Primer probe combination and kit

Abstract

The invention provides a primer probe combination and a kit, and relates to but is not limited to the field of biotechnology, the primer probe combination comprises at least two seed primer probe combinations: a first sub primer probe combination for detecting TERT promoter genes, a second sub primer probe combination for detecting FGFR3 genes, a third sub primer probe combination for detecting TERT promoter genes, a fourth sub primer probe combination for detecting TERT promoter genes, and a fifth sub primer probe combination for detecting TERT promoter genes. And the third sub primer probe combination is used for detecting the PIK3CA gene. Based on the scheme, a multi-target bladder cancer detection technology can be realized, so that the bladder cancer detection accuracy and sensitivity are improved.

Description

Technical Field

[0001] This disclosure relates to, but is not limited to, the field of biotechnology, and particularly to a primer-probe combination and kit. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system, posing a significant threat to human health. Currently, the standard for bladder cancer diagnosis is cystoscopy and pathological examination, but this has some drawbacks, such as causing urinary tract infections, low diagnostic rates, and urethral pain during cystoscopy. However, with the development of liquid detection technology, cell-free deoxyribonucleic acid (cfDNA) has been applied in tumors, and detection methods based on liquid biomarkers have been proposed.

[0003] Current detection methods primarily target the C228T mutation in the telomerase reverse transcriptase (TERT) gene. However, the accuracy and sensitivity of this gene test for bladder cancer diagnosis are relatively low. Summary of the Invention

[0004] This application provides a primer-probe combination and kit that can be used to implement a multi-target detection technique for bladder cancer, thereby improving the accuracy and sensitivity of bladder cancer detection.

[0005] Firstly, a primer-probe combination is provided, comprising at least two primer-probe combinations as follows:

[0006] The first primer-probe combination is used to detect the TERT promoter gene; the second primer-probe combination is used to detect the fibroblast growth factor receptor 3 (FGFR3) gene; and the third primer-probe combination is used to detect the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene.

[0007] In conjunction with the first aspect, in some implementations of the first aspect, the mutation site detected by the first sub-primer-probe combination includes at least one of C228T, C250T, and C228A; the mutation site detected by the second sub-primer-probe combination includes at least one of S248C, S249C, and Y353C; and the mutation site detected by the third sub-primer-probe combination includes at least one of H1047R, E542K, and E545K.

[0008] In conjunction with the first aspect, in certain implementations of the first aspect, the primer-probe combination described above satisfies any one or more of the following conditions: when the mutation site detected by the first sub-primer-probe combination includes C228T, the first sub-primer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of sequences derived from any one or more of SEQ ID NO: 1 to 4; when the mutation site detected by the first sub-primer-probe combination includes C250T, the first sub-primer-probe combination comprises sequences derived from SEQ ID NO: 1 to 4. At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of any one or more sequences from SEQ ID NO: 5 to 8; when the mutation site detected by the first sub-primer probe combination includes C228A, the first sub-primer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of sequences derived from any one or more sequences from SEQ ID NO: 9 to 12; when the mutation site detected by the second sub-primer probe combination includes S248C, the second sub-primer probe combination includes sequences derived from SEQ ID NO: 5 to 8. At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of any one or more sequences from SEQ ID NO: 13 to 16; when the mutation site detected by the second sub-primer probe combination includes S249C, the second sub-primer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of any one or more sequences from SEQ ID NO: 17 to 20; when the mutation site detected by the second sub-primer probe combination includes Y353C, the second sub-primer probe combination includes sequences derived from SEQ ID NO: 17 to 20. At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of any one or more sequences from SEQ ID NO: 21 to 24; when the mutation site to be detected by the third subprimer probe combination includes H1047R, the third subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of any one or more sequences from SEQ ID NO: 25 to 28;When the mutation site detected by the third sub-primer probe combination includes E542K, the third sub-primer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from any one or more of SEQ ID NO: 29 to 32; when the mutation site detected by the third sub-primer probe combination includes E545K, the third sub-primer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from any one or more of SEQ ID NO: 33 to 36.

[0009] In conjunction with the first aspect, in certain implementations of the first aspect, the primer-probe combination described above satisfies any one or more of the following conditions: when the mutation site detected by the first sub-primer-probe combination includes C228T, the first sub-primer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO:1 to 4; when the mutation site detected by the first sub-primer-probe combination includes C250T, the first sub-primer-probe combination comprises sequences derived from SEQ ID NO:1 to 4. At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences in SEQ ID NO: 5 to 8; when the mutation site detected by the first sub-primer probe combination includes C228A, the first sub-primer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO: 9 to 12; when the mutation site detected by the second sub-primer probe combination includes S248C, the second sub-primer probe combination includes sequences derived from SEQ ID NO: 9 to 12. At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences in SEQ ID NO: 13 to 16; when the mutation site detected by the second sub-primer probe combination includes S249C, the second sub-primer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences in SEQ ID NO: 17 to 20; when the mutation site detected by the second sub-primer probe combination includes Y353C, the second sub-primer probe combination includes sequences derived from SEQ ID NO: 17 to 20. At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of several sequences in SEQ ID NO:21 to 24; when the mutation site to be detected by the third subprimer probe combination includes H1047R, the third subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of several sequences derived from SEQ ID NO:25 to 28;When the mutation site detected by the third sub-primer-probe combination includes E542K, the third sub-primer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO:29 to 32; when the mutation site detected by the third sub-primer-probe combination includes E545K, the third sub-primer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO:33 to 36.

