algorithm, x is the number of observed variants, and m is the expected number of observed variants calculated by a known noise model. For example: if the base quality is 20, the corresponding m = total depth * 0.01, and if the base quality is 30, the corresponding m = total depth * 0.001. p represents the probability of obtaining more than x observed variants under the noise model, which is the false positive rate. Finally, the p value is converted to a Q value. Through the above calculation, the VQ value of the variant can be obtained. When VQ>20 is equivalent to a false positive rate of less than 20, the variant meets the VQ filtering requirements and is retained.

[0112] We selected four datasets for comparison, namely Titr, RAS, VP, and Myl. The calculation method of F1 is:

[0113]

[0114] like Figure 5 As shown, compared with Pvc, LoFreq, and VarDict, the algorithm has the highest recall rate, precision, and F1 value for SNVs and Indels of somatic and germlin.

[0115] Experimental Example 1 Optimization of primer sequences

[0116] Multiplex PCR is a widely used molecular biology technique for amplifying multiple targets in a single PCR experiment. In a multiplex assay, multiple target sequences can be amplified by using multiple primer pairs in the reaction mixture. As an extension of the practical application of PCR, this technique can save a lot of time and effort in the laboratory without affecting the practicality of the experiment.

[0117] The design of specific primer sets in multiplex PCR is crucial. The main technical difficulties are:

[0118] 1. Specificity of primer sequence: Multiple PCR amplification uses multiple pairs of primers to amplify multiple target sequences. It must be ensured that each pair of primers can only amplify the corresponding target sequence, and there should not be much homology between the amplified fragments to avoid nonspecific amplification.

[0119] 2. Parameter selection for primer design: Multiplex PCR detection involves a large number of primers, all of which are amplified in the same system. Therefore, it is required to select primers with appropriate parameters such as length, Tm value, and GC content.

[0120] 3. Avoidance of primer dimers: Check whether the designed primers form primer dimers. All primers should be present in the reaction mixture. The primers should not be complementary to each other, especially the 3′ complementarity, to avoid the formation of dimers. Dimerization will lead to nonspecific amplification.

[0121] (1) Primer screening for BRAF gene

[0122] The present invention finds the fragment files of 13 genes related to solid tumors according to NCBI, obtains suitable primers through Blast, selects primers with suitable parameters such as length, Tm value, GC content, etc., performs primer synthesis, and then performs detection. Take three pairs of primers of primer pair KST002 for detecting the BRAF gene chr7:140453103-140453221 region as an example to illustrate, screen out the optimal primer pair, and the specific primer sequences are shown in Table 6.

[0123] Table 6 Primer design for BRAF gene chr7: 140453103-140453221 region

[0124]

[0125] The above three sets of primers were used for amplification-library construction-sequencing-analysis. The final sequencing depth results of the target site are as follows: Figure 6 As shown. Figure 6 It can be seen that the sequencing depth of the target site of the first set of primer pairs is 131 reads, the sequencing depth of the target site of the second set of primer pairs is 2131 reads, and the sequencing depth of the target site of the third set of primer pairs is 1152 reads, which preliminarily indicates that the second set of primer pairs is more in line with the requirements for further testing.

[0126] (2) Primer screening for EGFR gene

[0127] The present invention finds the fragment files of 13 genes related to solid tumors according to NCBI, obtains suitable primers through Blast, selects primers with suitable parameters such as length, Tm value, GC content, etc., performs primer synthesis, and then performs detection. Take three pairs of primers of primer pair KST008 for detecting the chr7:55248975-55249100 region of EGFR gene as an example to illustrate, screen out the optimal primer pair, and the specific primer sequence is shown in Table 7.

[0128] Table 7 Primer design for EGFR gene chr7: 55248975-55249100 region

[0129]

[0130]

[0131] The above three sets of primers were used for amplification-library construction-sequencing-analysis. The final sequencing depth results of the target site are as follows: Figure 7 As shown. Figure 7It can be seen that the sequencing depth of the target site of the first set of primer pairs is 178 reads, the sequencing depth of the target site of the second set of primer pairs is 2978 reads, and the sequencing depth of the target site of the third set of primer pairs is 5043 reads, which preliminarily indicates that the third set of primer pairs is more in line with further detection requirements.

[0132] (3) Primer screening for KRAS gene

[0133] The present invention finds the fragment files of 13 genes related to solid tumors according to NCBI, obtains suitable primers through Blast, selects primers with suitable parameters such as length, Tm value, GC content, etc., performs primer synthesis, and then performs detection. Take three pairs of primers of the primer pair KST020 for detecting the chr12:25378550-25378658 region of the KRAS gene as an example to illustrate, screen out the optimal primer pair, and the specific primer sequences are shown in Table 8.

