Kit for EGFR gene multi-site combined detection

Abstract

The present invention provides a primer-probe combination and a kit for multi-site joint detection of EGFR genes. Based on the method of five-channel digital PCR, only two PCR reaction tubes and 20 ng of sample DNA are needed to detect 35 multiple multi-site mutations of EGFR genes, including cis-trans mutations of T790M and C797S and unknown E19del mutations. The copy number and mutation frequency of different mutant DNAs can be accurately quantified, which is of great significance for determining the clinical significance of mutations in disease progression, treatment response and drug resistance, and helps doctors formulate more individualized treatment strategies.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and specifically relates to a primer-probe combination and a kit for multi-site joint detection of EGFR gene based on a digital PCR platform. Background Art

[0002] Lung cancer is a very common type of cancer in the world, with about 2 million new cases each year. The World Health Organization (WHO) divides lung cancer into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), of which NSCLC accounts for more than 85%. In non-small cell lung cancer (NSCLC), especially adenocarcinoma, the mutation frequency of epidermal growth factor receptor (EGFR) gene is very high, especially in Asian non-smoking patients, with a mutation rate as high as 40%-60%. The main mutation forms of EGFR are concentrated in exons 18-21. Mutations cause activation of the downstream pathway of EGFR, uncontrolled cell growth, and thus lead to tumor occurrence. Among them, EGFR exon 19 deletion mutation (E19del) accounts for about 45% of all EGFR mutations, and exon 21 L858R point mutation accounts for about 40%, so they are called common EGFR mutations or classic EGFR mutations. Both mutations will lead to activation of the tyrosine kinase domain, and are therefore related to the sensitivity of tyrosine kinase inhibitors (TKIs). Therefore, these two mutations are often called EGFR sensitive mutations and can be used as effective predictive indicators for EGFR-TKIs targeted drug therapy. Among NSCLC patients with EGFR gene mutations, 2%-4% of patients carry exon 20 insertion mutations (E20ins), which is the third most common mutation after exon 19 deletion mutations and exon 21 L858R mutations. Different exon 20 insertion mutations have different resistance to EGFR-TKIs. Currently, there are corresponding targeted drugs for exon 20 insertion mutations, such as Amivantamab (JNJ-6372) and Mobocertinib (TAK-788). The incidence of S768I in patients with EGFR mutations is about 1.5%-3%, and it often occurs in combination with mutations at sites such as E19del, G719X, E709X, and L861X. Clinical studies have shown that for patients with S768I mutations, the clinical use of second-generation EGFR-TKIs can benefit patients' survival. The L861Q point mutation is located in exon 21, accounting for about 2% of EGFR mutations. It is another rare mutation that is highly sensitive to second-generation EGFR-TKIs and relatively sensitive to osimertinib. When patients become resistant to first-generation / second-generation EGFR inhibitors, approximately 50%-60% of patients can detect the drug-resistant mutation T790M mutation. This is one of the main reasons why patients no longer benefit from first-generation / second-generation EGFR inhibitors, also known as the "gatekeeper mutation", so it is mainly treated with third-generation EGFR-TKIs represented by osimertinib.However, 6-17 months after the T790M resistance mutation, the C797S mutation located on exon 20 occurs, which is an acquired resistance mutation of the third-generation EGFR-TKIs. Unfortunately, there is still no effective targeted drug for patients with sensitive mutations / T790M / C797S triple mutations.

[0003] Currently, the U.S. Food and Drug Administration (FDA) and the China State Administration for Market Regulation (CFDA) have clearly stipulated that EGFR-TKI treatment should select appropriate treatment subjects based on the results of EGFR gene testing, and only patients with EGFR sensitive mutations can undergo subsequent EGFR-TKI treatment. Therefore, before determining whether a patient is suitable for EGFR-TKI treatment, the mutation status of the EGFR gene must be tested first. At present, multiple mutation detection of the EGFR gene is generally achieved by detecting free nucleic acid molecules (cellfreeDNA, cfDNA) in blood samples or tissue samples through fluorescent quantitative PCR, ARMS-PCR, NGS and other technologies. Compared with the PCR method, although NGS technology has high throughput and high sensitivity, the operation process is cumbersome, the detection cycle is long, and the cost is high and the requirements for bioinformatics analysts are high. Therefore, for the sequence of known mutation sites, the mutation sites can be detected by PCR technologies such as fluorescent quantitative PCR and ARMS-PCR.

[0004] Fluorescence quantitative PCR mainly uses fluorescent probes to label PCR products, and uses the accumulation of fluorescent signals to monitor the PCR reaction process in real time. It can perform quantitative analysis of DNA based on the increment of DNA during the reaction time. ARMS-PCR is a method used to detect known mutations. Its basic principle is to design primers based on known point mutations. If the 3'-end base of the primer does not completely match the template, primer extension will be blocked. If the 3'-end base of the primer is complementary to the mutant template base, PCR will only amplify the mutant template, thereby achieving the purpose of detecting point mutations. However, both fluorescence quantitative PCR and ARMS-PCR can only achieve qualitative and quantitative detection of known mutations, but cannot accurately quantify the mutation frequency. In addition, when conducting joint detection of multi-site mutation sites, due to the limitation of the fluorescence channel, the reaction tubes are usually divided into four to eight reaction tubes, which not only reduces the throughput of the detection, but also increases the reagent cost of the detection, and requires the use of more precious clinical samples, such as blood samples or tissue samples. For example, patent CN 113755590A disclosed a reagent and kit for the combined detection of EGFR gene mutations in December 2021. The patent uses a method based on a fluorescent PCR technology platform combined with ARMS technology and Tagman probes to perform multi-site combined detection of exons 19 to 21 of the EGFR gene, but the patent can only detect 19 mutations of E19del, with a narrow coverage range; further, the patent requires the division of 32 mutations into eight fluorescent PCR detection reaction tubes in operation, which is not only complicated to operate, but also requires a total amount of cfDNA in the sample of 80 to 160 ng. However, the concentration of cfDNA in plasma is generally very low, usually less than 10 micrograms per milliliter of plasma; and the content of circulating tumor DNA (ctDNA) derived from tumor cells and tumor cell exosomes in cfDNA is only less than 1%, so this method is not suitable for samples with low tumor content and ctDNA samples. Therefore, patent CN 116751867A disclosed a primer and fluorescent probe for detecting EGFR mutant genes in September 2023. The 41 base mutation forms of the EGFR gene were specifically detected through three PCR tubes, reducing the total amount of sample cfDNA to 30ng; however, the patent cannot detect the cis / trans mutations of T790M and C797S on exon 20 of the EGFR gene. Studies have shown that the secondary C797S mutation of the EGFR gene and the T790M mutation have two different relationships, cis / trans. When T790M and C797S mutate on the same allele at the same time, that is, the two mutations are located on the same chromosome, this configuration is called cis; T790M and C797S are located on different alleles, that is, the two mutations are located on different chromosomes, and this configuration is called trans.Studies have shown that the cis / trans mutations of T790M and C797S are significantly correlated with the efficacy of targeted drugs: NSCLC patients with T790M / C797S trans mutations are resistant to third-generation EGFR-TKIs, but sensitive to the combination of first- and third-generation EGFR-TKIs; while patients with T790M / C797S cis mutations are insensitive to both single-drug and combined first- and third-generation EGFR-TKIs. Therefore, the detection of cis / trans mutations of T790M and C797S has important reference value for the clinical treatment and medication guidance of NSCLC. Patent CN 116837098 A, published in October 2023, provides a primer and fluorescent probe for detecting cis-trans mutations of T790M and C797S based on digital PCR detection technology. However, the patent still requires two PCR tubes during the detection process. Summary of the invention

[0005] In order to solve the problems of low detection sensitivity, low detection throughput, high sample cfDNA usage, incomplete detection sites, etc. in the prior art when performing multi-site joint detection of EGFR gene, the present invention provides a primer-probe combination and a kit for multi-site joint detection of EGFR gene. In the method based on quintuple channel digital PCR, only two PCR reaction tubes and 20ng sample DNA are needed to realize the detection of 35 multiple multi-site mutations of EGFR gene including cis-trans mutations of T790M and C797S and unknown E19del mutation, and the copy number and mutation frequency of different mutant DNA can be accurately quantified, which is of great significance for determining the clinical significance of mutations in disease progression, treatment response and drug resistance, and helps doctors formulate more individualized treatment strategies.

