Primer and probe for fgfr2 gene fusion detection and kit thereof

Abstract

The application discloses a primer and probe for FGFR2 gene fusion detection and a kit thereof. The application belongs to the field of biological detection. The kit for detecting FGFR2 gene fusion comprises a composition for detecting FGFR2 gene fusion. The composition comprises a primer F, primers R1-R8, a fusion detection probe and an internal standard probe. The nucleotide sequence of the primer F is shown as SEQ ID No:1. The nucleotide sequences of the primers R1-R8 are shown as SEQ ID No:2-SEQ ID No:9 respectively. The nucleotide sequence of the fusion detection probe is shown as SEQ ID No:10. The nucleotide sequence of the internal standard probe is shown as SEQ ID No:13. Through optimization of primer and probe design, the specificity and sensitivity of detection are improved, and a kit which is simple to operate, rapid and accurate is developed, thereby providing strong support for precise medical treatment of tumors.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to primers and probes for detecting FGFR2 gene fusion and their kits. Background Technology

[0002] The fibroblast growth factor receptor (FGFR) family mainly includes four types of receptors, FGFR1-FGFR4. FGFRs are transmembrane proteins with extracellular domains, transmembrane domains, and intracellular tyrosine kinase domains. The binding of FGF ligands to FGFRs initiates a cascade of intracellular signaling events, playing a crucial role in wound healing, angiogenesis, and tissue repair.

[0003] FGF / FGFR activating mutations, amplifications, or gene fusions can lead to persistent FGFR activation, promoting the progression of various tumors. Therefore, developing patient-accessible diagnostic reagents based on the FGFR2 target would provide a reliable basis for tumor diagnosis, enabling better differentiation of tumor subtypes and the development of personalized treatment plans.

[0004] Currently available methods for detecting FGFR2 gene fusions include DNA / RNA-based high-throughput sequencing (NGS), immunohistochemistry (IHC), in situ fluorescence hybridization (FISH), and reverse transcription polymerase chain reaction (RT-PCR).

[0005] NGS high-throughput sequencing technology offers high throughput and accuracy, enabling the simultaneous detection of multiple mutations and variants, and providing information on fusion partners and breakpoints.

[0006] Therefore, while NGS-based detection of FGFR2 gene fusions demonstrates good accuracy and effectiveness, the complex procedures involved, typically including library construction, enrichment, sequencing, data analysis, and variant annotation, result in lengthy processing times and require sophisticated data processing and analysis techniques, stringent quality control, and high overall economic costs. This imposes certain limitations on its clinical diagnostic and research applications.

[0007] IHC / FISH is a traditional method for detecting fusions. IHC is based on the specific recognition and binding of antibodies to antigens in tissue cells, using a colorimetric reaction on the antibody to locate, characterize, and quantify the antigens in the cells. IHC does not directly detect genetic changes in tumor cells, but rather detects FGFR proteins, using expression levels to determine positivity. However, fusion does not necessarily cause an increase in protein levels, leading to the possibility of false negatives. FISH technology designs fluorescent probes at both ends of the gene to be tested, and determines gene fusion by observing the degree of separation between the probes. FISH result interpretation relies on rigorously trained pathologists, and biopsy specimens often do not meet the required standards for tumor cells. Furthermore, IHC and FISH cannot determine the location of the fusion breakpoint, the specific fusion partner, or other information, making it impossible to discover new fusion partners. They also have low throughput, typically detecting only one gene fusion at a time. Due to the complexity of FGFR fusion partners, the traditional detection technology IHC / FISH has significant limitations in detecting FGFR fusions.

[0008] qRT-PCR technology utilizes changes in fluorescence signals to detect changes in the amount of amplification product in each cycle of the PCR amplification reaction in real time, thereby enabling qualitative and quantitative analysis of the starting template. Fluorescent labeling methods include dye-based methods and TaqMan probe methods; the TaqMan probe method is suitable for detecting mutations in target regions. In urothelial carcinoma, the FDA approved the therascreen® FGFR RGQ RT-PCR Kit (Qiagen) as a companion diagnostic kit for erdatinib. This kit uses qRT-PCR technology to qualitatively detect FGFR gene mutations in patient tumor tissue, including two FGFR2 fusions (FGFR2-BICC1 and FGFR2-CASP7). qRT-PCR probes need to be designed according to known mutation sites, can only detect target mutations, and have relatively low sensitivity and throughput.

[0009] Given the significant role of FGFR2 gene fusion detection in tumor diagnosis and treatment, and the limitations of existing detection methods, the development of simpler and more economical primers, probes, and kits for FGFR2 gene fusion detection has significant clinical application value and market potential.

[0010] Digital PCR is a highly sensitive molecular detection technique that can quantitatively detect specific DNA sequences in a sample, including mutations, gene amplifications, and gene fusions. Compared to conventional PCR, digital PCR has many advantages (Table 1), such as greater sensitivity to low-frequency mutations, rapid and accurate detection of small amounts of DNA molecules, better reproducibility, and a lower error rate.

[0011] Table 1. Comparison of technologies available for gene fusion detection Summary of the Invention

[0012] The main problem this invention aims to solve is how to efficiently and quantitatively detect FGFR2 gene fusions.

[0013] To address the aforementioned problems, this invention first provides a kit for detecting FGFR2 gene fusions.

[0014] The kit for detecting FGFR2 gene fusion provided by the present invention includes a composition for detecting FGFR2 gene fusion; the composition includes primer F, primers R1-R8, a fusion detection probe and an internal control probe; The nucleotide sequence of primer F is shown in SEQ ID No:1; The nucleotide sequences of primers R1-R8 are shown in SEQ ID No:2-SEQ ID No:9, respectively; The nucleotide sequence of the fusion detection probe is shown in SEQ ID No:10; The nucleotide sequence of the internal standard probe is shown in SEQ ID No:13.