[0134] Table 8 Primer design for KRAS gene chr12: 25378550-25378658 region

[0135]

[0136] The above three sets of primers were used for amplification-library construction-sequencing-analysis. The final sequencing depth results of the target site are as follows: Figure 8 As shown. Figure 8 It can be seen that the sequencing depth of the target site of the first set of primer pairs is 0 reads, the sequencing depth of the target site of the second set of primer pairs is 1821 reads, and the sequencing depth of the target site of the third set of primer pairs is 229 reads, which preliminarily indicates that the second set of primer pairs is more in line with further detection requirements.

[0137] (4) Primer screening of NRAS gene

[0138] The present invention finds the fragment files of 13 genes related to solid tumors according to NCBI, obtains suitable primers through Blast, selects primers with suitable parameters such as length, Tm value, GC content, etc., performs primer synthesis, and then performs detection. Take three pairs of primers of primer pair KST022 for detecting the chr1:115256505-115256584 region of NRAS gene as an example to illustrate, screen out the optimal primer pair, and the specific primer sequence is shown in Table 9.

[0139] Table 9 Primer design for NRAS gene chr1: 115256505-115256584 region

[0140]

[0141]

[0142] The above three sets of primers were used for amplification-library construction-sequencing-analysis. The final sequencing depth results of the target site are as follows: Fig. 9 As shown. Fig. 9 It can be seen that the sequencing depth of the target site of the first set of primer pairs is 7802 reads, the sequencing depth of the target site of the second set of primer pairs is 286 reads, and the sequencing depth of the target site of the third set of primer pairs is 3472 reads, which preliminarily indicates that the first set of primer pairs is more in line with the requirements for further testing.

[0143] (5) Primer screening for PIK3CA gene

[0144] The present invention finds the fragment files of 13 genes related to solid tumors according to NCBI, obtains suitable primers through Blast, selects primers with suitable parameters such as length, Tm value, GC content, etc., performs primer synthesis, and then performs detection. Take three pairs of primers of primer pair KST024 for detecting the chr3:178951997-178952097 region of the PIK3CA gene as an example to illustrate, screen out the optimal primer pair, and the specific primer sequences are shown in Table 10.

[0145] Table 10 Primer design for chr3:178951997-178952097 region of PIK3CA gene

[0146]

[0147] The above three sets of primers were used for amplification-library construction-sequencing-analysis. The final sequencing depth results of the target site are as follows: Fig.10 As shown. Fig.10 It can be seen that the sequencing depth of the target site of the first set of primer pairs is 12829 reads, the sequencing depth of the target site of the second set of primer pairs is 428 reads, and the sequencing depth of the target site of the third set of primer pairs is 6061 reads, which preliminarily indicates that the first set of primer pairs is more in line with the requirements for further testing.

[0148] Experimental Example 2 Optimization of primer concentration

[0149] According to Experimental Example 1, after screening and obtaining a primer set for detecting 13 genes related to solid tumors, the optimal primer concentration was explored: the primers were first mixed at a ratio of 1:1, and then amplified and constructed the library according to the method of Example 2, and then the concentration of each pair of primers was adjusted according to the sequencing depth of the offline data analysis.

[0150] A total of 5 groups of tests were conducted, and the primer concentrations for each group of tests are shown in Table 11 below:

[0151] Table 11

[0152]

[0153]

[0154] Table 12 Sequencing depth statistics of primer concentration optimization test

[0155]

[0156]

[0157] As shown in Table 12, a total of 5 rounds of primer adjustment were tested. The depth comparison of different primers during the test process is as follows: Fig.11 As shown, the sequencing depth of Test 5 was relatively uniform, and the final primer concentration corresponding to Test 5 is shown in Table 3.