[0006] To this end, the present invention provides a kit for multi-site joint detection of EGFR gene, the kit comprising a first reaction tube and / or a second reaction tube; the first reaction tube comprises at least one of E19del reagent, L858R reagent, S768I reagent, and T790M&C797S reagent; the second reaction tube comprises at least one of G719X reagent, E20ins reagent, and L861Q reagent; wherein the E19del reagent comprises primers with base sequences as shown in SEQ ID NOs.1 to 2, and a fluorescent probe with a base sequence as shown in SEQ ID NO.15; the L858R reagent comprises primers with base sequences as shown in SEQ ID NOs.3 to 4, and a fluorescent probe with a base sequence as shown in SEQ ID NO.16; the S768I reagent comprises primers with base sequences as shown in SEQ ID NOs.5 to 6, and a fluorescent probe with a base sequence as shown in SEQ ID NO.17; the T790M&C797S reagent comprises primers with base sequences as shown in SEQ ID NOs.7 to 8, and a fluorescent probe with a base sequence as shown in SEQ ID NO. NO.18-20; the G719X reagent includes primers with base sequences as shown in SEQ ID NO.9-10 and fluorescent probes with base sequences as shown in SEQ ID NO.21-24; the E20ins reagent includes primers with base sequences as shown in SEQ ID NO.11-12 and fluorescent probes with base sequences as shown in SEQ ID NO.25-29; the L861Q reagent includes primers with base sequences as shown in SEQ ID NO.13-14 and fluorescent probes with base sequences as shown in SEQ ID NO.30.

[0007] Preferably, the first reaction tube further comprises an internal reference probe having a base sequence as shown in SEQ ID NO.31; and the second reaction tube further comprises an internal reference probe having a base sequence as shown in SEQ ID NO.32 and / or SEQ ID NO.33. The internal reference probe having a base sequence as shown in SEQ ID NO.31 is E19de-ref, the internal reference probe having a base sequence as shown in SEQ ID NO.32 is E20-ref, and the internal reference probe having a base sequence as shown in SEQ ID NO.33 is E21-ref.

[0008] In one or more embodiments of the present invention, the endpoint sequence or non-endpoint sequence of the fluorescent probe and / or the internal reference probe has or does not have a modification, and the modification is selected from one of locked nucleic acid modification, peptide nucleic acid modification, and MGB modification. Here, the locked nucleic acid (LNA) modification, peptide nucleic acid (PNA) modification, and MGB (Minor groove binder) modification can increase the Tm value of the probe, and when the modified probe sequence does not match the template, it is difficult to complementarily anneal with the template, thereby improving the specificity of the probe binding to the template.

[0009] Preferably, the 5' end of the fluorescent probe and the internal reference probe is connected to a fluorescent group and / or the 3' end is connected to a quenching group. Preferably, the fluorescent group is selected from at least one of FAM, VIC, ROX, CY5, and CY5.5. Preferably, the quenching group is selected from at least one of BHQ1, BHQ2, BHQ3, and NFQ. Among them, the 3' end of the fluorescent probe connected to the quenching group NFQ is generally modified with an MGB group to improve the specificity of the probe binding to the template.

[0010] The present invention designs a kit for multi-site joint detection including primers, fluorescent probes, and internal reference probes for various mutations of the EGFR gene. The kit only requires two PCR reaction tubes and a total of 20 ng sample volume to simultaneously perform rapid, accurate, and sensitive joint detection of 35 mutations of the EGFR gene, including cis-trans mutations of T790M and C797S, as well as detection of unknown E19del mutations. Specifically, the detection sites of the 35 EGFR gene mutations involved in the present invention are shown in Table 1.

[0011] Table 1. Detection sites of EGFR gene mutations

[0012]

[0013]

[0014] Note: Table 1 lists 33 mutation types. The cis and trans forms of T790M and C797S are not included in Table 1 as two mutation types.

[0015] To achieve the above purpose, the inventor team creatively designed specific primers and probes based on the wild-type gene sequences and mutation sequences of exons 18, 19, 20, and 21 of the human EGFR gene published in the Cosmics database. Specifically, primers and fluorescent probes were designed for the eight EGFR mutation sites, namely, E19 deletion mutation, L858R mutation, S768I mutation, T790M mutation, C797S mutation, E20ins mutation, L861Q mutation, and G719X mutation, and then the primers were preliminarily screened using the SYBR method, and the primer combinations obtained by the screening were combined with the fluorescent probes and screened again, and the primer and fluorescent probe combinations obtained by the screening were adjusted and designed to obtain the optimal primer and fluorescent probe combination, and digital PCR verification was performed using wild-type samples or mutant samples as templates. Further, the primers and fluorescent probes obtained by the above screening are combined step by step with different site systems, first two sites are combined, and the specificity and accuracy of each site are tested. If the specificity or accuracy of the system after the combination or the fluorescence map partition is poor, the primer probe concentration or sequence is adjusted. After the two site combinations are determined, the detection system of the third site is added, and the specificity and accuracy of each site are continued to be tested. Similarly, if the specificity or accuracy of the system after the combination or the fluorescence map partition is poor, the primer probe concentration or sequence is adjusted. If a good detection effect is not formed after adjustment, the adjustment of the distribution of the reaction tubes is considered. After the multiple systems of each reaction tube are initially determined, the optimization of the multiple systems is performed, and the concentration and amplification program of each primer probe in the reaction system are systematically optimized. The distribution of the reaction tubes finally formed after multiple adjustments is shown in Table 2, and the primers, probe sequences, reaction systems, and reaction programs finally determined are shown in Tables 3, 4, 5, and 6.

[0016] As shown in Table 2, the 33 sequences including primers, fluorescent probes, and internal reference probes that were finally screened and confirmed were scientifically and rationally allocated to two PCR reaction tubes, namely the first reaction tube and the second reaction tube. In the first reaction tube, multiple E19 deletion mutations, L858R mutations, S768I mutations, T790M mutations, and C797S mutations of the EGFR gene were detected simultaneously, and the cis / trans linkage mode of T790M and C797S mutations was determined at the same time; the second reaction tube simultaneously detected multiple E20ins mutations, L861Q mutations, and G719X mutations (including three mutations of G719C, G719A, and G719S), and a total of at least 35 EGFR mutations were detected, and the copy number and mutation frequency of different mutant DNAs could be accurately quantified. It should be noted that the fluorescent probe used to detect the E19del mutation in the kit is not only designed for the E19del mutation mentioned in Table 1. From the principle of the detection method, it can be understood that the fluorescent probe for detecting the E19del mutation provided by the present invention can theoretically detect other unknown mutations of E19del, further expanding the detection range.