[0010] In conjunction with the first aspect, in certain implementations of the first aspect, the primer-probe combination described above satisfies any one or more of the following conditions: when the mutation site detected by the first sub-primer-probe combination includes C228T, the first sub-primer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO: 1 to 4; when the mutation site detected by the first sub-primer-probe combination includes C250T, the first sub-primer-probe combination comprises sequences derived from SEQ ID NO: 1 to 4. At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO:5 to 8; when the mutation site detected by the first sub-primer probe combination includes C228A, the first sub-primer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences derived from SEQ ID NO:9 to 12; when the mutation site detected by the second sub-primer probe combination includes S248C, the second sub-primer probe combination includes sequences derived from SEQ ID NO:9 to 12. At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO: 13 to 16; when the mutation site detected by the second sub-primer probe combination includes S249C, the second sub-primer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO: 17 to 20; when the mutation site detected by the second sub-primer probe combination includes Y353C, the second sub-primer probe combination includes sequences derived from SEQ ID NO: 17 to 20. At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO:21 to 24; when the mutation site to be detected by the third subprimer probe combination includes H1047R, the third subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO:25 to 28;When the mutation site detected by the third sub-primer-probe combination includes E542K, the third sub-primer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from all sequences in SEQ ID NO: 29 to 32; when the mutation site detected by the third sub-primer-probe combination includes E545K, the third sub-primer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from all sequences in SEQ ID NO: 33 to 36.

[0011] In conjunction with the first aspect, in certain implementations of the first aspect, the primer-probe combination described above satisfies any one or more of the following conditions: when the mutation site detected by the first sub-primer-probe combination includes C228T, the first sub-primer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO:1 to 4; when the mutation site detected by the first sub-primer-probe combination includes C250T, the first sub-primer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO:1 to 4. At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO:5 to 8; when the mutation site detected by the first subprimer probe combination includes C228A, the first subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO:9 to 12; when the mutation site detected by the second subprimer probe combination includes S248C, the second subprimer probe combination includes sequences selected from SEQ ID NO:9 to 12. At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO:13 to 16; when the mutation site detected by the second sub-primer probe combination includes S249C, the second sub-primer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO:17 to 20; when the mutation site detected by the second sub-primer probe combination includes Y353C, the second sub-primer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO:17 to 20. At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO:21 to 24; when the mutation site to be detected by the third subprimer probe combination includes H1047R, the third subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO:25 to 28;When the mutation site detected by the third sub-primer probe combination includes E542K, the third sub-primer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences selected from all sequences in SEQ ID NO: 29 to 32; when the mutation site detected by the third sub-primer probe combination includes E545K, the third sub-primer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences selected from all sequences in SEQ ID NO: 33 to 36.

[0012] In conjunction with the first aspect, in certain implementations of the first aspect, in the first, second, or third subprimer-probe combination, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group. The fluorescent reporter group is selected from at least one of FAM, HEX, TEXAS, CY3, CY5, TAMRA, TET, JOE, VIC, NED, and ROX, and the fluorescent quencher group is selected from at least one of TAMRA, BHQ1, and DABCY.

[0013] In conjunction with the first aspect, in certain implementations of the first aspect, in the first sub-probe combination, the second sub-primer-probe combination, or the third sub-primer-probe combination, the fluorescent reporter group labeled at the 5' end of the upstream probe is different from the fluorescent reporter group labeled at the 5' end of the downstream probe, while the fluorescent quencher groups labeled at the 3' ends of the upstream probe and the downstream probe are the same.

[0014] Secondly, a kit is proposed that includes primer-probe combinations in any possible implementation of the primer-probe combination design as described in the first aspect above.

[0015] In conjunction with the second aspect, in some implementations of the second aspect, the above-mentioned kit is used for bladder cancer detection, the test sample of the above-mentioned kit is urine, and the bladder cancer detection process is performed by a computer. Attached Figure Description

[0016] Figure 1 This is an amplification detection result curve of a specificity verification experiment proposed in the embodiments of this application;

[0017] Figure 2 This is another amplification detection result curve of the specificity verification experiment proposed in the embodiments of this application. Detailed Implementation

[0018] The technical solutions in this application will now be described with reference to the accompanying drawings.

[0019] This application will present various aspects, embodiments, or features relating to a system comprising multiple devices, components, modules, etc. It should be understood and appreciated that individual systems may include additional devices, components, modules, etc., and / or may not include all the devices, components, modules, etc. discussed in conjunction with the accompanying drawings. Furthermore, combinations of these approaches are also possible.

[0020] Furthermore, in the embodiments of this application, the words "exemplary," "for example," etc., are used to indicate that they are examples, illustrations, or descriptions. Any embodiment or design scheme described as "exemplary" in the embodiments of this application should not be construed as being better or more advantageous than other embodiments or design schemes. Specifically, the use of the term "exemplary" is intended to present the concept in a concrete manner.

[0021] The business scenarios described in the embodiments of this application are for the purpose of more clearly illustrating the technical solutions of the embodiments of this application, and do not constitute a limitation on the technical solutions provided in the embodiments of this application. As those skilled in the art will know, with the evolution of technology and the emergence of new business scenarios, the technical solutions provided in the embodiments of this application are also applicable to similar technical problems.

[0022] References to "one embodiment" or "some embodiments" as described in this specification mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized.

[0023] In this application embodiment, "at least one" refers to one or more, and "more than one" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, and B alone, where A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, at least one of a, b, or c can represent: a, b, c, ab, ac, bc, or abc, where a, b, and c can be single or multiple.

[0024] Bladder cancer is one of the most common malignant tumors of the urinary system, posing a significant threat to human health. Currently, the standard for bladder cancer diagnosis is cystoscopy and pathological examination, but this method also has some drawbacks, such as causing urinary tract infections, low diagnostic rates, and urethral pain during cystoscopy.

[0025] With the development of liquid detection technology, cfDNA has been applied in tumors, and detection methods based on liquid biomarkers have also been proposed.

[0026] The detection of liquid biomarkers plays a crucial role in medical diagnostics. Probes and primers are indispensable tools in this process, enabling the detection of target biomarkers through specific sequence design and hybridization reactions.

[0027] Primers and probes are closely related in molecular biology experiments. They each have unique definitions and functions, and they work together in experiments to achieve specific detection or amplification targets.

[0028] Primers are short DNA or ribonucleic acid (RNA) sequences used to initiate DNA replication. In amplification techniques such as polymerase chain reaction (PCR), primers bind to specific regions of the target DNA sequence, which are then replicated by DNA polymerase. By designing specific primer sequences, DNA fragments of target biomarkers can be selectively amplified.

[0029] A probe is a short, single-stranded DNA or RNA sequence that is perfectly complementary to a specific region of a target biomarker. During detection, the probe hybridizes with the target sequence to form a stable double-stranded structure. By labeling the probe (e.g., with fluorescent labeling), the visual detection of the target biomarker can be achieved.