[0158] Test Example 3 Methodological Verification of the Method of the Invention

[0159] 1.1 Sequencing error rate

[0160] To verify the error rate of this test, four standards from the Coriell Institute, GM24385, GM24149, GM24143, and GM24631, were selected. Each sample was tested in triplicate for a total of 12 tests. During the bioinformatics analysis, four known mutations in the test region were removed (see Table 13). Other sites (including SNPs) were set to be consistent with the reference sequence. All variant reads outside the reference sequence were considered to be caused by sequencing or other errors. The variation of 4002 bases in the test region was statistically analyzed. The results are shown in Table 14. Fig.12 and Fig.13 :

[0161] Table 13 Known mutation sites of GM24385, GM24149, GM24143, and GM24631

[0162] Serial number Gene chromosome Location Ref Mutations Proportion 1 EGFR chr7 55228053 A T 99.4％(10873X) 2 HRAS chr11 534242 A G 47.7％(10998X) 3 EGFR chr7 55249063 G A 39.8％(9615X) 4 EGFR chr7 55241727 G A 49.9％(13809X)

[0163] Note: GM24385 and GM24143 both contain mutation sites with sequence numbers 1, 2, and 3; GW24149 contains mutation sites with sequence numbers 1 and 3; GM24631 contains mutation sites with sequence numbers 1 and 4.

[0164] Table 14 Cumulative frequency of false matching rate

[0165]

[0166]

[0167] Fig.12 is the cumulative frequency of false matching rate, Fig.13is the frequency of mismatched bases at each site.

[0168] From the above data, when the confidence interval is set to 99%, the false matching rates of A, T, C, and G are 0-0.48%, 0-0.26%, 0-0.69%, and 0-0.79%, respectively. Therefore, in this test, 0.79% is selected to establish the analysis cutoff value to filter SNVs with a frequency lower than 0.79%, that is, when the frequency is lower than 0.79%, the SNV variation is not output.

[0169] 1.2 Blank detection limit

[0170] In order to confirm the blank detection limit of this test, NTC tests were performed 14 times in total, and the final sequencing data statistics are shown in Table 15 below:

[0171] Table 15 NTC sequencing data statistics

[0172] parameter Mapped_Reads On Target Mean Depth min depth max depth Uniformity Maximum 405 399 10 1 34 96 Minimum 53 36 0 0 5 40 average value 145 130 3 0 13 70 SD 112 115 3 0 8 21 X+3SD 482 474 12 2 38 134 X-3SD -191 -215 -6 -1 -12 6

[0173] According to statistical data, the final SNV mutation output standard for this test is established as follows:

[0174] 1. MAF ≥ 0.79%;

[0175] 2. Mutant Reads ≥ 12;

[0176] 3. When the Mapped Reads of the NTC sample is greater than 482 and the Mean Depth is greater than 12, it is determined that the samples in this batch are contaminated and the cause needs to be investigated and out-of-control measures need to be taken.

[0177] 1.3 DNA input amount

[0178] To confirm the DNA input amount for this test, two clinical samples with known qPCR results and one HorizonDiscovery standard, HD827 OncoSpan Reference Standard, were selected. The input amounts were set to 100 ng, 70 ng, 50 ng, 30 ng, 20 ng, 10 ng, and 5 ng, respectively. Each sample was replicated for 3 times. The sequencing data results were statistically shown as follows: Fig.13 shown.

[0179] QC

[0180] like Fig.14 As shown, there is no significant difference in the Mapped Reads, On Target, and Mean Depth of different samples when comparing different DNA input amounts.

[0181] Mutation analysis

[0182] The statistics of the variation results within the detection area are shown in Table 16 below:

[0183] Table 16 Mutation detection with different DNA input amounts

[0184]

[0185]

[0186] According to the SNV result judgment standard (MAF>0.79%), the MAF of T790M at 10ng and 5ng were 0.71% and 0.30% respectively, which should be judged as negative, that is, a false negative occurred. Based on this, this test confirms that the minimum DNA input amount is 20ng and the optimal input amount is 100ng. When the input amount is lower than 20ng, the data amount should be appropriately increased to avoid false negatives due to low-frequency mutations.

[0187] 1.4 Minimum detection limit

[0188] SNV detection limit

[0189] To determine the detection limit of SNV in this test, according to the blank limit standards in 1.2: 1. MAF ≥ 0.79%; 2. Mutant Reads ≥ 12, the positive sites of all samples were counted, and the positive and negative definitions of different samples are as follows:

[0190] 1. Select 4 standards GM24385, GM24149, GM24143, and GM24631 from the Coriell Institute for testing. Compare the results with the reference sequencing results. In this test area, the sites in Table 12 are true positives, and the detection of other positions is false positive:

[0191] 2. Select Horizon Discovery's standard product HD827 OncoSpan Reference Standard and Jingliang Gene Technology (Shenzhen) Co., Ltd.'s standard product GWOCTM1001 for testing. Compare the results with the reference sequencing results. In this test area, the variants listed in Table 17 below are true positives, and the variants detected in other positions are false positives.