[0015] Furthermore, the 5' ends of the fusion detection probe and the internal standard probe are labeled with fluorescent groups, and the fluorescent groups of the fusion detection probe and the internal standard probe are different, while the 3' ends are labeled with quenching groups.

[0016] Furthermore, the 5' end of the probe is one of the fluorescent groups FAM, HEX, VIC, TET, ROX, TAMRA, JOE, Cy3, Cy5, and Cyc5.5; the 3' end of the probe is labeled with BHQ1 or BHQ3.

[0017] In one specific embodiment, the 5' end of the FGFR2 probe is labeled FAM, and the 3' end is labeled BHQ1. The 5' end of the internal control probe is labeled Cy5, and the 3' end is labeled BHQ3.

[0018] In this article, the kit also includes droplet generating oil, reverse transcriptase, dNTPs, MgCl2, DNA polymerase, and PCR buffer.

[0019] This invention also claims protection for the compositions in the kit described above.

[0020] The application of the kits or compositions described above in the preparation of products for the detection or auxiliary detection of FGFR2 gene fusion is also within the scope of protection of this invention.

[0021] This invention also provides a method for detecting the presence of FGFR2 gene fusion in a sample to be tested, comprising the following steps: 1) Extract RNA from the sample to be tested; 2) Perform digital PCR detection on the RNA obtained in step 1) using the composition described above, and determine whether the sample to be tested contains FGFR2 gene fusion based on the fluorescence signal of the amplification product; if the amplification product detects a fluorescence signal, the sample to be tested contains FGFR2 gene fusion; if the amplification product does not detect a fluorescence signal, the sample to be tested does not contain FGFR2 gene fusion.

[0022] In a specific embodiment, a mixture was set as a positive control and water as a negative control; If the sample to be tested is positive, the positive control is positive, and the blank control is - or ND, then the sample to be tested is of the FGFR2 fusion gene type. If the test sample is negative, the positive control is positive and the blank control is "-" or "ND", or the test sample is "ND", the positive control is positive and the blank control is "ND", then the test sample is not of the FGFR2 fusion gene type. If the sample to be tested shows "ND" (not detected), the positive control is positive, and the blank control is "-", then the sample needs to be retested. During retesting, the sample volume can be increased for repeated testing. If the results are consistent, the mutation at that site was detected in the sample, indicating a weak positive mutation; otherwise, the mutation was not detected, or the result was below the detection limit.

[0023] This invention also provides a quantitative detection method for evaluating FGFR2 gene fusions in a test sample, comprising the following steps: 1) Extract RNA from the sample to be tested; 2) Perform digital PCR detection on the RNA obtained in step 1) using the kit described above. Quantify the FGFR2 gene fusion in the sample based on the fluorescence signal of the amplification product. The quantification formula is: Concentration of target nucleic acid in the sample (copy number / μL) = (1 / ) V (microdroplet) × ln{1 ( N Positive droplets / N Total droplets); in V Microdroplet: The volume (uL) of a single microdroplet; N Positive droplets: The number of droplets with amplified fluorescent signals; N Total droplets: The total number of droplets generated in the reaction; ln: Natural logarithm, used for concentration conversion based on Poisson distribution.

[0024] If a fluorescence signal is detected in the amplification product, it is determined that the sample to be tested contains FGFR2 gene fusion. The content of FGFR2 gene fusion in the sample to be tested can be obtained by directly counting the fluorescence signal of the amplification product or by calculating it using the Poisson distribution principle.

[0025] In one specific embodiment, depending on the type of sample to be tested, the PCR reaction system and reaction procedure may be as follows: 1) If the sample to be tested is an RNA sample, the reaction system is as follows: RT-dPCR MIX (probe)*3.5μL, FGFR2Fusion Assay (10×)0.7μL, RNA sample2.8μL, total volume7μL; the PCR reaction program can be: reverse transcription 50℃ for 15 minutes, pre-denaturation 95℃ for 5 minutes, (denaturation 95℃ for 15 seconds, annealing / extension 58℃ for 30 seconds) 45 cycles.

[0026] 2) If the sample to be tested is a plasmid, the reaction system is as follows: RT-dPCR MIX (probe)* 3.5 μL, FGFR2Fusion Assay (10×) 0.7 μL, restriction endonuclease (Alul) 0.1 μL, plasmid 2.7 μL, total volume 7 μL. The PCR reaction program can be as follows: plasmid: digestion at 37℃ for 5 minutes, pre-denaturation at 95℃ for 5 minutes, (denaturation at 95℃ for 15 seconds, annealing / extension at 58℃ for 30 seconds) for 45 cycles.

[0027] The FGFR2 Fusion Assay (10×) configuration includes: 5µM of each of the sequences shown in SEQ ID No:2-SEQ ID No:10; 10µM of the sequence shown in SEQ ID No:1; 0.5µM of each of the sequences shown in SEQ ID No:11 and SEQ ID No:12; and 0.25µM of the sequence shown in SEQ ID No:13.

[0028] Based on the fully automated microdroplet chip digital PCR system, the microfluidic principle is used to randomly distribute fluorescently labeled PCR reaction reagents into the chip in a large number of uniform and independent "water-in-oil" microdroplet arrays. After automated PCR amplification, the chip image is acquired to obtain the number of positive microdroplets, and the absolute copy number concentration of the target gene is obtained by the "Poisson distribution" formula.