[0017] Table 2. Mutation type settings for different reaction tubes

[0018]

[0019] The inventor team has specifically matched different fluorescent groups to the fluorescent probes designed for different mutation sites. In the same reaction system (i.e., the same reaction tube), multiple gene site mutations can be detected synchronously and in parallel with the help of probes with different fluorescent modifications for different mutation sites. As shown in Table 2, each reaction tube includes 5 different fluorescent groups, which are used to report the binding signals of the probe and the template at different points. It should be noted that in order to achieve accurate quantitative detection of the mutation frequency of each detection site of the EGFR gene, the inventor team designed a quantitative system for the total copy number of the four exons 18, 19, 20, and 21. There are detections of internal reference genes on exons 19, 20, and 21. When calculating the mutation frequencies of E19del, L858R, T790M, E20ins, L861Q, S768I, and G797S sites, the total copy number of each of exons 19, 20, and 21 is the detection copy number of the internal reference, and exon 18 adopts the design of wild-type competitive probe, and the total copy number of exon 18 is the wild-type copy number plus the mutant copy number. It can be seen from this that if a fluorescent signal is assigned to the detection of each point, it is impossible to achieve 35 mutation detections through two reaction tubes using conventional methods under the current limited digital PCR fluorescence channels. Therefore, the inventor team cleverly used the characteristics of the four quadrant distribution in the digital PCR fluorescence map. As shown in Table 2, the detection of E19del-WT and the detection of L858R-MU in reaction tube 1 share the fluorescence channel ROX, without affecting their respective detections at all. The principle is explained here as follows: First, the E19del reagent used to detect the E19del mutation is only equipped with a pair of primers, an internal reference probe (VIC signal) and a wild-type probe (ROX signal). This setting not only simplifies the reaction system, but also can theoretically detect unknown mutations of E19del, which is perfectly compatible with digital PCR technology. When detecting the E19del wild-type template, both the internal reference probe and the wild-type probe will bind to the template, and a VIC+ROX dual fluorescence signal will be generated after amplification. The scatter points of the dual fluorescence signal are distributed in the first quadrant (upper right corner) of the fluorescence graph (X-axis ROX, Y-axis VIC); when detecting the E19del homozygous mutation template, only the internal reference probe will bind to the template, and a VIC fluorescence signal will be generated after amplification. The scatter points of the single VIC signal are distributed in the fourth quadrant (upper left corner) of the fluorescence graph (X-axis ROX, Y-axis VIC); when detecting the E19del heterozygous mutation template, the internal reference probe will bind to each template, and the wild-type probe will only bind to the wild-type template. After amplification, two fluorescence signals will be generated, a single VIC signal and a VIC+ROX dual fluorescence signal, and the scatter points of the positive signal are distributed in the first quadrant (upper right corner) and the fourth quadrant (upper left corner) of the fluorescence graph.It can be seen that the single ROX signal position in the two-dimensional fluorescence map of ROX and VIC in the E19del detection system is idle, so the inventor team took advantage of this and set the detection fluorescence signal of the L858R mutation signal to ROX. When a single ROX signal appears, it is an L858R mutation. In reaction tube 2, the detection of E20ins-MU and the detection of L861Q-MU share the fluorescence channel FAM, and do not affect their respective detections at all. The principle is the same as the above-mentioned reaction tube 1. In a preferred embodiment of the present invention, the specific primers screened and confirmed by the kit, as well as the detailed sequences of the fluorescent probes and internal reference probes modified with different fluorescent / quenching groups are shown in Table 3.

[0020] Table 3. Primers and probes used to detect 35 mutation types of EGFR gene

[0021]

[0022]

[0023] Note: In Table 3, “+” indicates that the base to the right of “+” is modified with locked nucleic acid.

[0024] The present invention also provides the use of the kit in the preparation of a kit for the joint detection of multiple sites of the EGFR gene. It is understandable that the kit is not limited to the joint detection of the above-mentioned 35 EGFR gene mutation sites. The first reaction tube or the second reaction tube in the kit can be used alone for the detection of the corresponding mutation site, and any tube can be selected according to the needs for experiments, result analysis, and report issuance; the same reaction tube can also be improved by improving the existing primer and probe design methods so that it is also suitable for the detection of other mutation types such as single-base deletions, short fragment insertions or deletions. It is also understandable that the first reaction tube or the second reaction tube may include one or more primer-probe combination reagents for the detection of different mutation sites, which are used for the detection of one mutation site or the joint detection of multiple mutation sites.

[0025] The present invention also provides a method for in vitro non-diagnostic EGFR gene multi-site joint detection, comprising the following steps: extracting DNA from the sample to be tested as a template, performing a PCR reaction using the above-mentioned kit for EGFR gene multi-site joint detection, and making a judgment based on the fluorescent PCR amplification results.

[0026] Preferably, the method uses a fluorescent PCR instrument and / or a digital PCR instrument to perform a PCR reaction. Compared with conventional PCR, digital PCR separates the sample into a large number of partitions so that each partition can be subjected to a separate PCR reaction, and can achieve absolute quantification of mutants in the sample. The digital PCR instrument generally includes a chip-type digital PCR instrument, a droplet digital PCR instrument, and a magnetic bead digital PCR instrument. Droplet digital PCR (ddPCR) technology, such as Bio-Rad, provides an oil-in-water emulsion droplet system, in which droplets are formed in a water-oil emulsion to separate partitions of template DNA molecules, and the sample is divided into droplets of 20,000 nanoliters in size. This division enables thousands of independent amplification events to be measured in a single sample, avoiding competition between target nucleic acids for PCR resources, so as to achieve sensitive measurement of samples and absolute quantification of mutants. In a preferred embodiment of the present invention, the method uses a droplet digital PCR instrument to perform a PCR reaction, and the detection cycle only takes 6 to 8 hours, and the copy number and mutation frequency of different mutant DNA can be accurately quantified. Among them, the calculation method of the mutation frequency of each site is as follows:

[0027] E19del mutation frequency = (VIC copy number (tube 1) - ROX copy number (tube 1)) / VIC copy number (tube 1) × 100%; L858R mutation frequency = (ROX copy number (tube 1) - VIC copy number (tube 1)) / CY5.5 copy number (tube 2) × 100%; E20ins mutation frequency = FAM & VIC double mutation copy number (tube 2) / VIC copy number (tube 2) × 100%;

[0028] T790M mutation frequency = FAM copy number (tube 1) / VIC copy number (tube 2) × 100%;

[0029] G719X mutation frequency = CY5 copy number (tube 2) / (CY5 copy number (tube 2)+ROX copy number (tube 2)) × 100%; S768I mutation frequency = CY5.5 copy number (tube 1) / VIC copy number (tube 2) × 100%;

[0030] L861Q mutation frequency = single mutation FAM copy number (tube 2) / CY5.5 copy number (tube 2) × 100%;

[0031] In the formula, tube 1 refers to the first reaction tube, and tube 2 refers to the second reaction tube.

[0032] Preferably, the sample to be tested is selected from at least one of body fluids, tissues, in vitro cell cultures, and plasmids. Here, the sample to be tested should be understood as any substance containing or presumed to contain nucleic acids, including tissues or fluids separated from individuals, or purified parts thereof, such as body fluids, tissues, and in vitro cell cultures. The body fluids include plasma, serum, cerebrospinal fluid, lymph fluid, sputum, bile, synovial fluid, urine, tears, semen, mammary secretions, saliva, joint fluid, ascites, pleural effusion, amniotic fluid, etc. The tissues include whole blood, normal tissues, tumor tissues, and also paraffin-embedded tissues or postoperative frozen tissues.