[0192] Table 17 Known mutation sites of standard products HD827 and GWOCTM1001

[0193]

[0194] According to this principle, all sample detection results are statistically analyzed. The statistical results are shown in Fig.15 and Fig.16 .

[0195] Fig.15 This is the SNV distribution diagram of the detected samples.

[0196] Conclusion: Combined with the above data, when set to 95% confidence interval, the SNV detection limit of this test is: MAF>2.7%, Total reads ＞ 455; MAF < 2.7%, SNVs with Total reads < 455 were not reported.

[0197] Indel detection limit

[0198] Fig.16 This is the indel distribution diagram of the detected samples.

[0199] Conclusion: Based on the above data, the detection limit of inde1 in this test is: MAF ＞ 1.6%, Total reads>571; MAF<1.6%, indels with Total reads<571 are not included in the report.

[0200] 1.5 Precision

[0201] The precision of this test is the ability to obtain the same result by repeatedly testing the specimen. This validation includes intra-batch repeatability and inter-batch repeatability.

[0202] Methods: Horizon Discovery's standard HD827 OncoSpan Reference Standard and Shenzhen Jingliang Gene Technology Co., Ltd.'s standard GWOCTM1001 were selected for testing: HD827 OncoSpan Reference Standard was tested 4 times within the batch and 8 times between batches; GWOCTM1001 was tested 3 times within the batch and 1 time between batches. The data of repeated tests within and between batches of the two were summarized and statistically analyzed according to the above minimum detection limit standards: SNV: MAF>2.7%, Total Reads>455; indel: MAF>1.6%, Total Reads>571. The detected variation frequency was used as the horizontal axis and the expected frequency was used as the vertical axis for plotting. The results are shown in the figure. Fig.17 It can be seen that the detection consistency of each gene within and between batches is good.

[0203] 1.6 Accuracy

[0204] The accuracy of the present invention is defined as the consistency of the sample test results with the test results of other detection methods or known results. The present invention adopts the methodology of Example 2 and verifies the accuracy of the detection method of the present invention through 32 samples (1 NTC + 31 clinical samples with known results). The specific results are shown in Table 18 below.

[0205] Table 18 Comparison of accuracy verification results

[0206]

[0207]

[0208] As shown in Table 17, the validation results of 31 clinical samples were compared with the known results, and the results were completely consistent, indicating that the accuracy of this methodology for evaluating the detection of solid tumor-related gene mutations is 100%.