[0029] This invention also provides the application of the method described above in the detection or auxiliary detection of FGFR2 gene fusions.

[0030] This invention provides a technique for detecting FGFR2 fusion genes based on a fully automated microdroplet chip digital PCR system, used for the detection and analysis of RNA in patient FFPE samples and tissue samples. By adding specific primer compositions and probes designed for flanking the fusion site to a single PCR amplification system, eight FGFR2 fusion genes can be detected simultaneously.

[0031] In this invention, the gene fused with the FGFR2 gene can be any one or a combination of PPHLN1, AHCYL1, BICC1, TACC3, MGEA5, KIAA1598, CCDC6, and CASP7.

[0032] In this invention, the sample to be tested may be: FFPE sample from a bile duct cancer patient, tissue sample, recombinant vector containing FGFR2 gene fusion, etc.

[0033] In this study, RNA samples extracted from tissues and FFPE were used for detection, and the limit of detection (LOD) was determined using constructed plasmids.

[0034] The digital PCR method for FGFR2 gene fusion detection established in this invention requires only one manual operation and one chip consumable. The PCR system is dispersed using a monolayer microdroplet array, and the target DNA sequence is independently and stably amplified under the same temperature cycling conditions in 25,000-30,000 uniform microdroplets. This simplifies the workflow to the greatest extent, improves the repeatability and reproducibility of results, and thus significantly increases work efficiency. It can detect dozens of samples, offering flexible throughput. Furthermore, by optimizing primer and probe design, the specificity and sensitivity of the detection are improved, and a simple, rapid, and accurate kit has been developed, which will provide strong support for precision medicine in oncology. Attached Figure Description

[0035] Figure 1 The digital PCR method for detecting FGFR2 gene fusion in Example 1 was validated. Samples 1 and 2 were negative controls, 3 and 4 were positive controls, and 5, 6, and 7 were samples without fusion. The blue channel represents the FGFR2 fusion gene channel, and the red channel represents the internal control channel. The horizontal axis represents the sample number, the vertical axis represents the fluorescence intensity, and the horizontal line in the middle represents the threshold line. Above the threshold line represents positive droplet distribution, and below the threshold line represents negative droplet distribution. Figure 2Example 2: FGFR2 fusion assay in clinical samples. 1 is the negative control, 2 is the positive control, and 3-16 are the samples to be tested. The blue channel represents the FGFR2 fusion gene channel, and the red channel represents the internal control channel. The horizontal axis represents the sample number, and the vertical axis represents the fluorescence signal value. The horizontal line in the middle is the threshold line; above the threshold line represents positive droplet distribution, and below the threshold line represents negative droplet distribution.

[0036] Figure 3 This is for individual validation of the primer and probe gene sites in Example 2. Sequences 1-8 are individual FGFR2 fusion assay sequences, SEQ ID Nos: 2, 3, 14, 5, 15, 7, 16, and 9, respectively. The blue channel represents the FGFR2 fusion gene channel, and the red channel represents the internal control channel. The horizontal axis represents the sample number, and the vertical axis represents the fluorescence intensity. The horizontal line in the middle represents the threshold line; above the threshold line indicates positive droplet distribution, and below the threshold line indicates negative droplet distribution. Positive droplet distribution in the red channels (1-8) indicates that all RNA samples participated in the reaction; positive droplet distribution is present in the blue channels (3, 5, and 7); and negative droplet distribution is present in the blue channels (1, 2, 4, 6, and 8).

[0037] Figure 4 This is the screening of universal forward primers SEQ ID No:17-19 in Example 2. Primers 1-3 are universal forward primers 1, 2, and 3, respectively. The blue channel represents the FGFR2 fusion gene channel, and the red channel represents the internal control channel. The horizontal axis represents the sample number, the vertical axis represents the fluorescence intensity, and the horizontal line in the middle represents the threshold line. Above the threshold line is the distribution of positive droplets, and below the threshold line is the distribution of negative droplets.

[0038] Figure 5 The sequences BCC1, MGEA5, CCDC6, and SEQ ID Nos. 4, 6, and 8 in Example 2 were identified. Where 1 represents BCC1; 2 represents MGEA5; and 3 represents CCDC6. The blue channel represents the FGFR2 fusion gene channel, and the red channel represents the internal control channel. The horizontal axis represents the sample number, the vertical axis represents the fluorescence intensity, and the middle horizontal line represents the threshold line. Above the threshold line is the distribution of positive droplets, and below the threshold line is the distribution of negative droplets.

[0039] Figure 6This section describes the screening of probes in Example 2. In the diagram, 1 and 2 represent the positive and negative RNA detection results for FGFR2 fusion probe 1, respectively; 3 and 4 represent the positive and negative RNA detection results for FGFR2 fusion probe 2, respectively; 5 and 6 represent the positive and negative RNA detection results for FGFR2 fusion probe 3, respectively; and 7 and 8 represent the positive and negative RNA detection results for FGFR2 fusion probe 4, respectively. The blue channel represents the FGFR2 fusion gene channel, and the red channel represents the internal control channel. The horizontal axis represents the sample number, the vertical axis represents the fluorescence intensity, and the middle horizontal line represents the threshold line. Above the threshold line indicates positive droplet distribution, and below the threshold line indicates negative droplet distribution.

[0040] Figure 7 This is for plasmid construction verification in Example 3. 1-8 are eight positive plasmids with sequences SEQ ID No: 23-30. The blue channel represents the FGFR2 fusion gene channel, and the red channel represents the internal control channel. The horizontal axis represents the sample number, the vertical axis represents the fluorescence intensity, and the middle horizontal line represents the threshold line. Above the threshold line is the distribution of positive droplets, and below the threshold line is the distribution of negative droplets.