[0033] In one or more embodiments of the present invention, the PCR reaction system generally includes the above-mentioned kit for multi-site joint detection of EGFR gene, PCR reaction premix, and DNA extracted from the sample to be tested. Among them, the DNA extracted from the sample to be tested generally refers to DNA extracted from paraffin-embedded tissue (FFPE) samples, or cfDNA extracted from plasma samples, or mutant cell line DNA, or plasmid DNA. The PCR reaction premix generally includes reaction reagents well known to those skilled in the art such as PCR buffer, dNTP and TaqDNA polymerase. In a single reaction tube, that is, the first reaction tube or the second reaction tube, the amount of the sample to be tested is not less than 10ng, preferably 10 to 150ng, and specifically can be 10ng, 11ng, 12ng, 13ng, 20ng, 30ng, 40ng, 50ng, 60ng, 70ng, 80ng, 90ng, 100ng, 120ng, 140ng, 150ng.

[0034] In a preferred embodiment of the present invention, the PCR reaction comprises: mixing the kit for multi-site joint detection of EGFR gene, PCR reaction premix and DNA extracted from the sample to be tested according to the repeatedly optimized PCR reaction system provided in Table 4 and Table 5, and performing digital PCR reaction under the PCR reaction program provided in Table 6. Based on the five-fold fluorescent signal channel, the method provided by the present invention can quickly, accurately and sensitively perform joint detection of 35 mutations of EGFR gene. The detection process only requires two PCR reaction tubes and 20ng sample DNA, achieving a DNA sample dosage as low as 10ng / reaction tube. Compared with the prior art, it not only has high throughput and low detection cost, but also greatly reduces the dosage of precious clinical samples.

[0035] Table 4. Multiple reaction system of the first reaction tube

[0036]

[0037]

[0038] Table 5. Multiple reaction system of the second reaction tube

[0039] Reagent name Sample volume (ul) 2xPCR reaction master mix 15 G719X-F(20uM) 1.2 G719X-R(20uM) 1.2 G719X-WP(10uM) 0.3 G719A-MP(10uM) 0.5 G719C-MP(10uM) 0.3 G719S-MP(10uM) 0.3 E20ins-F(20uM) 1.2 E20ins-R(20uM) 1.2 E20ins-ref-P(10uM) 0.5 E20ins-MT1-P(10uM) 0.2 E20ins-MT2-P(10uM) 0.2 E20ins-MT3-P(10uM) 0.3 E20ins-MT4-P(10uM) 0.3 E20ins-MT5-P(10uM) 0.5 L861Q-F(20uM) 1.2 L861Q-R(20uM) 1.2 L861Q-MP(10uM) 0.75 E21-ref-P(10uM) 0.75 DNA (10ng) 1 <![CDATA[H2O]]> to30ul

[0040] Table 6. Reaction procedure

[0041]

[0042] After the PCR reaction is performed using a multiple droplet digital PCR instrument, a judgment is made based on the fluorescent PCR amplification results. The judgment criteria are as follows: under the premise that the positive and negative quality control is under control: (1) if there are continuous positive droplets (single fluorescence value) ≥ 3, it is judged as positive; (2) if the positive droplets are ≤ 2, it is necessary to increase the input amount to improve the post-detection judgment; (3) if the number of positive droplets = 0, it is judged as negative. Among them, the method for judging the cis / trans linkage of T790M and C797S mutations is: judging by the digital PCR fluorescence image of the first reaction tube, when the sample is cis-linked with T790M and C797S mutations, there is only a double mutation of FAM+CY5 in the FAM-CY5 two-dimensional fluorescence image; when the sample is trans-linked with T790M and C797S mutations, there is only a single FAM mutation and a single CY5 mutation in the FAM-CY5 two-dimensional fluorescence image; when the sample is cis+trans-linked with T790M and C797S mutations, there are both a single FAM mutation and a double mutation of FAM+CY5 in the FAM-CY5 two-dimensional fluorescence image.

[0043] Beneficial effects of the present invention: The kit for multi-site joint detection of EGFR gene provided by the present invention is based on the method of five-fold fluorescent channel digital PCR. Only two PCR reaction tubes are needed to detect at least 35 EGFR mutations, and the cis / trans linkage of T790M and C797S mutations and unknown E19del mutations can be detected. At the same time, the copy number and mutation frequency of different mutant DNAs can be accurately quantified, which improves the convenience of detection, reduces the detection cost, and ensures the sensitivity and accuracy of detection. Secondly, the present invention reduces the total amount of sample DNA to 20ng, so that it can be used not only for the detection of tissue samples, but also for the detection of cfDNA samples, solving the problem of small amount of clinical cfDNA samples. The detection technology is based on a digital PCR platform, which has the advantages of low detection abundance, high specificity, simple operation, fast time, and simple and intuitive interpretation. It can improve the detection performance of gene mutation sites, thereby better assisting scientific research and clinical detection needs, and is of great significance for guiding doctors in medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The results of fluorescence PCR detection of T790M site in Example 1, wherein (a) is the wild template and (b) is the mutant standard.

[0045] Figure 2 It is a two-dimensional fluorescence scatter plot of digital PCR of T790M site in Example 1, wherein (a) is a wild template, (b) is a mutant plasmid, and (c) is a mutant standard.

[0046] Figure 3 The two-dimensional fluorescence scatter plot of digital PCR of T790M site in Example 1, wherein (a) is clinical sample 1,

[0047] (b) is clinical sample 2.

[0048] Figure 4 These are the fluorescence detection results of the C797S site in Example 1, where (a) is the wild-type template, (b) is the C797S (c.2389T>A) site mutation standard product CBP10341P, and (c) is the C797S (c.2390G>C) site mutation standard product CBP10342P.

[0049] Figure 5 It is a two-dimensional fluorescence scatter plot of digital PCR of C797S site in Example 1, wherein (a) is the wild template of C797S (c.2389T>A), (b) is the wild template of C797S (c.2390G>C), (c) is the mutation standard product CBP10341P of C797S (c.2389T>A), and (d) is the mutation standard product CBP10342P of C797S (c.2390G>C).

[0050] Figure 6 It is the two-dimensional fluorescence scatter plot of digital PCR of C797S (c.2389T>A) site in Example 1, where (a) is clinical sample 3, (b) is clinical sample 4, (c) is clinical sample 5, and (d) is clinical sample 6.

[0051] Figure 7 This is a two-dimensional fluorescence scatter plot of the wild-type template digital PCR in the multiplex system in Example 1.

[0052] Figure 8 This is a two-dimensional fluorescence scatter plot of the digital PCR of the multiple system mutation standard T790M-plasmid / CBP10341P / CBP10342P in Example 1.

[0053] Fig. 9 This is the two-dimensional fluorescence scatter plot of the PC-9 digital PCR of the multiple system mutation standard in Example 1

[0054] Fig.10 This is a two-dimensional fluorescence scatter plot of digital PCR of clinical sample 2 in the multiplex system in Example 1.

[0055] Fig.11This is a two-dimensional fluorescence scatter plot of digital PCR of clinical sample 4 in the multiplex system in Example 1.

[0056] Fig.12 This is a two-dimensional fluorescence scatter plot of digital PCR of clinical sample 6 in the multiplex system in Example 1.