[0030] Taking PCR amplification as an example, during the PCR amplification process, specific primer sequences can be designed to selectively amplify DNA fragments of target biomarkers. Simultaneously, combining this with labeling technologies such as fluorescent probes allows for real-time monitoring of the PCR amplification process, improving the sensitivity and accuracy of detection.

[0031] Current detection methods primarily target the C228T mutation in the TERT gene, specifically by using primer-probe combinations to detect this mutation. The C228T mutation refers to the substitution of cytosine (C) for thymine (T) at position 228 of the TERT gene promoter region.

[0032] However, medical research shows that TERT promoter mutations account for 46% of bladder cancer patients, while C228T accounts for only 64% of TERT promoter mutations in bladder cancer patients. Therefore, especially for the early detection of bladder cancer, the accuracy and sensitivity of diagnosing whether a patient has bladder cancer based on the detection results of C228T mutations in the TERT gene are relatively low.

[0033] In view of this, embodiments of this application propose a primer-probe combination that can be used to realize a multi-target detection technique for bladder cancer, thereby improving the accuracy and sensitivity of bladder cancer detection.

[0034] The primer-probe combination may include at least two of the following primer-probe combinations: a first primer-probe combination for detecting the TERT promoter gene, a second primer-probe combination for detecting the FGFR3 gene, and a third primer-probe combination for detecting the PIK3CA gene.

[0035] It should be noted that the above primer-probe combination may include at least two of the following: a combination of the first, second and third primer-probe combinations, or a combination of all three.

[0036] A medical study involving 295 bladder cancer patients found that 93% of them carried at least one mutated gene, with an average of 2.6 clinically significant variants per patient. The most frequently mutated genes were, in descending order: TERT (46.7%), CDKN2A (34%), FGFR3 (21%), PIK3CA (20%), and ERBB2 (17%). TERT, FGFR3, and PIK3CA are the most frequently mutated genes in early-stage bladder cancer. Therefore, simultaneous mutation testing of TERT, FGFR3, and PIK3CA genes can further improve the accuracy of bladder cancer detection.

[0037] In some possible embodiments, the primer sequence length of the above primer-probe combination is in the range of [19bp, 20bp], while the primer sequence length of a common single primer-probe combination for detecting the TERT gene is usually in the range of [16bp, 18bp]. The primer-probe combination proposed in this application has a slightly longer sequence length, which can effectively reduce non-specific amplification and make the amplification concentration of the target fragment higher.

[0038] Based on the above technical solution, at least two of the first, second, and third sub-primer probe combinations are combined to obtain a primer probe combination. This primer probe combination can simultaneously detect at least two gene mutations related to bladder cancer among the TERT, FGFR3, and PIK3CA genes. Compared with primer probe combinations that only detect TERT gene mutations, the primer probe combination proposed in this application has a higher detection hit rate, stronger specificity, and higher sensitivity.

[0039] In some possible embodiments, the mutation sites detected by the first sub-primer-probe combination include at least one of C228T, C250T, and C228A; the mutation sites detected by the second sub-primer-probe combination include at least one of S248C, S249C, and Y353C; and the mutation sites detected by the third sub-primer-probe combination include at least one of H1047R, E542K, and E545K.

[0040] The various mutation sites mentioned above and their corresponding gene sequences (which can be simply referred to as sequences) can be represented by Tables 1, 2, and 3 below. Table 1 shows the mutation sites and corresponding gene sequences of the TERT gene, Table 2 shows the mutation sites and corresponding gene sequences of the FGFR3 gene, and Table 3 shows the mutation sites and corresponding gene sequences of the PIK3CA gene.

[0041] Table 1

[0042]

[0043] Table 2

[0044]

[0045] Table 3

[0046]

[0047] Based on the above technical solutions, the primer-probe combination proposed in this application can simultaneously detect up to nine gene sequences that may mutate during carcinogenesis, ensuring the comprehensiveness of mutated gene detection and thereby increasing the detection accuracy and sensitivity of the primer-probe combination.

[0048] The gene sequences of the primers and probes included in the first, second, and third sub-primer-probe combinations can be represented by Table 4 below.

[0049] For example, the SEQ ID NO:X mentioned in the embodiments of this application is used to indicate the sequence shown in the Xth row of the corresponding column of primer and probe sequences in Table 4, where X is a positive integer; for example, SEQ ID NO:1 represents the primer and probe sequence corresponding to TF1 (i.e., in the same row) in Table 4.

[0050] Table 4

[0051]

[0052] In some possible embodiments, the above primer-probe combination satisfies any one or more of the following conditions:

[0053] When the mutation site to be detected by the first subprimer-probe combination includes C228T, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequence derived from any one or more of SEQ ID NO: 1 to 4.

[0054] When the mutation site to be detected by the first subprimer-probe combination includes C250T, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequence derived from any one or more of SEQ ID NO: 5 to 8.

[0055] When the mutation site to be detected by the first subprimer-probe combination includes C228A, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequence derived from any one or more of SEQ ID NO: 9 to 12.

[0056] When the mutation site to be detected by the second subprimer probe combination includes S248C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequence derived from any one or more of SEQ ID NO: 13 to 16.

[0057] When the mutation site to be detected by the second subprimer probe combination includes S249C, the second subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequence derived from any one or more of SEQ ID NO: 17 to 20.

[0058] When the mutation site to be detected by the second subprimer probe combination includes Y353C, the second subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequence derived from any one or more of SEQ ID NO:21 to 24.

[0059] When the mutation site to be detected by the third subprimer probe combination includes H1047R, the third subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequence derived from any one or more of SEQ ID NO: 25 to 28.

[0060] When the mutation site detected by the third subprimer probe combination includes E542K, the third subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of any one or more sequences in SEQ ID NO:29 to 32;

[0061] When the mutation site to be detected by the third subprimer-probe combination includes E545K, the third subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from any one or more of SEQ ID NO: 33 to 36.

[0062] In some possible embodiments, the primer-probe combination described above may further satisfy any one or more of the following conditions:

[0063] When the mutation site to be detected by the first subprimer-probe combination includes C228T, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO:1 to 4.