[0041] Figure 8 This example demonstrates the application of the method in Example 5 in detecting FGFR2 gene fusions in clinical samples. 1 represents the negative control; 2 represents the positive control; and 3-8 represent samples RNA1, RNA2, WLXI-5, WBZ1-10, 10, and 11, respectively. The blue channel represents the FGFR2 fusion gene channel, and the red channel represents the internal control channel. The horizontal axis represents the sample number, the vertical axis represents the fluorescence intensity, and the horizontal line in the middle represents the threshold line. Above the threshold line represents positive droplet distribution, and below the threshold line represents negative droplet distribution. Detailed Implementation

[0042] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0044] This invention designs specific primers and fluorescent probe sets on both sides of the fusion site, and selects the optimal sequences to improve the sensitivity and specificity of detection. The 5' end gene is the FGFR2 gene (nucleotide sequence is Genbank number: NM_000141.5, update date: 21-NOV-2025), and the specific information of each fusion gene at the 3' end is shown in Table 2.

[0045] Table 2. Detection targets and primer / probe sequences for the FGFR2 fusion gene

[0046] The information on the internal control primers and probes of this invention is as follows: Internal control upstream primer: 5'-AGATTTGGACCTGCGAGCG-3' (SEQ ID No: 11); Internal control downstream primer: 5'-GAGCGGCTGTCTCCACAAGT-3' (SEQ ID No: 12); Internal control probe: 5'-TTCTGACCTGAAGGCTCTGCGCG-3' (SEQ ID No:13).

[0047] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0048] The samples used in the following examples were all surgical samples from Tsinghua Chang Gung Hospital, and some samples were not replicated after use. Paraffin-embedded samples or frozen samples from patients with cholangiocarcinoma were selected. Paraffin-embedded samples were prepared using the Qiagen RNeasyDPS FFPE Kit-73604 according to the instructions, and tissue samples were prepared using the Qiagen AllPrep DNA / RNA Mini Kit-80204 according to the instructions. This biological material is available to the public from the applicant and is intended solely for replicating the experiments of this invention; it may not be used for any other purpose.

[0049] Ethical Statement Each participant signed an informed consent form. This study was approved by the Beijing Tsinghua Chang Gung Medical Ethics Committee (Ethics Review Approval Document: Review No. 24213-0-01).

[0050] Research subjects All study subjects were Chinese adults.

[0051] Sample inclusion criteria FFPE slides and frozen samples of surgically removed tumor tissue from newly diagnosed patients with intrahepatic cholangiocarcinoma or hilar cholangiocarcinoma.

[0052] The inclusion criteria for patients with intrahepatic cholangiocarcinoma (hereinafter referred to as "cases") are as follows: The specific diagnostic criteria are as follows: Intrahepatic cholangiocarcinoma: The surgical resection specimen was found to be a lesion within the liver parenchyma, not in the hilum. Pathological diagnosis: Intrahepatic cholangiocarcinoma. Histological types: Adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, sarcomatoid carcinoma. Histological classification: G1, G2, G3, G4. Immunohistochemical results: CK7(+), CK19(+), HepPar-1(-), CK20(-), CDX-2(-), Ki-67 (10-60%) fusion and non-fusion. Hilar cholangiocarcinoma: The surgical resection specimen was found to be from the extrahepatic bile duct. Pathological diagnosis: Hilar cholangiocarcinoma. Histological type: Predominantly adenocarcinoma. Histological classification: G1, G2, G3, G4. Immunohistochemical results: Typical results show characteristics of bile duct epithelial differentiation. CK7(+), CK19(+), MUC1(+), CA19-9(+), CEA(+), CDX2 (-). The patient has not received radiotherapy or chemotherapy, and this has been confirmed by pathology.

[0053] Example 1: Establishment of a digital PCR method for detecting FGFR2 gene fusions (all sequences are final sequences after screening). This invention uses RNA from FFPE and tissue samples of patients with cholangiocarcinoma as the detection target, and utilizes a fully automated microdroplet chip digital PCR system to detect the FGFR2 fusion gene. The specific steps are as follows: 1. Design of specific primers and fluorescent probes Information on the FGFR2 gene fusion site is shown in Table 2. First, specific primers and fluorescent probes were designed on both sides of the fusion site (sequence information is detailed in Table 2, 5'FAM-CAGTTGGTAGAAGACTTGGATCG-3'BHQ1). The 5' end of the probe in Table 2 is attached with a FAM fluorescent label, the fluorescent channel of which is the Blue channel. The 3' end is fused with BHQ1. The fusion gene in the sample RNA was detected by one-step RT-PCR technology.

[0054] The kit includes primers and fluorescent probes containing conserved regions of human endogenous genes (sequence information is detailed in Table 3). The internal control probe (5'Cy5-TTCTGACCTGAAGGCTCTGCGCG-3' BHQ3) has a Cy5 fluorescent label at its 5' end (Red channel) and is fused to its 3' end with BHQ3, serving as an internal control to monitor the effectiveness of the sample and the detection system. Additionally, the kit includes positive and negative controls (purified water) to ensure quality control during the detection process and prevent false positives and false negatives.

[0055] The positive control consisted of 9 sequences: 8 plasmids and the FGFR2 positive plasmid internal control, mixed 1:1.