[0057] Fig.13 This is the two-dimensional fluorescence scatter plot of digital PCR of clinical samples P0109984, P1107459, P1109449, and P0101236 in Example 7.

[0058] Fig.14 This is the two-dimensional fluorescence scatter plot of digital PCR of clinical samples P0103097, P0103407, P0101090, and P0101160 in Example 7.

[0059] Fig.15 This is the two-dimensional fluorescence scatter plot of digital PCR of clinical samples P0101289, P0102130, P1108682, and P1109009 in Example 7.

[0060] Fig.16 This is the two-dimensional fluorescence scatter plot of digital PCR of clinical samples P1109231, WT-1, and WT-2 in Example 7. DETAILED DESCRIPTION

[0061] The following is an explanation of the embodiments of the present invention by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. In the embodiments of the present invention, the methods used are all conventional methods unless otherwise specified, and the reagents used can be obtained from commercial sources.

[0062] Example 1: Primer and probe screening and validation process for T790M and C797S cis / trans site detection 1. Extraction of FFPE sample DNA and plasma sample cfDNA

[0063] (I) DNA extraction from FFPE samples

[0064] The genomic DNA was extracted from paraffin-embedded tissue using a silica gel membrane adsorption kit (Biospin paraffin-embedded tissue genomic DNA extraction kit). The concentration and purity of the DNA were detected using a spectrophotometer (Thermo Fisher) to ensure that the A260 / A280 of the DNA was between 1.8 and 2.0 and the A260 / A230 was greater than 1.7. The concentration of the DNA was detected using Qubit 3.0 (ThermoFisher).

[0065] (II) Extraction of cfDNA from plasma samples

[0066] Collect 10 mL of whole blood sample in an anticoagulant tube and centrifuge at 1600 g for 10 minutes. After the centrifugation, gently remove the blood collection tube; transfer the supernatant plasma to the prepared 2 mL sterile centrifuge tube to avoid aspirating the white film layer during the aspiration process, and centrifuge the 2 mL centrifuge tube containing plasma for a second time at 16000×g for 10 minutes; finally, transfer the supernatant after centrifugation to a new 2 mL sterile centrifuge tube, taking care not to aspirate the precipitate; use a silica membrane adsorption kit (Qiagen) to extract cfDNA from plasma, and use a spectrophotometer (Thermo Fisher) to detect the concentration and purity of DNA to ensure that the A260 / A280 of DNA is between 1.8-2.0 and the A260 / A230 is greater than 1.7. At the same time, use Qubit 3.0 (Thermo Fisher) to detect the concentration of DNA.

[0067] 2. Design and synthesis of primers and probes

[0068] For the T790M and C797S cis / trans sites, the primer probe design software Primer Express 3.0.1 was used to design common primers and their respective wild type / mutant probes. The designs are as follows:

[0069] Table 7. Primer probe design

[0070]

[0071] 3. Primer Screening

[0072] Different upstream and downstream primer combinations were tested. The template used was healthy human gDNA. The amplification reagent was Kangwei's 2xGoldstar probe mix. The PCR amplification conditions were 95°C pre-denaturation for 30 seconds, 95°C denaturation for 15 seconds, 60°C annealing for 34 seconds, and 40 cycles. The fluorescence signal was collected at the end of the 60°C annealing step. The instrument was ABI 7500 fluorescence quantitative PCR instrument, and the amplification system was shown in Table 8 below. The primer combination with better specificity was screened, and the optimal primer combination was initially screened out as follows: T790M&C797S-F1 / R2, T790M&C797S-F2 / R1, T790M&C797S-F1 / R4, T790M&C797S-F2 / R3, T790M&C797S-F1 / R3, and the detection Ct value was shown in Table 9 below.

[0073] Table 8. Primer screening amplification system

[0074] Ingredients Addition amount 2xPCR reaction premix (Kangwei) 10μl T790M&C797S upstream primer (10uM) 1μl T790M&C797S downstream primer (10uM) 1μl Template gDNA 20ng Sterile double distilled water to20ul Total volume 20ul

[0075] Table 9. Primer screening test Ct values

[0076]

[0077] 4. Screening of primer-probe combinations

[0078] (I) Screening and verification of primer-probe combinations at the T790M locus

[0079] 1. T790M site primer-probe combination fluorescence PCR screening

[0080] A. Experimental description: The above 5 pairs of primers were tested in combination with 6 wild-type probes and 6 mutant probes, using healthy human gDNA and mutant standard products as templates, and the amplification reagent was AG's 2X Pro Taq HS Probe Premix. The PCR amplification conditions were 95℃ pre-denaturation for 30sec, 95℃ denaturation for 15sec, 60℃ annealing for 34sec, 40 cycles, and the fluorescence signal was collected at the end of the 60℃ annealing step. The instrument was ABI 7500 fluorescence quantitative PCR instrument, and the amplification system was shown in Table 10 below.

[0081] Table 10. Site combination test fluorescence system

[0082] Ingredients Addition amount 2xPCR Master Mix (AG) 10μl Upstream primer (10uM) 1μl Downstream primer (10uM) 1μl WT probe (10uM) 0.5μl MT probe (10uM) 0.5μl Template gDNA 10ng Sterile double distilled water to20ul Total volume 20ul

[0083] B. Result description: After a series of fluorescence PCR experiments, the optimal combination of primers and probes was obtained: primer pair: T790M&C797S-F2 / R1, wild probe: T790M&C797S-WP6, T790M mutation probe: T790M-MP3. The optimal combination detection results are shown in the attached Figure 1 , Figure 1 a The template used was healthy human gDNA. Figure 1 b The template used was the DNA of the T790M mutant cell line: NCI-H1975, with a mutation frequency of 77.68%.

[0084] 2. Digital PCR verification of primer-probe combination at T790M locus

[0085] A. Experimental description: Use healthy human gDNA, mutant plasmid, mutant standard NCI-H1975, clinical sample 1, and clinical sample 2 as templates, use the optimal combination of primers and probes, and prepare the digital PCR system according to Table 11. The amplification reagent is Xinyi 2x microdroplet digital PCR reaction premix. The specific sample droplet preparation experimental process is as follows: Place the microdroplet generation chip into the microdroplet generation chip adapter; Cover the adapter cover, and use an 8-channel pipette to add 30μl of aqueous reaction system and 180μl of microdroplet generation oil to the corresponding solution wells. Note that the aqueous reaction system should be added first and then the microdroplet generation oil; Cover the microdroplet chip sealing pad; Open the Xinyi Droplet Chip; The cover of the Maker sample preparation instrument was opened, and the 8-tube strips were placed in. After loading, the DropMaker program was started to generate droplets. The generated droplets were placed in a PCR instrument (Bio-rad) and PCR amplification was performed according to the program in Table 12. The amplified droplets were analyzed using a Xinyi biochip analyzer to obtain data such as fluorescence scatter plots, copy numbers, and mutation rates.

[0086] Table 11. Digital PCR reaction system

[0087] Ingredients Addition amount 2xPCR reaction premix (Xinyi) 15μl Upstream primer (10uM) 2.4μl Downstream primer (10uM) 2.4μl WT probe (10uM) 0.75μl MT probe (10uM) 0.75μl Template gDNA 10ng Sterile double distilled water to30ul Total volume 30ul

[0088] Table 12. Digital PCR program

[0089]

[0090] B. Instructions for mutation frequency calculation: T790M mutation frequency = FAM fluorescence copy number / ROX fluorescence copy number.

