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CN-122012702-A - Primer probe combination and kit

CN122012702ACN 122012702 ACN122012702 ACN 122012702ACN-122012702-A

Abstract

The application provides a primer probe combination and a kit, which relate to the field of biotechnology, and the primer probe combination comprises at least 2 seed primer probe combinations, namely a first seed primer probe combination for detecting a TERT promoter gene, a second seed primer probe combination for detecting an FGFR3 gene and a third seed primer probe combination for detecting a PIK3CA gene. Based on the scheme, the method can be used for realizing a multi-target bladder cancer detection technology, so that the detection accuracy and sensitivity of bladder cancer are improved.

Inventors

  • LV JIAHAO

Assignees

  • 北京京东方技术开发有限公司
  • 京东方科技集团股份有限公司

Dates

Publication Date
20260512
Application Date
20241112

Claims (10)

  1. 1. A primer probe combination comprising at least 2 seed primer probe combinations of: A first primer probe combination for detecting telomerase reverse transcriptase TERT promoter gene, a second primer probe combination for detecting fibroblast growth factor receptor 3FGFR3 gene, and a third primer probe combination for detecting phosphatidylinositol 3 kinase catalytic subunit PIK3CA gene.
  2. 2. The primer probe combination of claim 1, wherein the first sub-primer probe combination is used for detecting mutation points comprising at least one of C228T, C T and C228A, the second sub-primer probe combination is used for detecting mutation points comprising at least one of S248C, S249C and Y353C, and the third sub-primer probe combination is used for detecting mutation points comprising at least one of H1047R, E542K and E545K.
  3. 3. The primer probe combination of claim 2, wherein any one or more of the following conditions are satisfied: When the mutation point for detection of the first sub-primer probe combination comprises the 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 a sequence derived from any one or several of SEQ ID NOs 1 to 4; When the mutation point for detection of the first sub-primer probe combination comprises the 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 a sequence derived from any one or several of SEQ ID NOs 5 to 8; When the mutation point for detection of the first sub-primer probe combination comprises the C228A, 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 a sequence derived from any one or several of SEQ ID NOs 9 to 12; When the mutation site for detection of the second sub-primer probe combination comprises the S248C, the second sub-primer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97% or 100% of a sequence derived from any one or several of SEQ ID NOs 13 to 16; when the mutation site for detection of the second sub-primer probe combination comprises the S249C, the second sub-primer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97% or 100% of a sequence derived from any one or several of SEQ ID NOs 17 to 20; when the mutation site for detection of the second sub-primer probe combination comprises the Y353C, the second sub-primer probe combination comprises at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97% or 100% of a sequence derived from any one or several of SEQ ID NOs 21 to 24; When the mutation point for detection of the third sub-primer probe combination comprises the H1047R, 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 a sequence derived from any one or several of SEQ ID NOs 25 to 28; When the mutation point for detection of the third sub-primer probe combination comprises the 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 a sequence derived from any one or several of SEQ ID NOs 29 to 32; when the mutation point for detection of the third sub-primer probe combination comprises 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 any one or several sequences of SEQ ID NOs 33 to 36.
  4. 4. A primer probe combination according to claim 2 or 3, wherein any one or more of the following conditions are met: When the mutation point for detection of the first sub-primer probe combination comprises the 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 sequence derived from the numbers of SEQ ID NOs 1 to 4; When the mutation point for detection of the first sub-primer probe combination comprises the 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 the sequence derived from the number of items in SEQ ID NOs 5 to 8; When the mutation point for detection of the first sub-primer probe combination comprises the C228A, 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 sequence derived from the items of SEQ ID NOs 9 to 12; When the mutation site for detection of the second sub-primer probe combination comprises the S248C, the second 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 sequence derived from the items of SEQ ID NOs 13 to 16; when the mutation site for detection of the second sub-primer probe combination comprises the S249C, the second 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 sequence derived from the items of SEQ ID NOs 17 to 20; When the mutation site for detection of the second sub-primer probe combination comprises the Y353C, the second 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 sequence derived from the number of items in SEQ ID NOs 21 to 24; When the mutation point for detection of the third sub-primer probe combination comprises the H1047R, 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 sequence derived from the number of items in SEQ ID NOs 25 to 28; When the mutation point for detection of the third sub-primer probe combination comprises the 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 sequence derived from the items of SEQ ID NOs 29 to 32; When the mutation point for detection of the third sub-primer probe combination comprises 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 sequence derived from the several items of SEQ ID NOs 33 to 36.