[0056] To verify the effectiveness of the primers and probes, plasmids containing fusion gene sites of PPHLN1, AHCYL1, BICC1, TACC3, MGEA5, KIAA1598, CCDC6, and CASP7 were constructed. Information for eight of these plasmids is as follows: The structures of the recombinant vectors PPHLN1, AHCYL1, BICC1, TACC3, MGEA5, KIAA1598, CCDC6, and CASP7 are described below: These recombinant vectors were obtained by inserting any of the DNA fragments described in SEQ ID No:23-SEQ ID No:30 between the BamHI and BamHI restriction sites of the starting vector pUC57-KANA (Shanghai Newp Biotechnology Co., Ltd., V012365), while keeping other sequences of the vector pUC57-KANA unchanged.

[0057] The structure of the FGFR2 positive plasmid is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence SEQ ID No:31 between the two restriction sites of BamHI and BamHI in the starting vector pUC57-KANA (Shanghai Newp Biotechnology Co., Ltd., V012365), while keeping the other sequences of the vector pUC57-KANA unchanged.

[0058] The amplification efficiency and specificity of the primers and probes were initially verified using plasmid DNA carrying eight fusion sites (containing specific gene sequences SEQ ID No:23-SEQ ID No:30).

[0059] 2. Components of the test kit The kit for detecting the FGFR2 fusion gene contains all the reagents listed in Table 3. Additional consumables and reagents to be prepared by the customer include: rubychip (Beijing DeepBlue Cloud Technology Co., Ltd., catalog number: C16011), 1.5 mL RNase-Free centrifuge tubes, PCR reaction tubes, and nucleic acid extraction reagents (Qiagen, catalog numbers: 73604, 80204).

[0060] In addition, the single plasmid sample also has an enzyme digestion reagent: NEB: AlulR0137S, RT-dPCR MIX (probe) (Beijing Shenlanyun Technology Co., Ltd., catalog number 15000701k).

[0061] Table 3. Components of the kit for FGFR2 fusion gene detection

[0062] Note: Components from different batches of the kit should not be used interchangeably.

[0063] The preparation method for the FGFR2 Fusion Assay (10×) is shown in Table 4: 5 µM of each of the sequences shown in SEQ ID No:2-SEQ ID No:10, 10 µM of the sequence shown in SEQ ID No:1, 0.5 µM of each of the sequences shown in SEQ ID No:11 and 12, and 0.25 µM of the sequence shown in SEQ ID No:13. The template for the positive control, namely the 8 plasmids from step 1, and the FGFR2 positive plasmid internal control, totaling 9 plasmid DNAs, are mixed 1:1.

[0064] Table 4. Preparation method of FGFR2 Fusion Assay (10×)

[0065] 3. Testing Applicable Models Apexbio Biotechnology (Suzhou) Co., Ltd. manufactures the Naica EL digital PCR analysis system; Naica CNG PCR amplification instrument and Apexbio Naica CN10 biochip analysis instrument; and Stilla Technologies' naica® fully automated microdroplet chip digital PCR system, naica® six-channel microdroplet chip digital PCR system, and Nio+ fully automated microdroplet digital PCR integrated system.

[0066] 4. PCR reaction system and procedure for detecting the FGFR2 fusion gene The PCR reaction system for detecting the FGFR2 fusion gene is as follows (7µL): RT-dPCR MIX (probe)* 3.5µL, FGFR2 Fusion Assay (10×) 0.7µL, and RNA sample to be tested 2.8µL.

[0067] The reaction procedure was as follows: first, droplets were generated; reverse transcription was performed at 50°C for 15 minutes; pre-denaturation was performed at 95°C for 5 minutes; denaturation was performed at 95°C for 15 seconds, followed by annealing / extension at 58°C for 30 seconds, for 45 cycles; and then decompression was performed.

[0068] After the PCR reaction is complete, input the sample information and exposure time, acquire the signal, and save the data. The exposure time for the Blue channel (FAM) is 80 milliseconds, and the exposure time for the Red channel (CY5) is 50 milliseconds. Set the threshold and export the analysis results.

[0069] The results are as follows: (1) Validity determination a. If the total number of droplets in the reaction wells is ≥10000, the test results are valid and further analysis can be performed.

[0070] b. If the total number of droplets in the reaction well is <10000, and the result can be interpreted after further analysis (Table 5), the result of that reaction well is still valid; otherwise, please retest. The positive and negative controls of the kit must meet the following requirements; otherwise, the experiment is considered invalid.

[0071] Table 5. Criteria for determining the validity of an experiment

[0072] Note: "+" indicates a positive droplet count ≥ 2, "-" indicates a positive droplet count of 0, and "ND" indicates a positive droplet count of 1.

[0073] a. Retest. There could be several reasons why a positive control might not be detected, such as: incorrect pipetting, use of expired reagents, or adding the wrong reagent; b. If the blank control result is indeterminate (ND), a comprehensive analysis should be conducted in conjunction with the results of the test sample.

[0074] c. Retesting. A positive result in the blank control indicates false-positive contamination with one or more reagents. All possible causes need to be ruled out, such as incorrect placement of PCR tubes or pipetting errors. An assessment should be conducted based on the investigation results, and retesting at the extraction stage may be necessary.

[0075] (2) Judgment of the results of the test sample If the sample to be tested is positive, the positive control is positive and the blank control is "-" or "ND", then the sample to be tested is of the FGFR2 fusion gene type. If the test sample is negative, the positive control is positive and the blank control is "-" or "ND", or the test sample is "ND", the positive control is positive and the blank control is "ND", then the test sample is not of the FGFR2 fusion gene type. If the sample to be tested shows "ND" (not detected), the positive control is positive, and the blank control is "-", then the sample needs to be retested. During retesting, the sample volume can be increased for repeated testing. If the results are consistent, the mutation at that site was detected in the sample, indicating a weak positive mutation; otherwise, the mutation was not detected, or the result was below the detection limit.