[0091] C. Result description: When the T790M site primer-probe combination was used for digital PCR detection using wild-type template, only ROX fluorescence was detected. When the mutant plasmid template was used for detection, both FAM and ROX fluorescence were detected, indicating good specificity of the probe. The two-dimensional fluorescence scatter plot is shown in Figure 2. Figure 2 a, 2b; When using mutation standards and clinical samples for testing, the mutation frequencies obtained from data analysis were consistent with expectations, indicating the accuracy of primer-probe combination detection. Figure 2 c, 3a, and 3b respectively show the fluorescence two-dimensional scatter plots of the T790M site mutation standard and two clinical samples detected by digital PCR. The calculated copy number and mutation frequency data of all samples are shown in Table 13.

[0092] Table 13. T790M site digital PCR detection data

[0093] Sample No. Total number of droplets FAM copy number ROX copy number Mutation frequency NGS reference frequency WT 37099 0 6085.5 / / plasmid 39671 1426.6 1426.6 100％ 100％ NCI-H1975 33582 4313.5 5315.5 81.15％ 77.68％-78.14％ Clinical Sample 1 54570 169.1 1684.3 10.04％ 12.66％ Clinical Sample 2 48984 335.2 3522.3 9.52％ 11.47％

[0094] (II) Screening and verification of primer-probe combination for C797S locus

[0095] 1. Fluorescence PCR screening of primer-probe combination at C797S locus

[0096] A. Experimental description: The above five pairs of primers were tested in combination with six wild-type probes, four C797S (c.2389T>A) mutant probes or three C797S (c.2390G>C) mutant probes, respectively, using healthy human gDNA, mutation standard CBP10341P, and mutation standard CBP10342P as templates, and the amplification reagent was AG's 2X Pro Taq HS Probe Premix. The PCR amplification conditions were 95°C pre-denaturation for 30 seconds, 95°C denaturation for 15 seconds, 60°C annealing for 34 seconds, and 40 cycles. The fluorescence signal was collected at the end of the 60°C annealing step. The instrument was ABI 7500 fluorescence quantitative PCR instrument, and the amplification system was shown in Table 4.

[0097] B. Result description: For the C797S (c.2389T>A) mutation, the optimal combination of primers and probes is: primer pair: T790M&C797S-F2 / R1, wild probe: T790M&C797S-WP6, C797S (c.2389T>A) mutation probe: C797S-2389-MP4; for the C797S (c.2390G>C) mutation, the optimal combination of primers and probes is: primer pair: T790M&C797S-F2 / R1, wild probe: T790M&C797S-WP6, C797S (c.2390G>C) mutation probe: C797S-2390-MP3; the optimal combination detection results are shown in the attached Figure 4 : Figure 4 a shows the C797S site (c.2389T>A) mutation and C797S (c.2390G>C) mutation, using the gDNA of a healthy person as the template for the test results, only the ROX fluorescence amplification peak is obvious; Figure 4 The templates used in b and 4c are C797S (c.2389T>A) mutation standard CBP10341P and C797S (c.2390G>C) mutation standard CBP10342P, respectively, and the results of detection using their respective optimal combination systems are shown in the figure. The VIC fluorescence has a clear amplification peak.

[0098] 2. Digital PCR verification of primer-probe combination at C797S locus

[0099] A. Experimental description: Healthy human gDNA, mutation standard CBP10341P, mutation standard CBP10342P, mutation standard PC-9 and 4 clinical samples were used as templates, and the optimal combination of primers and probes was used. The digital PCR system was prepared according to Table 11. The amplification reagent was Xinyi 2x microdroplet digital PCR reaction premix. The specific sample droplet preparation experimental process and PCR reaction process were the same as above.

[0100] B. Instructions for calculating mutation frequency: C797S mutation frequency = CY5 fluorescence copy number / ROX fluorescence copy number.

[0101] CResult Description: Figure 5 The fluorescence two-dimensional scatter plots of the primer-probe combination system for C797S site (c.2389T>A) mutation and C797S (c.2390G>C) mutation were respectively detected by digital PCR using wild template and respective mutant standards (CBP10341P / CBP10342P). Only ROX fluorescence was detected when the wild template was detected, while both CY5 and ROX fluorescence were detected when the mutant template was detected, indicating good specificity of the probe; Figure 6 The primer-probe combination systems for the C797S site (c.2389T>A) mutation and the C797S (c.2390G>C) mutation are shown respectively, and the detection fluorescence two-dimensional scatter plots of digital PCR of 2 clinical samples with corresponding mutations are shown. The copy number and mutation rate data of all samples are calculated as shown in Table 14. The mutation frequency detection results of the mutant template are consistent with expectations, indicating the accuracy of the primer-probe combination detection.

[0102] Table 14. C797S site digital PCR detection data

[0103] Sample No. Total number of droplets CY5 copy number ROX copy number Mutation rate NGS reference frequency 2389WT 44582 0 5871.1 2390WT 38651 0 5669.8 CBP10341P 38110 3869.5 3876.8 100％ 100％ CBP10341P 37080 3058.5 3065.2 100％ 100％ Clinical Sample 3 47677 182.7 1309.3 13.95％ 11.23％ Clinical Sample 4 46288 257.7 2520.8 10.22％ 13.95％ Clinical Sample 5 46490 283.3 1417.4 19.99％ 15.12％ Clinical Sample 6 49655 150.9 933.6 16.16％ 16.86％

[0104] (III) Digital PCR verification of T790M+C797S primer-probe combination

[0105] After a series of screening and verification, the optimal combinations of primers and probes for the T790M mutation site, C797S (c.2389T>A) mutation site and C797S (c.2390G>C) mutation site were determined, and the specific information is shown in Table 15. The above triple reactions were mixed into one tube for verification of the multiplex digital PCR reaction. The templates used for verification included the wild type, mutation standards at the three sites, and mutation clinical samples at the three sites, which were used to verify the specificity and accuracy of the primer-probe combination, where the accuracy included the accuracy of the mutation frequency and the accuracy of cis-trans detection.

[0106] Table 15. Primer probe information

[0107]

[0108]

[0109] 1. Specificity

[0110] A. Experimental description: Use healthy human gDNA, T790M mutant plasmid, mutation standard CBP10341P, and mutation standard CBP10342P as templates, use the primer probe combination in Table 15, and prepare the digital PCR system according to Table 16. The amplification reagent is Xinyi 2x microdroplet digital PCR reaction premix. The specific sample droplet preparation experimental process and PCR reaction process are the same as above.

[0111] Table 16. Multiplex digital PCR reaction system

[0112] Ingredients Addition amount 2xPCR reaction premix (Xinyi) 15μl T790M&C797S-F2(10uM) 2.4μl T790M&C797S-R1(10uM) 2.4μl T790M&C797S-WP6(10uM) 0.75μl T790M-MP3(10uM) 0.75μl C797S-2389-MP4(10uM) 0.75μl C797S-2390-MP3(10uM) 0.75μl Template gDNA 10ng Sterile double distilled water To30ul Total volume 30ul

[0113] B. Mutation frequency and cis-trans linkage judgment instructions:

[0114] T790M mutation frequency = FAM fluorescence copy number / ROX fluorescence copy number.