[0064] When the mutation site to be detected by the first subprimer-probe combination includes C250T, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO: 5 to 8.

[0065] When the mutation site to be detected by the first subprimer-probe combination includes C228A, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO: 9 to 12.

[0066] When the mutation site to be detected by the second subprimer probe combination includes S248C, the second subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from several sequences in SEQ ID NO: 13 to 16;

[0067] When the mutation site to be detected by the second subprimer probe combination includes S249C, the second subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from several sequences in SEQ ID NO: 17 to 20;

[0068] When the mutation site to be detected by the second subprimer probe combination includes Y353C, the second subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from several sequences in SEQ ID NO:21 to 24;

[0069] When the mutation site to be detected by the third subprimer probe combination includes H1047R, the third subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from several sequences in SEQ ID NO: 25 to 28;

[0070] When the mutation site to be detected by the third subprimer probe combination includes E542K, the third subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from several sequences in SEQ ID NO:29 to 32;

[0071] When the mutation site to be detected by the third subprimer-probe combination includes E545K, the third subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from several sequences in SEQ ID NO:33 to 36.

[0072] In some possible embodiments, the primer-probe combination described above may further satisfy any one or more of the following conditions:

[0073] When the mutation site to be detected by the first subprimer probe combination includes C228T, the first subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO:1 to 4.

[0074] When the mutation site to be detected by the first subprimer-probe combination includes C250T, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO: 5 to 8.

[0075] When the mutation site to be detected by the first subprimer probe assembly includes C228A, the first subprimer probe assembly comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences in SEQ ID NO: 9 to 12.

[0076] When the mutation site to be detected by the second subprimer probe combination includes S248C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO: 13 to 16;

[0077] When the mutation site to be detected by the second subprimer probe combination includes S249C, the second subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO: 17 to 20;

[0078] When the mutation site to be detected by the second subprimer probe combination includes Y353C, the second subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO:21 to 24;

[0079] When the mutation site to be detected by the third subprimer probe combination includes H1047R, the third subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO: 25 to 28;

[0080] When the mutation site to be detected by the third subprimer probe combination includes E542K, the third subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO:29 to 32;

[0081] When the mutation site to be detected by the third subprimer probe combination includes E545K, the third subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO:33 to 36.

[0082] It should be noted that "derived from" in the embodiments of this application can be understood as mutation, substitution, deletion or modification of nucleotides, etc.

[0083] In some possible embodiments, the primer-probe combination described above may further satisfy any one or more of the following conditions:

[0084] When the mutation site to be detected by the first subprimer-probe combination includes C228T, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO:1 to 4;

[0085] When the mutation site to be detected by the first subprimer-probe combination includes C250T, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO: 5 to 8;

[0086] When the mutation site to be detected by the first subprimer probe combination includes C228A, the first subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO: 9 to 12;

[0087] When the mutation site to be detected by the second subprimer probe combination includes S248C, the second subprimer probe combination includes at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO:13 to 16;

[0088] When the mutation site to be detected by the second subprimer probe combination includes S249C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO:17 to 20;

[0089] When the mutation site to be detected by the second subprimer probe combination includes Y353C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO:21 to 24;

[0090] When the mutation site to be detected by the third subprimer probe combination includes H1047R, the third subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO:25 to 28;

[0091] When the mutation site to be detected by the third subprimer probe combination includes E542K, the third subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO:29 to 32;

[0092] When the mutation site detected by the third subprimer probe combination includes E545K, the third subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO:33 to 36.

[0093] It should be noted that the term "selected from" in the embodiments of this application can be understood as selecting specific genes, fragments or information from gene sequences, or screening gene sequences based on specific conditions.

[0094] In some possible embodiments, the primer-probe combination proposed in this application may simultaneously include at least one sequence derived from Table 4 and at least one sequence selected from Table 4.

[0095] For example, referring to Table 4, when the mutation site to be detected by the first sub-primer probe combination includes C228T, the upstream primer TF1 of the first sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:1, the downstream primer TR1 of the first sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:2, the upstream probe TP1-WT of the first sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:3, and the downstream probe TP1-MUT of the first sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:4. This part of the primer probe can be denoted as T1, where the probe suffix WT is used to represent the wild-type gene sequence and the probe suffix MUT is used to represent the mutant gene sequence.

[0096] When the mutation site to be detected by the first sub-primer probe combination includes C250T, the upstream primer TF2 of the first sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:5, the downstream primer TR2 of the first sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:6, the upstream probe TP2-WT of the first sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:7, and the downstream probe TP2-MUT of the first sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:8, and this part of the primer probe can be referred to as T2;

[0097] When the mutation site to be detected by the first sub-primer probe combination includes C228A, the upstream primer TF3 of the first sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:9, the downstream primer TR3 of the first sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:10, the upstream probe TP3-WT of the first sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:11, and the downstream probe TP3-MUT of the first sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:12. This part of the primer probe can be referred to as T3.

[0098] For example, referring to Table 4, when the mutation site to be detected by the second sub-primer probe combination includes S248C, the upstream primer FF1 of the second sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:13, the downstream primer FR1 of the second sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:14, the upstream probe FP1-WT of the second sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:15, and the downstream probe FP1-MUT of the second sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:16, and this part of the primer probe can be referred to as F1;

[0099] When the mutation site to be detected by the second sub-primer probe combination includes S249C, the upstream primer FF2 of the second sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:17, the downstream primer FR2 of the second sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:18, the upstream probe FP2-WT of the second sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:19, and the downstream probe FP2-MUT of the second sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:20, and this part of the primer probe can be referred to as F2;

[0100] When the mutation site to be detected by the second sub-primer probe combination includes Y353C, the upstream primer FF3 of the second sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:21, the downstream primer FR3 of the second sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:22, the upstream probe FP3-WT of the second sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:23, and the downstream probe FP3-MUT of the second sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:24, and this part of the primer probe can be referred to as F3.