  5. 5. The primer probe combination of any one of claims 2 to 4, wherein any one or more of the following conditions are satisfied: When the mutation point for detection of the first sub-primer probe combination comprises the 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 the entire sequence in SEQ ID NOs 1 to 4; When the mutation point for detection of the first sub-primer probe combination comprises the 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% derived from the entire sequence in SEQ ID NOs 5 to 8; when the mutation point for detection of the first sub-primer probe combination comprises the C228A, 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 the entire sequence in SEQ ID NOs 9 to 12; When the mutation site for detection of the second sub-primer probe combination comprises the S248C, the second 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 total sequence derived from SEQ ID NOs 13 to 16; when the mutation site for detection of the second sub-primer probe combination comprises the S249C, the second 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 total sequence derived from SEQ ID NOs 17 to 20; When the mutation site for detection of the second sub-primer probe combination comprises the Y353C, the second 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 the entire sequence in SEQ ID NOs 21 to 24; When the mutation point for detection of the third sub-primer probe combination comprises the H1047R, 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 the entire sequence in SEQ ID NOs 25 to 28; When the mutation point for detection of the third sub-primer probe combination comprises the 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% derived from the entire sequence in SEQ ID NOs 29 to 32; When the mutation point for detection of the third sub-primer probe combination comprises 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 the entire sequence in SEQ ID NOs 33 to 36.
  6. 6. The primer probe combination of any one of claims 2 to 5, wherein any one or more of the following conditions are satisfied: When the mutation point for detection of the first sub-primer probe combination comprises the 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 total sequence selected from SEQ ID NOs 1 to 4; When the mutation point for detection of the first sub-primer probe combination comprises the 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 the total sequence selected from SEQ ID NOs 5 to 8; when the mutation point for detection of the first sub-primer probe combination comprises the C228A, 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 total sequence selected from SEQ ID NOs 9 to 12; when the mutation site for detection of the second sub-primer probe combination comprises the S248C, the second 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 total sequence selected from SEQ ID NOs 13 to 16; when the mutation site for detection of the second sub-primer probe combination comprises the S249C, the second 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 total sequence selected from the group consisting of SEQ ID NOs 17 to 20; When the mutation site for detection of the second sub-primer probe combination comprises the Y353C, the second 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 total sequence selected from SEQ ID NOs 21 to 24; When the mutation point for detection of the third sub-primer probe combination comprises the H1047R, 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 total sequence selected from SEQ ID NOs 25 to 28; When the mutation point for detection of the third sub-primer probe combination comprises the 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 total sequence selected from SEQ ID NOs 29 to 32; When the mutation point for detection of the third sub-primer probe combination includes the E545K, the third 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 total sequence selected from the group consisting of SEQ ID NOs 33 to 36.
  7. 7. The primer probe combination of any one of claims 3 to 6, wherein in the first, second, or third primer probe combination, the 5 'end of the probe is labeled with a fluorescence reporter group, the 3' end of the probe is labeled with a fluorescence quencher group selected from at least one of FAM, HEX, TEXAS, CY, CY5, TAMRA, TET, JOE, VIC, NED, and ROX, and the fluorescence quencher group is selected from at least one of TAMRA, BHQ1, and DABCY.
  8. 8. The primer probe combination of claim 7, wherein 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 an upstream probe is different from the fluorescent reporter group labeled at the 5' end of a downstream probe, and the fluorescent quenching groups labeled at the 3' ends of the upstream probe and the downstream probe are the same.
  9. 9. A kit comprising a primer probe combination according to any one of claims 1 to 8.
  10. 10. The kit according to claim 9, wherein the kit is used for detecting bladder cancer, the detection sample of the kit is urine, and the detection of bladder cancer is performed by a computer.