[0115] C797S mutation frequency = CY5 fluorescence copy number / ROX fluorescence copy number;

[0116] The basis for judging cis-trans linkage: when the sample is cis-linked with T790M and C797S mutations, there are only double mutations of FAM+CY5 in the FAM-CY5 two-dimensional fluorescence map; when the sample is trans-linked with T790M and C797S mutations, there are only single FAM mutations and single CY5 mutations in the FAM-CY5 two-dimensional fluorescence map; when the sample is cis+trans-linked with T790M and C797S mutations, there are both single FAM mutations and double mutations of FAM+CY5 in the FAM-CY5 two-dimensional fluorescence map.

[0117] C. Result explanation: When the multiplex system uses healthy human gDNA as wild-type template for digital PCR detection, only ROX fluorescence appears, and no other nonspecific fluorescence appears, such as Figure 7 When T790M mutant plasmid, mutation standard CBP10341P, and mutation standard CBP10342P were used as templates for digital PCR detection, the fluorescence of each mutation probe and ROX fluorescence were displayed simultaneously, indicating good specificity of the primer-probe combination. Figure 8 , the copy number and mutation frequency data are shown in Table 17.

[0118] Table 17. Multiplex system specific digital PCR detection data

[0119]

[0120] 2. Accuracy

[0121] A. Experimental description: Use mutation standard PC-9, clinical sample 2, clinical sample 4, and clinical sample 6 as templates, use the primer probe combination in Table 15, and prepare the digital PCR system according to Table 16. The amplification reagent is Xinyi 2x micro-droplet digital PCR reaction premix. The specific sample droplet preparation experimental process and PCR reaction process are the same as above. At the same time, the detection template mutation standard PC-9, clinical sample 4, and clinical sample 6 were sent to Shanghai Bochen Biotechnology Co., Ltd. for cis-trans verification comparison using the Bio-rad QX200 digital PCR system.

[0122] B. Result description: When the multiplex system uses mutation standard PC-9, clinical sample 2, clinical sample 4, and clinical sample 6 as templates for digital PCR detection, the mutation frequencies calculated from the data are consistent with the NGS mutation frequencies, indicating the accuracy of the multiplex system of this embodiment in detecting mutation frequencies in samples; mutation standard PC-9, clinical sample 4, and clinical sample 6 use the Bio-rad QX200 digital PCR system to detect mutation frequencies and cis-trans results, which are consistent with the detection results of the multiplex system of this embodiment, indicating the accuracy of the multiplex system of this patent in detecting C797S cis-trans judgment in samples. The detection data of all samples are shown in Tables 18 and 19 below, and the detection two-dimensional fluorescence scatter plot is shown in Tables 18 and 19 below. Figures 9 to 12 .

[0123] Table 18. Comparison of multiplex system accuracy digital PCR test data and NGS

[0124]

[0125] Table 19. Comparison of multiplex system accuracy digital PCR detection cis-trans results with Bio-rad QX200

[0126]

[0127] Example 2: L861Q site primer probe screening process

[0128] 1. Design of primers and probes

[0129] For the L861Q site, the primer probe design software Primer Express 3.0.1 was used to design common primers and wild type / mutation probes, respectively. The design details are shown in Table 20.

[0130] Table 20. Primers and wild type / mutant probes for L861Q locus

[0131]

[0132] 2. Test results of primer probe

[0133] First, the SYBR method was used to perform preliminary primer screening on a fluorescent quantitative PCR instrument (results not shown), and then the better primer combinations screened out were combined with probes for further screening. The test results after combining with the probes showed (Table 21) that NTC did not peak but the wild-type template targets all peaked, indicating that the specificity of this batch of primer and probe combinations was poor.

[0134] Table 21. L861Q site primers and wild type / mutation probe test results

[0135]

[0136]

[0137] The probe sequences were then adjusted to design the second batch of probes. The final test results (Table 22) showed that the test primer-probe combination F6 / R7-RP1 / MP6 had good specificity and target detection effects.

[0138] Table 22. Final test results of L861Q site primers and wild type / mutant probes

[0139]

[0140] 3. Digital PCR Validation

[0141] The screened primer-probe combination F6 / R7-RP1 / MP6 was used for digital PCR detection verification, and the verification results are shown in Table 23. The verification results on digital PCR showed that the screened primer-probe combination had good specificity and detection effect on digital PCR.

[0142] Table 23. Digital PCR validation of primer-probe combination F6 / R7-RP1 / MP6 at L861Q locus

[0143] Sample No. Sample Type Total number of droplets ROX copy number CY5 copy number Mutation rate NGS reference mutation rate R1101122 wild type 27692 2623.8 0 0.00％ 0.00％ P1101114 wild type 48956 3136.8 0 0.00％ 0.00％ P1101115 wild type 47560 3174.5 2.8 0.09％ 0.00％ P1101119 wild type 47006 3310.5 1.4 0.04％ 0.00％ P1101120 wild type 31291 3396 2.4 0.07％ 0.00％ P1101126 wild type 48115 3516.7 0 0.00％ 0.00％ P1120030 Mutant 35019 1376.8 125.1 9.09％ 11.28％ P1119137 Mutant 42152 499.9 57.1 11.42％ 15.61％

[0144] Example 3: Brief description of the G719X site primer probe screening and verification process

[0145] 1. Design of primers and probes

[0146] For the G719X site, common primers and wild type / mutation probes were designed using the primer probe design software Primer Express 3.0.1. The design results are shown in Table 24.

[0147] Table 24. G719X primers and wild type / mutant probes

[0148]

[0149] 2. Test results of primer probe

[0150] First, the SYBR method was used to perform preliminary primer screening on a fluorescent quantitative PCR instrument (results not shown), and then the selected better primer combinations were combined with probes for further screening. The results showed that when the three subtype sites were detected for NTC templates, all combinations had no peaks (results not shown), and when the wild-type templates were detected, the WT probe signals all had peaks (Table 25), and most of the MT signals had no peaks, indicating that most primer probe combinations had good specificity, and overall the specificity of probe MT-P1 was better than that of probe MT-P2.

[0151] Table 25. G719X primer and wild type / mutant probe test results

[0152]

[0153]

[0154] Note: Ud = Undetermined

[0155] 3. Digital PCR Validation

[0156] The primer-probe combination F3 / R2-WT-P1 / MT-P1 (G719C, G719A, G719S) with better performance after screening was first used to perform digital PCR detection verification using clinical samples. The verification results are shown in Table 26.

[0157] The test results showed that the results of the single-site duplex system and the three-site multiplex system of the G719A site were consistent with the NGS results, the results of the single-site duplex system of the G719C site were consistent with the NGS results, the results of the three-site multiplex system had a large difference in individual mutation frequencies between the NGS results, and the results of the single-site duplex system and the three-site multiplex system of the G719S site had a large difference in mutation frequencies between individual samples and the NGS results.

[0158] Table 26. Digital PCR validation of primer-probe combination F3 / R2-WT-P1 / MT-P1 at G719X locus

[0159]

[0160] Table 27. Digital PCR verification of F2 / R3-WT-P2 / MT-P1 at G719X site

[0161]

[0162]

[0163] Table 28. Digital PCR verification of F2 / R2-WT-P2 / MT-P2 at G719X site

[0164]

[0165] The test results showed that the results of the single-site duplex system and the three-site multiplex system of the primer probe combination F2 / R2-WT-P2 / MT-P2 were consistent with the NGS results. The specificity of the primer probe combination F2 / R2-WT-P2 / MT-P2 was verified by using the wild-type template to detect the three-site multiplex system. The test results are shown in Table 29. The test results showed that the primer probe combination F2 / R2-WT-P2 / MT-P2 had good specificity.