[0101] For example, referring to Table 4, when the mutation site to be detected by the third sub-primer probe combination includes H1047R, the upstream primer PF1 of the third sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:25, the downstream primer PR1 of the third sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:26, the upstream probe PP1-WT of the third sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:27, and the downstream probe PP1-MUT of the third sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:28, and this part of the primer probe can be referred to as P1;

[0102] When the mutation site to be detected by the third sub-primer probe combination includes E542K, the upstream primer PF2 of the third sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:29, the downstream primer PR2 of the third sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:30, the upstream probe PP2-WT of the third sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:31, and the downstream probe PP2-MUT of the third sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:32, and this part of the primer probe can be referred to as P2;

[0103] When the mutation site detected by the third sub-primer probe combination includes E545K, the upstream primer PF3 of the third sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:33, the downstream primer PR3 of the third sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:34, the upstream probe PP3-WT of the third sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:35, and the downstream probe PP3-MUT of the third sub-primer probe combination may include the primer probe sequence shown in SEQ ID NO:36. This part of the primer probe can be referred to as P3.

[0104] It should be understood that when the first sub-primer-probe combination is used simultaneously to detect mutation sites C228T, C250T, and C228A, the first sub-primer-probe combination may include all types of upstream and downstream probes mentioned in the above embodiments. That is, the first sub-primer-probe combination is derived from or selected from at least a portion of the corresponding sequences of primers T1, T2, and T3 in Table 4. The same applies to the first sub-primer-probe combination used to simultaneously detect other mutation site combinations. When the second sub-primer-probe combination is used simultaneously to detect mutation sites S248C, S249C, and Y353C, the second sub-primer-probe combination may include all types of upstream and downstream probes mentioned in the above embodiments. The upstream probe, i.e., the second sub-primer probe combination, is derived from or selected from at least a portion of the corresponding sequences of primer probe F1, primer probe F2, and primer probe F3. The same applies to the second sub-primer probe combination used to simultaneously detect other mutation site combinations. When the third sub-primer probe combination is used to simultaneously detect mutation sites H1047R, E542K, and E545K, the third sub-primer probe combination can include all types of upstream and downstream probes mentioned in the above embodiments. That is, the third sub-primer probe combination is derived from or selected from at least a portion of the corresponding sequences of primer probe P1, primer probe P2, and primer probe P3. The same applies to the third sub-primer probe combination used to simultaneously detect other mutation site combinations.

[0105] Based on the above technical solution, while introducing multiple sub-primer-probe combinations for detecting different genes into the primer-probe combination, each sub-primer-probe combination also introduces detection probes for wild-type genes and mutant genes. This allows the detection results of the primer-probe combination to be used for self-testing. That is, when a mutant gene is detected, the corresponding wild-type gene should not respond, and vice versa. This ensures that there are no abnormalities in gene amplification, thereby increasing the reliability of the detection results.

[0106] In some possible embodiments, in the primer-probe combination proposed in the embodiments of this application, the probe portion may also use only the mutant gene sequence.

[0107] In some possible embodiments, in the first, second, or third subprimer-probe combination described above, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group.

[0108] It should be understood that the above probes include upstream probes and downstream probes.

[0109] Furthermore, the labeling design of the aforementioned probe is based on the principle of fluorescence resonance energy transfer (FRET) and is applied in quantitative real-time PCR (qPCR) detection. The fluorescent reporter group is located at the 5' end of the probe. When the 5' end of the probe is irradiated with excitation light of a suitable wavelength, it absorbs light energy and transitions to a high-energy excited state. It then returns to the ground state by emitting light (fluorescence). This fluorescence signal serves as a key signal for detecting the presence of the target DNA sequence. The fluorescent quencher group is located at the 3' end of the probe and is typically a non-fluorescent chromophore. When the fluorescent quencher group is sufficiently close to the fluorescent reporter group, it absorbs the fluorescence energy emitted by the reporter group and releases it as heat, causing the fluorescence signal to weaken or disappear. This quenching effect persists when the probe has not hybridized with the target DNA sequence, thereby reducing background fluorescence.

[0110] When a probe hybridizes with a target DNA sequence, the probe's secondary structure changes (e.g., the hairpin loop structure of a molecular beacon probe opens), increasing the distance between the fluorescent reporter group and the fluorescent quencher group, exceeding the range of fluorescence resonance energy transfer. At this point, the fluorescent reporter group emits fluorescence at its own wavelength when exposed to excitation light, and is no longer absorbed by the quencher group. This enhancement of the fluorescence signal is proportional to the amount of PCR product, thus allowing for accurate detection of the presence and quantity of the target DNA sequence.

[0111] In some possible embodiments, the fluorescent reporter group may be selected from at least one of FAM, HEX, TEXAS, CY3, CY5, TAMRA, TET, JOE, VIC, NED and ROX; the fluorescent quencher group may be selected from at least one of TAMRA, BHQ1 and DABCY.

[0112] In some possible embodiments, in the first sub-probe combination, the second sub-primer-probe combination, or the third sub-primer-probe combination, the fluorescent reporter group labeled at the 5' end of the upstream probe is different from the fluorescent reporter group labeled at the 5' end of the downstream probe, while the fluorescent quencher groups labeled at the 3' ends of the upstream and downstream probes are the same.

[0113] For example, the 5' ends of the wild-type probes TP1-WT, TP2-WT, and TP3-WT can be labeled with the fluorescent reporter group FAM, the 5' ends of the mutant probes TP1-MUT, TP2-MUT, and TP3-MUT can be labeled with the fluorescent reporter group HEX, and the 3' ends of all probes can be labeled with the fluorescent quencher group BHQ1.

[0114] For example, the 5' ends of the wild-type probes FP1-WT, FP2-WT, and FP3-WT can be labeled with the fluorescent reporter group FAM, the 5' ends of the mutant probes FP1-MUT, FP2-MUT, and FP3-MUT can be labeled with the fluorescent reporter group HEX, and the 3' ends of all probes can be labeled with the fluorescent quencher group BHQ1.

[0115] For example, the 5' ends of the wild-type probes PP1-WT, PP2-WT, and PP3-WT can be labeled with the fluorescent reporter group FAM, the 5' ends of the mutant probes PP1-MUT, PP2-MUT, and PP3-MUT can be labeled with the fluorescent reporter group HEX, and the 3' ends of all probes can be labeled with the fluorescent quencher group BHQ1.