Description

Primer probe combination and kit Technical Field The embodiments of the present disclosure relate to, but are not limited to, the field of biotechnology, and in particular, to a primer probe combination and a kit. Background Bladder cancer is one of the most common malignant tumors of the urinary system, and becomes a serious disease threatening human health. The current standard for bladder cancer diagnosis is cystoscopy and pathological examination, but there are also some disadvantages such as urinary tract infection, low diagnosis rate, urethra pain caused by cystoscopy operation, etc. With the development of liquid detection technology, cell free deoxyribonucleic acid (cell-free deoxyribonucleic acid, cfDNA) is applied to tumors, and a detection mode based on liquid biomarkers is provided. In the current detection mode, C228T mutation of telomerase reverse transcriptase (TERT) gene of a subject is mainly detected, but the accuracy and sensitivity of diagnosing bladder cancer with respect to the detection result of the gene are low. Disclosure of Invention The application provides a primer probe combination and a kit, which can be used for realizing a technology for detecting bladder cancer by multiple targets, thereby improving the detection accuracy and sensitivity of bladder cancer. In a first aspect, there is provided a primer probe combination comprising at least 2 seed primer probe combinations of: A first sub-primer probe combination for detecting a TERT promoter gene, a second sub-primer probe combination for detecting a fibroblast growth factor receptor 3 (fibroblast growth factor receptor, fgfr 3) gene, and a third sub-primer probe combination for detecting a phosphatidylinositol 3 kinase catalytic subunit (phosphatidylinositol-4, 5-bisphosphate 3-KINASE CATALYTIC subsuit alpha, PIK3 CA) gene. With reference to the first aspect, in certain implementations of the first aspect, the mutation point for detection by the first sub-primer probe combination includes at least one of C228T, C T and C228A, the mutation point for detection by the second sub-primer probe combination includes at least one of S248C, S249C and Y353C, and the mutation point for detection by the third sub-primer probe combination includes at least one of H1047R, E542K and E545K. With reference to the first aspect, in certain implementations of the first aspect, any one or more of the above primer probe combinations satisfies the condition that, when the mutation point for detection of the first primer probe combination comprises C228T, the first primer 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 several of SEQ ID NOs 1 to 4, and when the mutation point for detection of the first primer probe combination comprises C250T, the first primer probe combination comprises at least 20%, 20% of the sequence derived from any one or several of SEQ ID NOs 5 to 8, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97% or 100%, where the mutation site for detection of the first sub-primer probe combination comprises C228A, 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 any one or several sequences of SEQ ID NOs 9 to 12, where the mutation site for detection of the second sub-primer probe combination comprises S248C, the second sub-primer probe combination comprises at least 20%, at least 20% of the sequence derived from any one or several sequences of SEQ ID NOs 13 to 16, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97% or 100%, where the mutation site for detection of the second sub-primer probe combination comprises S249C, the second 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 any one or several sequences of SEQ ID NOs 17 to 20, where the mutation site for detection of the second sub-primer probe combination comprises Y353C, the second sub-primer probe combination comprises at least 20%, at least 20% of the sequence derived from any one or several sequences of SEQ ID NOs 21 to 24, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97% or 100%, where the mutation point for detection of the third sub-primer probe combination comprises H1047R, 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 any one or several sequences of SEQ ID NOs 25 to 28, where the mutation point for detection of the third sub-primer probe combination comprises E542K, the third sub-primer probe combination comprises at least 20%, at least 20% of the sequence derived from any one or several sequences of SEQ ID NOs 29 to 32, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 97% or 100%, where the mutation point for detection of