[0166] Table 29. G719X site F2 / R2-WT-P2 / MT-P2 specificity verification

[0167] Clinical samples Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 ROX copy number 3525.5 3871.6 3580.5 3478.1 1994.5 2701.6 CY5 copy number 0 0 0 3.1 0 1.5 Mutation rate (%) 0.00％ 0.00％ 0.00％ 0.09％ 0.00％ 0.06％ NGS mutation rate (%) 0％ 0％ 0％ 0％ 0％ 0％

[0168] Example 4: Brief description of the L858R site primer probe screening and verification process

[0169] 1. Design of primers and probes

[0170] For the L858R site, the primer probe design software Primer Express 3.0.1 was used to design common primers and wild type / mutation probes, respectively. The design results are shown in Table 30.

[0171] Table 30. Primers and wild type / mutant probes for L858R locus

[0172]

[0173]

[0174] 2. Test results of primer probe

[0175] First, the SYBR method was used for preliminary primer screening on a fluorescent quantitative PCR instrument, and then the selected better primer combinations were combined with probes for further screening. The test results after combining with the probes showed (Table 31) that NTC did not peak but the wild-type template targets all peaked, indicating that the specificity of this batch of primer and probe combinations was poor.

[0176] Table 31. L858R site primers and wild type / mutant probe test results

[0177]

[0178] The probe sequences were then adjusted to design the second batch of probes. The final test results (Table 32) showed that the following four sets of primer-probe combinations had good specificity and target detection effects.

[0179] Table 32. Final test results of L858R site primers and wild type / mutant probes

[0180]

[0181] 3. Digital PCR Validation

[0182] First, the specificity of the mutation probe was verified in a single-plex system, and the wild-type template was used for detection. The results showed that the specificity of the four primer-probe combinations was good (Table 33).

[0183] Table 33. Specificity verification of L858R locus primer-probe combination in single-plex system

[0184]

[0185] Then, the cfDNA standard extracted from the cell line H1975 with L858R mutation (Table 34) and the clinical sample with L858R mutation (Table 36) were used to test the effect of mutation probe detection, and the test results are shown in Table 35 and Table 37. The test results showed that the primer probe combinations F6 / R6+MP4 / RP1 and F6 / R6+MP4 / RP1 were both good.

[0186] Table 34. cfDNA standards extracted from cell line H1975

[0187]

[0188] Table 35. Effect of L858R site primer probe combination on cfDNA standard detection

[0189]

[0190]

[0191] Table 36. Clinical samples of L858R mutation

[0192]

[0193] Table 37. Detection effect of L858R site primer-probe combination on clinical samples

[0194]

[0195] Example 5: Brief description of the S768I site primer probe screening and verification process

[0196] 1. Design of primers and probes

[0197] For the S768I site, common primers and wild type / mutation probes were designed using primer probe design software Primer Express 3.0.1. The design details are shown in Table 38.

[0198] Table 38. Primers for S768I site and wild type / mutant probes

[0199]

[0200]

[0201] 2. Test results of primer probe

[0202] First, the SYBR method was used to perform preliminary primer screening on a fluorescent quantitative PCR instrument, and then the selected better primer combination was combined with the probe for further screening. The test results after combining with the probe showed (Table 39) that neither NTC nor the wild-type template target had a peak, and the mutant template detection effect was good.

[0203] Table 39. S768I site primers and wild type / mutant probe test results

[0204]

[0205] 3. Digital PCR Validation

[0206] The best selected primer combination F3 / R4 was combined with different probes to test wild-type and mutant templates. The test results showed that the primer-probe combinations F3 / R4+MP1 and F3 / R4+MP3 had better specificity and detection performance (Table 40).

[0207] Table 40. Digital PCR validation of S768I site primers and wild type / mutant probes

[0208]

[0209]

[0210] The above primer-probe combination was verified using clinical samples with S768I mutation (Table 41). The test results showed that the two primer-probe combinations F3 / R4+MP1 and F3 / R4+MP3 at the S768I site had good specificity and detection effect (Table 42).

[0211] Table 41. Clinical samples of S768I mutation

[0212]

[0213] Table 42. Detection effect of primer-probe combination of S768I site on clinical samples

[0214]

[0215] Example 6: Multiple site combination process

[0216] First, 8 kinds of EGFR mutation sites, namely E19 deletion mutation, L858R mutation, S768I mutation, T790M mutation, C797S mutation, E20ins mutation, L861Q mutation, G719X mutation, the optimal primer combination (reference examples 1 to 5) screened out is gradually combined according to the distribution of the reaction tubes initially set, and the two sites are combined first to test the specificity and accuracy of each site. If the specificity or accuracy of the system after the combination or the fluorescence map partition is poor, the primer probe concentration or sequence is adjusted. After the two site combinations are determined, the detection system of the third site is added to continue to test the specificity and accuracy of each site. Similarly, if the specificity or accuracy of the system after the combination or the fluorescence map partition is poor, the primer probe concentration or sequence is adjusted. If a good detection effect is not formed after adjustment, the adjustment of the distribution of the reaction tubes is considered. After the multiple systems of each reaction tube are preliminarily determined, the multiple systems are optimized, and the concentration and amplification program of each primer probe in the reaction system are systematically optimized. The final distribution of the reaction tubes after multiple adjustments is shown in Table 2. The final reaction system and primer probe sequences are shown in Tables 3, 4, 5, and 6.

[0217] Example 7: Detection of clinical samples

[0218] The kit for multi-site joint detection of EGFR gene provided by the present invention (Table 3) was used to detect 15 paraffin-embedded tissue samples of patients with positive EGFR gene mutation. The droplet digital PCR reaction was performed according to the PCR reaction system provided in Table 4 and Table 5 and the PCR reaction program provided in Table 6, and the test results were compared with the sequencing results. Figures 13 to 16 ) is 100% consistent with the NGS sequencing method (Table 43, Table 44). Based on the five-fold fluorescent signal channel, the detection method provided by the present invention only requires two PCR reaction tubes and 20ng sample DNA to detect 35 EGFR gene multiple multi-site mutations including cis-trans mutations of T790M and C797S and the newly discovered E19del mutation, achieving a DNA sample dosage as low as 10ng / reaction tube. Compared with the existing technology, it not only has high throughput and low detection cost, but also greatly reduces the amount of precious clinical samples; and can accurately quantify the copy number and mutation frequency of different mutant DNAs, which is of great significance in determining the clinical significance of mutations in disease progression, treatment response and drug resistance, and helps doctors formulate more individualized treatment strategies.

[0219] Table 43 Summary of sample test results

[0220]

[0221]

[0222] Table 44 Comparison with NGS test results

[0223]

[0224] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope of the present invention.

Claims

1. A kit for multi-site combined detection of EGFR gene, comprising a first reaction tube and a second reaction tube as shown in the following table:

2. Use of the kit according to claim 1 in the preparation of a kit for multi-site combined detection of EGFR gene.

3. A method for in vitro non-diagnostic EGFR gene multi-site joint detection, characterized in that: The following steps are involved: DNA is extracted from the sample to be tested as a template, a PCR reaction is performed using the kit described in claim 1, and judgment is made based on the fluorescent PCR amplification result.

4. The method according to claim 3, characterized in that Perform PCR reactions using a fluorescent PCR instrument and / or a digital PCR instrument.

5. The method according to claim 3, characterized in that The sample to be tested is selected from at least one of body fluids, tissues, in vitro cell cultures, and plasmids.

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