[0076] With negative controls, positive controls, and 3 samples of non-fusion to be tested, the results are shown in Table 6 and... Figure 1As shown: 1 and 2 are negative controls, with both blue and red channels showing negative droplet distribution; 3 and 4 are positive controls, both showing positive droplet distribution; 5, 6, and 7 are non-fusion test samples, with blue channels showing negative droplet distribution and red channels showing positive droplet distribution. The results demonstrate that the sequence of this test reagent is accurate and qualified.

[0077] Table 6. Summary of droplets and copy numbers used in digital PCR for FGFR2 gene fusion detection method validation.

[0078] Specificity analysis of primers and probes used in digital PCR for FGFR2 gene fusion detection in Example 2 and Example 1. 1. Primer specificity analysis Refer to the following literature: 1) Daniela S, Bojan L, Agrin M, et al. Massive parallel sequencing uncovers actionable FGFR2-PPHLN1 fusion and ARAF mutations in intrahepatic cholangiocarcinoma. [J]. Nature communications, 2015, 6 (Jan.): 6087. DOI: 10.1038 / ncomms7087. 2) Fangda L, N. MP, J. DD. Functions of FGFR2 corrupted bytranslocations in intrahepatic cholangiocarcinoma[J]. Cytokine and GrowthFactor Reviews, 2019, 52 56-67. DOI: 10.1016 / j.cytogfr.2019.12.005. 3) Yi-Mi W , Fengyun S , Shanker K , et al. Identification of targetable FGFR gene fusions in diverse cancers.[J]. Cancer discovery, 2013, 3 (6): 636-47. DOI: 10.1158 / 2159-8290.CD-13-0050. 4) JMB, DMC, BJE, et al. Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma. [J]. PLoS genetics, 2014, 10 (2): e1004135. DOI: 10.1371 / journal.pgen.1004135. 5) SJR, Kai W, Laurie G, et al. New routes to targeted therapy of intrahepatic cholangiocarcinomas revealed by next-generation sequencing. [J]. The oncologist, 2014, 19 (3): 235-42. DOI: 10.1634 / theoncologist.2013-0352. 6) MIS, Meijuan L, Karthikeyan M, et al. Validation and Characterization of FGFR2 Rearrangements in Cholangiocarcinoma With Comprehensive Genomic Profiling.[J].The Journal of Molecular Diagnostics:JMD,2022,24(4):DOI:10.1016 / J.JMOLDX.2021.12.012. Primers designed in Table 7 were used as a control to compare the specificity of primers designed in Table 2 above. Sample information is shown in Table 8.

[0079] The reaction system, reaction procedure, and result determination of the experiment were carried out in accordance with Example 1.

[0080] Table 7. Primer and probe sequence information for FGFR2 fusion assay in clinical samples.

[0081] Table 8. Test Sample Information

[0082] Table 8 shows the information for the samples to be tested, and the test results are shown in Table 9 below. Figure 2 As shown: 1 is the negative control, with negative droplet distribution in both blue and red channels; 2 is the positive control, with positive droplet distribution in both blue and red channels; 3-16 are the test samples, with positive droplet distribution in both blue and red channels. The results demonstrate that the test reagent is positive in all samples except the negative control, indicating an excessively high FGFR2 positivity rate, which may indicate nonspecific amplification.

[0083] Table 9. Summary of droplets and copy numbers tested in clinical samples using the FGFR2 fusion assay.

[0084] 2. Individual validation of primers, probes, and gene loci. RNA sample No. 7, which was negative for FGFR2 fusion, was used as the test sample. The primers for each FGFR2 fusion site were different. The primer and probe information (Table 7) and the reaction procedure were performed according to Example 1.

[0085] The results are shown in Table 10 and Figure 3 As shown: The red channels (1-8) indicate positive droplet distribution, meaning all RNA samples participated in the reaction. Blue channels (3, 5, and 7) also show positive droplet distribution, while blue channels (1, 2, 4, 6, and 8) show negative droplet distribution. These results demonstrate that the RNA-FGFR2-7 fusion negative sample exhibited non-specific amplification with primers 3, 5, and 7. Specifically, this indicates non-specific amplification in the FGFR2-BICC1, FGFR2-MGEA5, and FGFR2-CCDC6 combinations.

[0086] Table 10. Summary of droplet number and copy number concentration for individual validation of primer and probe gene sites.

[0087] 3. Optimization of three fusion gene sites: FGFR2-BICC1, FGFR2-MGEA5, and FGFR2-CCDC6. Optimization was performed on three fusion gene sites: FGFR2-BICC1, FGFR2-MGEA5, and FGFR2-CCDC6. Primer sequences for FGFR2-PPHLN1, FGFR2-AHCYL1, FGFR2-TACC3, FGFR2-KIAA1598, and FGFR2-CASP7 were confirmed, as shown in Table 11 below.

[0088] Table 11. List of gene sites without nonspecific amplification

[0089] Three primers, FGFR2-BICC1, FGFR2-MGEA5, and FGFR2-CCDC6, were screened. Primer and probe information is shown in Tables 12 and 13 below for two combinations. Two rounds of experiments were conducted to verify the results of RNA-FGFR2-7 fusion negative samples using the primers and probes in Tables 12 and 13. The reaction system, reaction procedure, and result interpretation were the same as in Example 1.