[0116] Based on the above technical solution, by labeling the 5' ends of the upstream and downstream probes with fluorescent reporter groups of corresponding colors, the detection results of multiple genes from the primer-probe combination can be distinguished from each other and will not be confused, making the detection results more obvious and easier to observe.

[0117] In some possible embodiments, the annealing temperature of the primer-probe combination proposed in this application can be in the range of [60℃, 65℃], which is slightly higher than that of the primer-probe combination for ordinary single gene detection at present (usually in the range of [55℃, 60℃]). Based on this, it can reduce the non-specific binding of the primer-probe combination without affecting the binding efficiency of the primers in the primer-probe combination.

[0118] In some possible embodiments, the GC content of the primer-probe combination proposed in this application is within the range of [40%, 60%], thereby ensuring the initiation efficiency of the primer-probe combination. Here, GC content refers to the ratio of guanine (G) and cytosine (C) among the four bases of DNA (adenine A, guanine G, thymine T, and cytosine C).

[0119] It should be understood that the higher the GC content, the higher the DNA density, and the less likely the substance is to denature due to heating or alkali. Therefore, this property can be used for DNA separation or determination.

[0120] In some possible embodiments, the primer-probe combination proposed in this application has a ΔG value of less than 6 at the 3′ end of the primer, which is higher than the upper limit of the ΔG value of primer-probe combinations for common single-gene detection at present (usually less than 9). Based on this, the 3′ end of the primer with a lower ΔG value can reduce mismatches and primer dimer formation, thereby improving the specificity and efficiency of the PCR reaction.

[0121] Furthermore, this application also proposes a kit that includes any primer-probe combination proposed in this application.

[0122] In some possible embodiments, the above-described kit is used for bladder cancer detection, the test sample of the kit is urine, and the bladder cancer detection process is performed by a computer.

[0123] In some possible embodiments, the kit described above also includes reagents required for performing fluorescent PCR.

[0124] In some possible embodiments, the reagents required for fluorescent PCR using the above kit can be adapted to the specific type of sample to be tested.

[0125] For example, the reagents required for performing fluorescent PCR may include PCR reaction solution and Taq enzyme, wherein the PCR reaction solution includes the primer-probe combination and PCR reaction buffer described in this application; the reagents required for performing fluorescent PCR may also include DNA extraction solution, positive control and negative control.

[0126] Furthermore, this application illustrates the detection process and detection effect of the primer-probe combination proposed in the embodiments of this application through the following experiments.

[0127] The following preparations are required before testing:

[0128] S101, Obtain the PCR reaction solution and the primer-probe combination for fluorescence PCR amplification. The PCR reaction solution can be the reaction solution of Taq Pro U+MultipleProbe qPCR Mix, and the primer-probe combination can include the primer-probe T1 and primer-probe F1 mentioned in the above embodiments.

[0129] S102, according to the wild-type gene sequences and mutant gene sequences corresponding to primer probe T1 and primer probe F1 as shown in Tables 1 and 2 above, plasmids were synthesized and serially diluted with ultrapure water to obtain plasmid samples of different concentrations, which were then stored at -20℃.

[0130] S103. Based on the gene sequences of primer probe T1 and primer probe F1 as shown in Table 4 above, primer probes are synthesized to obtain a primer probe combination proposed in this application embodiment. The primers are diluted with ultrapure water, and the concentrations of each upstream and downstream primer are 10 μmol / L, and the concentrations of wild-type probes and mutant probes are 10 μmol / L.

[0131] The procedure for fluorescent PCR detection is as follows:

[0132] S201: Prepare the test solution according to the proportions shown in Table 5 below.

[0133] Table 5

[0134] Components Dosage (per person) / uL PCR reaction solution 13 upstream primer 1 Downstream primer 1 probe 0.5 <![CDATA[dH2O]]> Fill up to 20uL

[0135] S203: After vortexing the above test solution, dispense 2.5 μL into each of eight tubes and centrifuge for 3-5 seconds.

[0136] S205: Add 10 μL of the plasmid sample obtained in the above preparation work to each tube in an eight-tube array, and centrifuge for 3-5 seconds.

[0137] S207: Place the above eight tubes into a real-time quantitative PCR instrument and perform amplification according to the operating parameters in Table 6 below.

[0138] Table 6

[0139]

[0140] The PCR instrument can be an ABI 7500.

[0141] S209: Obtain the cycle threshold value (Ct), which is the number of cycles required for the fluorescence signal in each reaction tube to reach the set threshold. When the Ct value is ≤37.00, the result is positive. When 37.00 < Ct value ≤40.00, the above steps S201 to S207 need to be repeated. If the Ct value of the second test is still less than 40.00, the result is positive; otherwise, the result is negative.

[0142] The primer-probe combination sensitivity detection procedure proposed in this application is as follows:

[0143] S301: Prepare the premixed solution according to Table 5 above.

[0144] S303: Plasmids containing the wild-type and mutant genes of the TERT promoter gene and the FGFR3 gene were serially diluted with ultrapure water to obtain five concentrations of plasmid samples: 1×10⁵ copies / mL (L1), 1×10³ copies / mL (L2), 500 copies / mL (L3), 200 copies / mL (L4), and 100 copies / mL (L5). The samples were stored at -20℃ for later use.

[0145] S305: The sensitivity of primers T1 and F1 in the primer-probe combination was verified to obtain the enhanced detection Ct value.

[0146] Taking samples L1 and L2 as examples, the amplification detection Ct values ​​obtained after sensitivity verification of these two samples are shown in Table 7 below.

[0147] Table 7

[0148]

[0149] S307: Plasmids containing mutant genes of TERT promoter, FGFR3 and PIK3CA were serially diluted with ultrapure water to obtain four different concentrations of plasmid samples: 1×103 copies / mL, 500 copies / mL, 200 copies / mL and 100 copies / mL. The samples were stored at -20℃ for later use.

[0150] S309: The sensitivity of primers T1, F1 and P1 in the primer-probe combination was verified to obtain the enhanced detection Ct value.

[0151] The augmented detection Ct values ​​obtained after sensitivity verification based on the samples obtained from S307 are shown in Table 8 below.