[0209] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A primer set for detecting solid tumor-related gene mutations, characterized in that: The primer set includes primer pairs for at least two genes in the mutations in 40 regions of 13 related genes, and the primer pairs are selected from the following: A primer pair for detecting BRAF gene mutation, the primer pair being selected from: an upstream primer having a sequence as shown in SEQ ID NO.1 and a downstream primer having a sequence as shown in SEQ ID NO.2; an upstream primer having a sequence as shown in SEQ ID NO.3 and a downstream primer having a sequence as shown in SEQ ID NO.4; A primer pair for detecting EGFR gene mutation, the primer pair being selected from: an upstream primer having a sequence as shown in SEQ ID NO.5 and a downstream primer having a sequence as shown in SEQ ID NO.6; an upstream primer having a sequence as shown in SEQ ID NO.7 and a downstream primer having a sequence as shown in SEQ ID NO.8; an upstream primer having a sequence as shown in SEQ ID NO.9 and a downstream primer having a sequence as shown in SEQ ID NO.10; an upstream primer having a sequence as shown in SEQ ID NO.11 and a downstream primer having a sequence as shown in SEQ ID NO.12; an upstream primer having a sequence as shown in SEQ ID NO.13 and a downstream primer having a sequence as shown in SEQ ID NO.14; an upstream primer having a sequence as shown in SEQ ID NO.15 and a downstream primer having a sequence as shown in SEQ ID NO.16; an upstream primer having a sequence as shown in SEQ ID NO.17 and a downstream primer having a sequence as shown in SEQ ID NO.18; A primer pair for detecting a GNA11 gene mutation, the primer pair comprising: an upstream primer having a sequence as shown in SEQ ID NO.19 and a downstream primer having a sequence as shown in SEQ ID NO.20; A primer pair for detecting a GNAQ gene mutation, the primer pair comprising: an upstream primer having a sequence as shown in SEQ ID NO.21 and a downstream primer having a sequence as shown in SEQ ID NO.22; A primer pair for detecting a GNAS gene mutation, the primer pair being selected from: an upstream primer having a sequence as shown in SEQ ID NO.23 and a downstream primer having a sequence as shown in SEQ ID NO.24; an upstream primer having a sequence as shown in SEQ ID NO.25 and a downstream primer having a sequence as shown in SEQ ID NO.26; A primer pair for detecting HRAS gene mutation, the primer pair being selected from: an upstream primer having a sequence as shown in SEQ ID NO.27 and a downstream primer having a sequence as shown in SEQ ID NO.28; an upstream primer having a sequence as shown in SEQ ID NO.29 and a downstream primer having a sequence as shown in SEQ ID NO.30; A primer pair for detecting IDH1 gene mutation, the primer pair comprising: an upstream primer having a sequence as shown in SEQ ID NO.31 and a downstream primer having a sequence as shown in SEQ ID NO.32; A primer pair for detecting IDH2 gene mutation, the primer pair comprising: an upstream primer having a sequence as shown in SEQ ID NO.33 and a downstream primer having a sequence as shown in SEQ ID NO.34; A primer pair for detecting KRAS gene mutation, wherein the primer pair is selected from: an upstream primer having a sequence as shown in SEQ ID NO.35 and a downstream primer having a sequence as shown in SEQ ID NO.36; an upstream primer having a sequence as shown in SEQ ID NO.37 and a downstream primer having a sequence as shown in SEQ ID NO.38; an upstream primer having a sequence as shown in SEQ ID NO.39 and a downstream primer having a sequence as shown in SEQ ID NO.40; A primer pair for detecting NRAS gene mutation, wherein the primer pair is selected from: an upstream primer having a sequence as shown in SEQ ID NO.41 and a downstream primer having a sequence as shown in SEQ ID NO.42; an upstream primer having a sequence as shown in SEQ ID NO.43 and a downstream primer having a sequence as shown in SEQ ID NO.44; an upstream primer having a sequence as shown in SEQ ID NO.45 and a downstream primer having a sequence as shown in SEQ ID NO.46; A primer pair for detecting a PIK3CA gene mutation, the primer pair being selected from: an upstream primer having a sequence as shown in SEQ ID NO.47 and a downstream primer having a sequence as shown in SEQ ID NO.48; an upstream primer having a sequence as shown in SEQ ID NO.49 and a downstream primer having a sequence as shown in SEQ ID NO.50; an upstream primer having a sequence as shown in SEQ ID NO.51 and a downstream primer having a sequence as shown in SEQ ID NO.52; an upstream primer having a sequence as shown in SEQ ID NO.53 and a downstream primer having a sequence as shown in SEQ ID NO.54; an upstream primer having a sequence as shown in SEQ ID NO.55 and a downstream primer having a sequence as shown in SEQ ID NO.56; an upstream primer having a sequence as shown in SEQ ID NO.57 and a downstream primer having a sequence as shown in SEQ ID NO.58; an upstream primer having a sequence as shown in SEQ ID NO.59 and a downstream primer having a sequence as shown in SEQ ID NO.60; an upstream primer having a sequence as shown in SEQ ID NO.61 and a downstream primer having a sequence as shown in SEQ ID NO.62; an upstream primer having a sequence as shown in SEQ ID NO.63 and a downstream primer having a sequence as shown in SEQ ID NO. A downstream primer as shown in SEQ ID NO.64; an upstream primer as shown in SEQ ID NO.65 and a downstream primer as shown in SEQ ID NO.66; an upstream primer as shown in SEQ ID NO.67 and a downstream primer as shown in SEQ ID NO.68; A primer pair for detecting a TERT gene mutation, the primer pair being selected from: an upstream primer having a sequence as shown in SEQ ID NO.69 and a downstream primer having a sequence as shown in SEQ ID NO.70; an upstream primer having a sequence as shown in SEQ ID NO.71 and a downstream primer having a sequence as shown in SEQ ID NO.72; an upstream primer having a sequence as shown in SEQ ID NO.73 and a downstream primer having a sequence as shown in SEQ ID NO.74; A primer pair for detecting ERBB2 gene mutation, wherein the primer pair is selected from: an upstream primer with a sequence as shown in SEQ ID NO.75 and a downstream primer with a sequence as shown in SEQ ID NO.76; an upstream primer with a sequence as shown in SEQ ID NO.77 and a downstream primer with a sequence as shown in SEQ ID NO.78; an upstream primer with a sequence as shown in SEQ ID NO.79 and a downstream primer with a sequence as shown in SEQ ID NO.