[0090] Table 12. List of information on universal forward primers for non-specific amplification gene sites.

[0091] Table 13. List of gene locus screening information for non-specific amplification gene loci

[0092] The results are shown in Table 14 and Figure 4 As shown: the blue channel represents the FGFR2 fusion gene channel, and the red channel represents the internal control channel. The horizontal axis represents the sample number, and the vertical axis represents the fluorescence intensity. The middle horizontal line is the threshold line; above the threshold line is positive droplet distribution, and below the threshold line is negative droplet distribution. The presence of positive droplet distribution in red channels 1-3 indicates that all RNA samples participated in the reaction, while the presence of positive droplet distribution in blue channels 1-3 demonstrates that non-specific amplification still occurred in the RNA-FGFR2 fusion negative sample (sample number 7) using three universal primers redesigned and synthesized for the FGFR2-BICC1, FGFR2-MGEA5, and FGFR2-CCDC6 fusion genes.

[0093] The experimental results are shown in Table 15 based on Table 13. Figure 5 As shown: the blue channel is the FGFR2 fusion gene channel, the red channel is the internal control channel, the horizontal axis is the sample number, the vertical axis is the fluorescence intensity, the middle horizontal line is the threshold line, above the threshold line is positive droplet distribution, and below the threshold line is negative droplet distribution; the presence of positive droplet distribution in red channels 1-3 indicates that all RNA samples participated in the reaction, while the absence of positive droplet distribution in blue channels 1-3 indicates that the redesigned and synthesized downstream primers for the three fusion genes FGFR2-BICC1, FGFR2-MGEA5, and FGFR2-CCDC6 (Table 13) showed no non-specific amplification in the RNA-FGFR2-7 fusion negative sample. The primer sequences for the three fusion genes FGFR2-BICC1, FGFR2-MGEA5, and FGFR2-CCDC6 are shown in Table 13.

[0094] Table 14. Copy number concentrations for screening using universal forward primers SEQ ID No:17-SEQ ID No:19

[0095] Table 15. Copy number concentration of gene site screening information for non-specific amplification gene sites.

[0096] Downstream primers were redesigned and synthesized for three fusion genes: FGFR2-BICC1, FGFR2-MGEA5, and FGFR2-CCDC6, and digital PCR detection was performed. Compared with combination 1, the primers designed in combination 2 are more specific.

[0097] In summary, the primer sequences for the three fusion genes FGFR2-BICC1, FGFR2-MGEA5, and FGFR2-CCDC6 were confirmed as follows: BICC1 primer 2: 5'-CGATCTTCAGTTTTGATG-3'; MGEA5 primer 2: 5'-GTCTGATAGTATAAACTTTGG-3'; and CCDC6 primer 2: 5'-AGGGTTTCTTTCTCCTTC-3'.

[0098] 3. Probe screening The test probes 1-4 in Table 16 below were designed for probe screening. The reaction system, reaction procedure, and result interpretation were performed according to Example 1.

[0099] Table 16. Probe Screening Sequence List

[0100] The experiment was conducted according to Table 16. The RNA-FGFR2-7 fusion negative sample and the positive control were selected as the test specimens. The results are shown in Table 17 and... Figure 6 As shown: the blue channel represents the FGFR2 fusion gene channel, and the red channel represents the internal control channel. The horizontal axis represents the sample number, and the vertical axis represents the fluorescence intensity. The horizontal line in the middle is the threshold line; above the threshold line is positive droplet distribution, and below the threshold line is negative droplet distribution. The presence of positive droplet distribution in all red channels 1-8 indicates that all samples participated in the reaction. Channels 1-8 show positive droplet distribution in all samples except sample 2. The results demonstrate that for negative RNA samples, only probe 1 failed to detect the FGFR2 fusion signal, while probes 2-4 showed varying degrees of amplification. Therefore, probe 1 was chosen as the probe for the FGFR2 fusion gene.

[0101] Table 17. Copy number concentration of probe screening

[0102] Example 3: Application of digital PCR for FGFR2 gene fusion detection The detection method established in Example 1 was used to verify the human FGFR2 gene fusion site of the positive plasmid DNA.

[0103] First, the amplification efficiency and specificity of the primers and probes were initially verified using DNA from plasmids (PPHLN1, AHCYL1, BICC1, TACC3, MGEA5, KIAA1598, CCDC6, and CASP7) constructed in Example 1, which carried eight fusion sites.

[0104] 2. Absolute Quantitative Analysis of a Fully Automated Microdroplet Chip Digital PCR System The synthesized plasmid was linearized using restriction endonuclease (Alul), and absolute quantification was performed using a fully automated microdroplet chip digital PCR system. The reaction system, reaction procedure, and result interpretation were performed according to the plasmid sample described in the invention.

[0105] The results are shown in Table 18 and Figure 7 As shown: the blue channel is the FGFR2 fusion gene channel, the red channel is the internal control channel, the horizontal axis is the sample number, the vertical axis is the fluorescence intensity, the middle horizontal line is the threshold line, the above the threshold line is the positive droplet distribution, and the below the threshold line is the negative droplet distribution; no positive droplet distribution is found in the red channels 1-8, and positive droplet distribution is found in all plasmids 1-8.

[0106] Quantitative results show (Table 18 and Figure 7 The reagent was used to detect FGFR2 fusion positive in 8 positive plasmid DNAs. The 8 plasmids showed signals only in the FAM channel and the quantitative concentrations were as expected, which proves the effectiveness of the primers and probes and the successful construction of the plasmid templates, which can be used for subsequent sensitivity detection.