[0152] Table 8

[0153]

[0154] Based on the validation results in Tables 7 and 8 above, it can be seen that for plasmid samples with concentrations of 1×10⁵ copies / mL and 1×10³ copies / mL, primer-probe combinations T1 and F1 can detect mutations; for plasmid samples with concentrations of 500 copies / mL, 200 copies / mL, and 100 copies / mL, primer-probe combinations T1, F1, and P1 can still detect mutations. These results demonstrate that the primer-probe combination of this application has high sensitivity for bladder cancer mutation detection.

[0155] The procedure for the specific detection experiment of the primer-probe combination proposed in this application embodiment is as follows:

[0156] S401: Prepare the premixed solution according to Table 5 above.

[0157] S403: Use ultrapure water to dilute the plasmids containing the wild-type genes of the TERT promoter gene and the FGFR3 gene respectively to obtain plasmid samples with a concentration of 1×103 copies / mL, and store them at -20℃ for later use.

[0158] S405: The specificity of primers T1 and F1 in the primer-probe combination was verified to obtain the enhanced detection Ct value.

[0159] The augmented detection Ct values ​​obtained after specificity verification of the samples obtained based on S403 are shown in Table 9 below.

[0160] Table 9

[0161]

[0162] In this application, mutant plasmid samples use wild-type probes, and wild-type plasmid samples use mutant probes; specifically, TW uses TP1-MUT, FW uses FP1-MUT, and PW uses PP1-MUT; TMU uses TP1-WT, FMU uses FP1-WT, and PMU uses PP1-WT. Furthermore, embodiments of this application also provide... Figure 1 This is a graph showing the amplification and detection results of this experiment.

[0163] Figure 1 This is an amplification detection result curve of a specificity verification experiment proposed in the embodiments of this application.

[0164] refer to Figure 1 As shown, the vertical axis represents the number of relative fluorescence units (RFUs), and the horizontal axis represents the number of PCR reaction cycles.

[0165] RFU (Relative Fluorescence Unit) is a unit of measurement used to measure fluorescence intensity. In qPCR experiments, fluorescent dye is added to the reaction system. As the PCR reaction proceeds, the DNA template is amplified, and the fluorescence signal gradually increases. The intensity of this fluorescence signal can be quantified as the RFU value. A higher RFU value indicates stronger fluorescence intensity, which usually also means a greater quantity of PCR products. Furthermore, in the PCR reaction, the DNA template is continuously amplified in a thermal cycler through denaturation, annealing, and extension steps. Each cycle represents one DNA replication process, and the cycles value increments by 1 with each cycle.

[0166] It's important to note that qPCR results are typically an S-shaped amplification curve, depicting the change in RFU (Reactive Fluorescence) value as cycles increase. This curve can be divided into three phases: the baseline phase, the exponential growth phase, and the plateau phase. The baseline phase is the initial stage of the PCR reaction, where the fluorescence signal is very low and almost constant. The fluorescence signal during the baseline phase mainly comes from background fluorescence and non-specific fluorescence in the reaction system. The exponential growth phase occurs as the DNA template is continuously amplified, and the fluorescence signal begins to increase rapidly. During this phase, the RFU value exhibits an exponential relationship with cycles, meaning that the RFU value increases exponentially with each cycle. The plateau phase occurs after the PCR reaction has reached a certain number of cycles. Due to the consumption of primers, enzymes, and other components in the reaction system, as well as the saturation of the DNA template, the rate of increase in the fluorescence signal begins to slow down and eventually stabilizes. During the plateau phase, the change in RFU value is no longer significant, but it can still remain at a relatively high level.

[0167] Therefore, the amplification detection result curve of qPCR experiments provides a direct understanding of the PCR reaction process and results. Furthermore, the shape and characteristics of the amplification curve can be used to assess the quality of the PCR reaction, amplification efficiency, and the presence of non-specific amplification. In addition, by calculating parameters such as the Ct value (cycle threshold), PCR products can be quantitatively analyzed, and differences between different samples can be compared.

[0168] refer to Figure 1 As shown in Table 9, the amplification curves of all samples were not S-shaped, and no Ct values ​​were detected in the detection channels corresponding to each fluorescent reporter group. This indicates that the primer-probe combination did not undergo non-specific binding. Therefore, the primer-probe combination proposed in this application embodiment has no cross-reaction with wild-type or mutant genes and has good specificity.

[0169] Taking the detection of TERT promoter gene mutation frequency as an example, the experimental procedure for detecting TERT promoter gene mutation frequency using the primer-probe combination proposed in this application is as follows:

[0170] S501: Prepare the premixed solution according to Table 5 above.

[0171] S503: The plasmids containing the wild-type gene of the TERT promoter were diluted with ultrapure water to obtain plasmid samples with a concentration of 1×103 copies / mL. Then, they were mixed in proportion to obtain plasmid mixed samples with mutation frequencies of 0%, 0.5%, 1%, 2% and 5%, respectively. They were stored at -20℃ for later use.

[0172] S505: The mutation frequency of the TERT promoter gene of primer T1 in the primer-probe combination was verified to obtain the augmented detection Ct value.

[0173] The augmented detection Ct values ​​obtained after verifying the mutation frequency of the TERT promoter gene based on the samples obtained from S503 are shown in Table 10 below.

[0174] Table 10

[0175] sample Ct value 0% of mutant samples No Ct 0.5% of mutant samples 29.28 1% of mutant samples 27.57 2% of mutant samples 26.44 5% of mutant samples 25.05

[0176] Furthermore, embodiments of this application also provide Figure 2 This is a graph showing the amplification and detection results of this experiment.

[0177] Figure 2 This is another amplification detection result curve of the specificity verification experiment proposed in the embodiments of this application.

[0178] in, Figure 2 The meaning of the x and y coordinates in the figure Figure 1 The same applies, so I won't repeat it here.

[0179] refer to Figure 2 As shown in Table 10, the primer-probe combination proposed in this application can detect the mutated gene normally, and the higher the mutation frequency of the mutated sample, the lower the corresponding Ct value, which is in line with the expected experimental results.