80.

2. The primer set for detecting solid tumor-related gene mutations according to claim 1, characterized in that: The primer set includes primer pairs for NRAS and PIK3CA genes.

3. The primer set for detecting solid tumor-related gene mutations according to claim 2, characterized in that: The primer set also includes at least one of the primer pairs for BRAF, EGFR, KRAS, GNA1 1, GNAQ, GNAS, HRAS, IDH1, IDH2, TERT, and ERBB2 genes.

4. The primer set for detecting solid tumor-related gene mutations according to claim 1, characterized in that: The primer set includes primer pairs for BRAF, EGFR, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KRAS, NRAS, PIK3CA, TERT, and ERBB2 genes.

5. Use of the primer set according to any one of claims 1 to 4 in preparing a product for detecting the solid tumor-related gene mutation.

6. The use according to claim 5, characterized in that The product is a test kit.

7. A kit for detecting solid tumor-related gene mutations, characterized in that: The kit comprises the primer set according to any one of claims 1 to 4.

8. A method for detecting solid tumor-related gene mutations, characterized in that: The method comprises the following steps: using the DNA of the sample to be tested as a template, performing multiplex PCR amplification using the primer set according to any one of claims 1 to 4 or the kit according to claim 7, constructing a library, and sequencing.

9. The method according to claim 8, characterized in that The DNA includes gDNA and / or ctDNA; and / or, The multiplex PCR amplification includes a first round of PCR amplification and a second round of PCR amplification; the reaction system of the first round of PCR amplification includes the following components: Multiplex PCR Master Mix, DMSO, the primer pair mixture and DNA template; the reaction system of the second round of PCR amplification includes the following components: Multiplex PCR Master Mix, the product of the first round of PCR amplification, and a linker; and / or, The method for constructing the library is an amplicon capture method; and / or, The sequencing was based on the Illumina sequencing platform.

10. The method according to claim 9, characterized in that The concentration of the primer pairs SEQ ID NO.1-2, SEQ ID NO.25-26, SEQ ID NO.53-54, SEQ ID NO.77-78, and SEQ ID NO.79-80 in the reaction system of the first round of PCR amplification is 0.035uM respectively; The concentration of the primer pairs SEQ ID NO.3-4, SEQ ID NO.17-18, SEQ ID NO.35-36, SEQ ID NO.39-40, SEQ ID NO.47-48, and SEQ ID NO.63-64 in the reaction system of the first round of PCR amplification is 0.06 uM respectively: The concentration of the primer pairs SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.15-16, SEQ ID NO.19-20, SEQ ID NO.41-42, SEQ ID NO.55-56, and SEQ ID NO.57-58 in the reaction system of the first round of PCR amplification is 0.05 uM respectively; The concentration of the primer pairs SEQ ID NO.11-12, SEQ ID NO.29-30, SEQ ID NO.45-46, SEQ ID NO.49-50, SEQ ID NO.51-52, and SEQ ID NO.59-60 in the reaction system of the first round of PCR amplification is 0.045 uM respectively; The concentration of the primer pairs SEQ ID NO.13-14, SEQ ID NO.21-22, SEQ ID NO.33-34, SEQ ID NO.37-38, and SEQ ID NO.67-68 in the reaction system of the first round of PCR amplification is 0.04uM respectively; The concentration of the primer pair SEQ ID NO. 23-24 in the reaction system of the first round of PCR amplification is 0.055 uM respectively; The concentration of the primer pair SEQ ID NO. 27-28 in the reaction system of the first round of PCR amplification is 0.075 uM respectively; The concentration of the primer pairs SEQ ID NO.31-32, SEQ ID NO.61-62, and SEQ ID NO.65-66 in the reaction system of the first round of PCR amplification is 0.03uM respectively; The concentration of the primer pairs SEQ ID NO.43-44, SEQ ID NO.69-70, SEQ ID NO.71-72, and SEQ ID NO.73-74 in the reaction system of the first round of PCR amplification is 0.065uM respectively; The concentration of the primer pair SEQ ID NO. 75-76 in the reaction system of the first round of PCR amplification is 0.02 uM respectively; and / or, The reaction procedure of the first round of PCR amplification is: 1) 95°C, 5 min; 2) 95°C, 10 s; 60°C, 30 s; 72°C, 15 s; 20 cycles; 3) 72°C, 1 min; The reaction procedure of the second round of PCR amplification was: 1) 95° C., 10 min; 2) 95° C., 30 s; 65° C., 90 s; 10 cycles.

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