[0107] Table 18. Copy number concentration for plasmid construction validation

[0108] Example 4: Sensitivity validation of digital PCR method for detecting FGFR2 gene fusions Based on the Clinical and Laboratory Standards Institute (CLSI) EP17-A2 standard, "Guideline for Approval of Limit of Detection and Limit of Quantitation Determination Protocols," sensitivity testing was performed using the kit from Example 1, the plasmid from Example 3, and negative samples.

[0109] Sample preparation is as follows: LOB: ≥30 negative control replicate samples to achieve a 95% confidence level. LOD for 8 plasmid points: five independently prepared low-level samples (L1, L2, L3, L4 and L5, namely 1×LOB, 2×LOB, 3×LOB, 4×LOB, 5×LOB) are tested by Crystal Digital PCR™, with at least 6 replicates for each sample.

[0110] The plasmid reaction system, reaction procedure, and result interpretation are all in accordance with Example 3; the reaction system, reaction procedure, and result interpretation of RNA samples are all in accordance with Example 1.

[0111] For FGFR2 fusion gene, the following LOB and LOD results are obtained. Distribution assumption for statistical analysis: the low concentration region approximately follows normal distribution; core methods: quantile estimation, pooled variance; correction mechanism: Cp coefficient compensates for small sample deviation. Positive detection rate: 95% at LoD, ≤5% when lower than LoB, and uncertain when between the two. Interpretation of target X concentration: when C[X] ≤ LoB, the target is not detected; when LoB<C[X]<LoD, the target is detected but cannot be quantified; when C[X] ≥ LoD, the target is detected and can be quantified. The results are shown in Tables 19 to 21 below.

[0112] Table 19, Copy number concentration detected in LOB test

[0113] Table 20, Summary table of LOD detection at each fusion site (unit: copies / µL)

[0114] Table 21, Summary of LOB and LOD

[0115] The results are shown in Table 21: when C[X] ≤ 0.29, the target is not detected; when 0.29<C[X]<1.22, the target is detected but cannot be quantified; when C[X] ≥ 1.22, the target is detected and can be quantified.

[0116] Example 5, Application of the method established by the present invention in detecting FGFR2 gene fusion in clinical samples The samples to be tested and clinical diagnosis information are shown in Table 22 below, and the clinical diagnosis results (Table 22) are obtained by next-generation sequencing detection method.

[0117] The reaction system, reaction procedure, and result interpretation are all in accordance with Example 1, and the detection results are shown in Table 23 and Figure 8The reagents were used to test 6 FFPE RNA samples: the clinical fusion gene WLXI1-5 in sample WLXI1-5 was positive and the result was also positive, while the rest were negative, which was consistent with the clinical judgment.

[0118] Table 22, Sample Information Table

[0119] Table 23. Application of the method in detecting FGFR2 gene fusion in clinical samples

[0120] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A kit for detecting FGFR2 gene fusions, characterized in that, The kit includes a composition for detecting FGFR2 gene fusion; the composition includes primer F, primers R1-R8, a fusion detection probe, and an internal control probe; The nucleotide sequence of primer F is shown in SEQ ID No:1; The nucleotide sequences of primers R1-R8 are shown in SEQ ID No:2-SEQ ID No:9, respectively; The nucleotide sequence of the fusion detection probe is shown in SEQ ID No:10; The nucleotide sequence of the internal standard probe is shown in SEQ ID No:

13.

2. The reagent kit according to claim 1, characterized in that, The 5' ends of the fusion detection probe and the internal standard probe are labeled with fluorescent groups, and the fluorescent groups of the fusion detection probe and the internal standard probe are different, while the 3' ends are labeled with quenching groups.

3. The reagent kit according to claim 2, characterized in that, The 5' end of the probe is one of the fluorescent groups FAM, HEX, VIC, TET, ROX, TAMRA, JOE, Cy3, Cy5, and Cyc5.5; the 3' end of the probe is labeled with BHQ1 or BHQ3.

4. The kit according to any one of claims 1-3, characterized in that, The kit also includes droplet-generating oil, reverse transcriptase, dNTPs, MgCl2, DNA polymerase, and PCR buffer.

5. The composition according to any one of claims 1-4.

6. The use of the kit according to any one of claims 1-4 or the composition according to claim 5 in the preparation of products for the detection or auxiliary detection of FGFR2 gene fusion.

7. A method for detecting the presence of FGFR2 gene fusion in a sample to be tested, characterized in that, Includes the following steps: 1) Extract RNA from the sample to be tested; 2) The RNA obtained in step 1) is subjected to digital PCR detection using the kit described in claim 4. The presence of FGFR2 gene fusion in the sample is determined based on the fluorescence signal of the amplification product. If the amplification product detects a fluorescence signal, the sample contains FGFR2 gene fusion. If the amplification product does not detect a fluorescence signal, the sample does not contain FGFR2 gene fusion.

8. A quantitative detection method for evaluating FGFR2 gene fusions in a test sample, characterized in that, Includes the following steps: 1) Extract RNA from the sample to be tested; 2) The RNA obtained in step 1) is subjected to digital PCR detection using the kit described in claim 4, and the fusion of the FGFR2 gene in the sample to be tested is quantified based on the fluorescence signal of the amplification product. If a fluorescence signal is detected in the amplification product, it is determined that the sample to be tested contains FGFR2 gene fusion. The content of FGFR2 gene fusion in the sample to be tested can be obtained by directly counting the fluorescence signal of the amplification product or by calculating it using the Poisson distribution principle.

9. The application of the method of claim 7 or 8 in the detection or auxiliary detection of FGFR2 gene fusion.

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