[0180] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A primer-probe combination, characterized in that, Includes at least two of the following primer-probe combinations: The first primer-probe combination is used to detect the TERT promoter gene of telomerase reverse transcriptase; the second primer-probe combination is used to detect the fibroblast growth factor receptor 3FGFR3 gene; and the third primer-probe combination is used to detect the PIK3CA gene of phosphatidylinositol 3-kinase catalytic subunit.

2. The primer-probe combination according to claim 1, characterized in that, The mutation sites detected by the first sub-primer-probe combination include at least one of C228T, C250T, and C228A; the mutation sites detected by the second sub-primer-probe combination include at least one of S248C, S249C, and Y353C; and the mutation sites detected by the third sub-primer-probe combination include at least one of H1047R, E542K, and E545K.

3. The primer-probe combination according to claim 2, characterized in that, Meet any one or more of the following conditions: When the mutation site detected by the first subprimer-probe combination includes the C228T, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequence derived from any one or more of SEQ ID NO: 1 to 4. When the mutation site detected by the first subprimer-probe combination includes the C250T, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequence derived from any one or more of SEQ ID NO: 5 to 8. When the mutation site detected by the first subprimer-probe combination includes C228A, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequence derived from any one or more of SEQ ID NO: 9 to 12. When the mutation site detected by the second subprimer probe combination includes S248C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from any one or more of SEQ ID NO: 13 to 16. When the mutation site detected by the second subprimer probe combination includes S249C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from any one or more of SEQ ID NO: 17 to 20. When the mutation site detected by the second subprimer probe combination includes Y353C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequence derived from any one or more of SEQ ID NO: 21 to 24. When the mutation site detected by the third subprimer-probe combination includes H1047R, the third subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequence derived from any one or more of SEQ ID NO: 25 to 28. When the mutation site detected by the third subprimer-probe combination includes the E542K, the third subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from any one or more of SEQ ID NO: 29 to 32. When the mutation site to be detected by the third sub-primer probe combination includes the E545K, the third sub-primer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from any one or more of SEQ ID NO: 33 to 36.

4. The primer-probe combination according to claim 2 or 3, characterized in that, Meet any one or more of the following conditions: When the mutation site detected by the first subprimer-probe combination includes the C228T, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO:1 to 4. When the mutation site detected by the first subprimer-probe combination includes the C250T, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO: 5 to 8. When the mutation site detected by the first subprimer-probe combination includes C228A, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO: 9 to 12. When the mutation site detected by the second subprimer probe combination includes S248C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO:13 to 16. When the mutation site detected by the second subprimer probe combination includes S249C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO:17 to 20. When the mutation site detected by the second subprimer probe combination includes Y353C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from several sequences in SEQ ID NO: 21 to 24; When the mutation site detected by the third subprimer-probe combination includes H1047R, the third subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from several sequences in SEQ ID NO: 25 to 28; When the mutation site detected by the third subprimer-probe combination includes the E542K, the third subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO:29 to 32; When the mutation site to be detected by the third sub-primer probe combination includes the E545K, the third sub-primer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of the sequences derived from SEQ ID NO:33 to 36.

5. The primer-probe combination according to any one of claims 2 to 4, characterized in that, Meet any one or more of the following conditions: When the mutation site detected by the first subprimer-probe combination includes the C228T, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO: 1 to 4. When the mutation site detected by the first subprimer-probe combination includes the C250T, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO: 5 to 8. When the mutation site detected by the first subprimer-probe combination includes C228A, the first subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO: 9 to 12. When the mutation site detected by the second subprimer probe combination includes S248C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO: 13 to 16; When the mutation site detected by the second subprimer probe combination includes S249C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO: 17 to 20; When the mutation site detected by the second subprimer probe combination includes Y353C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO: 21 to 24; When the mutation site detected by the third subprimer probe combination includes H1047R, the third subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO: 25 to 28; When the mutation site to be detected by the third subprimer-probe combination includes the E542K, the third subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO:29 to 32; When the mutation site to be detected by the third sub-primer probe combination includes the E545K, the third sub-primer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% derived from all sequences in SEQ ID NO:33 to 36.

6. The primer-probe combination according to any one of claims 2 to 5, characterized in that, Meet any one or more of the following conditions: When the mutation site detected by the first subprimer probe combination includes the C228T, the first subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO:1 to 4. When the mutation site detected by the first subprimer probe combination includes the C250T, the first subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO: 5 to 8; When the mutation site detected by the first subprimer probe combination includes C228A, the first subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO: 9 to 12; When the mutation site detected by the second subprimer probe combination includes S248C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO: 13 to 16; When the mutation site detected by the second subprimer probe combination includes S249C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO: 17 to 20; When the mutation site detected by the second subprimer probe combination includes Y353C, the second subprimer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO:21 to 24; When the mutation site detected by the third subprimer-probe combination includes H1047R, the third subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO: 25 to 28; When the mutation site to be detected by the third subprimer-probe combination includes the E542K, the third subprimer-probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO:29 to 32; When the mutation site detected by the third sub-primer probe combination includes E545K, the third sub-primer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97%, or 100% of all sequences selected from SEQ ID NO:33 to 36.

7. The primer-probe combination according to any one of claims 3 to 6, characterized in that, In the first, second, or third subprimer-probe combination, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group. The fluorescent reporter group is selected from at least one of FAM, HEX, TEXAS, CY3, CY5, TAMRA, TET, JOE, VIC, NED, and ROX, and the fluorescent quencher group is selected from at least one of TAMRA, BHQ1, and DABCY.

8. The primer-probe combination according to claim 7, characterized in that, In the first sub-probe combination, the second sub-primer-probe combination, or the third sub-primer-probe combination, the fluorescent reporter group labeled at the 5' end of the upstream probe is different from the fluorescent reporter group labeled at the 5' end of the downstream probe, while the fluorescent quencher group labeled at the 3' ends of the upstream probe and the downstream probe are the same.

9. A reagent kit, characterized in that, Includes the primer-probe combination as described in any one of claims 1 to 8.

10. The reagent kit according to claim 9, characterized in that, The kit is used for bladder cancer detection. The test sample for the kit is urine, and the bladder cancer detection process is performed by computer.

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