Combined auxiliary diagnostic method, kit, system and use of point mutation and methylation of bladder cancer driver genes
View Patent ↗Disclosed are the method, kit, system and use for screening or detecting or assisting in the diagnosis of bladder cancer, which are able to simultaneously detect FGFR3, TERT, PLEKHS1 point mutations and OTX1, NID2 and NRN1 methylation levels, thereby realizing the combined detection of gene point mutation and methylation, reducing the demand for samples, experimental operation steps and detection cycle, and improving the specificity and sensitivity of detection.
1 . A detection reagent for screening bladder cancer by targeting driver genes related to the pathway of bladder cancer, comprising specific primers and probes designed for detection of regions in a template, wherein the regions to be detected comprise at least one of a gene point mutation or a gene methylation;
wherein the gene point mutation comprises a FGFR3 point mutation, a TERT point mutation, and a PLEKHS1 point mutation,
wherein the gene methylation comprises OTX1 methylation, NID2 methylation, and NRN1 methylation,
wherein the region to be detected for the FGFR3 point mutation is the sequence set forth in SEQ ID NO: 1; the region to be detected for the TERT point mutation is the sequence set forth in SEQ ID NO: 2; and the region to be detected for the PLEKHS1 point mutation is the sequence set forth in SEQ ID NO: 3, wherein no transformation or sequence alteration of the gene point mutation sequences is required;
wherein the region to be detected for the OTX1 methylation is the sequence set forth in SEQ ID NO: 4; the region to be detected for the NID2 methylation is the sequence set forth in SEQ ID NO: 5; and the region to be detected for the NRNI methylation is the sequence set forth in SEQ ID NO: 6;
wherein the specific primers and probes designed for the regions to be detected in the template are the following primer pairs and probes:
the forward primer, reverse primer, and probe for the FGFR3 point mutation are the sequences set forth in SEQ ID NOs: 10, 11 and 12 respectively;
the forward primer, reverse primer, and probe for the TERT point mutation are the sequences set forth in SEQ ID NOs: 16, 17 and 18 respectively;
the forward primer, reverse primer, and probe for the PLEKHS1 point mutation are the sequences set forth in SEQ ID NOs: 22, 23 and 24 respectively;
the forward primer, reverse primer, and probe for the OTX1 methylation are the sequences set forth in SEQ ID NOs: 25, 29 and 30 respectively;
the forward primer, reverse primer, and probe for the NID2 methylation are the sequences set forth in SEQ ID NOs: 31, 34 and 35 respectively;
the forward primer, reverse primer, and probe for the NRN1 methylation are the sequences set forth in SEQ ID NOs: 36, 38 and 37 respectively;
wherein the detection reagent further comprises primers and a probe designed for an internal reference gene, wherein the internal reference gene is ATCB; and
wherein the primers and probe designed for the internal reference gene are the sequences set forth in SEQ ID NOs: 41-43.
2 . The detection reagent according to claim 1 , wherein the specific primers and probes for detecting the gene point mutation and/or gene methylation are further distributed in different reaction tubes, the reaction tubes are respectively a reaction tube A and a reaction tube B,
wherein the reaction tube A is used to detect the gene point mutation point mutations, and a composition thereof is as follows:
Reagent Name
Reaction Volume (μL)
Primer&Probe A
2
2× Gold 360 Master Mix
25
PCR enhancer
1
nucleic acid to be detected
5
NF-H 2 O
17
total volume
50
wherein, Primer&Probe A is a mixture composed of the primer pairs and probes for detection of the point mutations in FGFR3\TERT\PLEKHS1 and the internal reference gene primers and probe; and
wherein the reaction tube B is used to detect gene methylation, and the composition thereof is as follows:
Reagent Name
Reaction Volume (μL)
Primer&Probe B
2
2× Gold 360 Master Mix
25
PCR enhancer
1
purified nucleic acid after heavy salt
5
transformation
NF-H 2 O
17
total volume
50
wherein, Primer&ProbeB is a mixture composed of the primer pairs and probes for detection of methylation in OTX1\NRN1\NID2 and internal reference gene primers and probes.
3 . The detection reagent according to claim 1 , comprising the following components:
Reagent Name
Effective Components
Primer&Probe A
A mixture of primer pairs and probes for
detection of the point mutations in FGFR3.
TERT, and PLEKHS1 and the internal
reference gene
Primer&Probe B
A mixture of primer pairs and probes for
detection of methylation in OTX1, NRN1,
and NID2 and the internal reference gene
DNA polymerase
DNA polymerase, dNTPs, Mg 2+ , Tris-HCl,
Master Mix
NaCl and nuclease-free water
PCR enhancer
DTT and/or BSA
point mutation positive
adenovirus packaging positive mutant
quality control
plasmid
methylation positive
methylation-positive nucleic acid after
quality control
methylase transformation
negative control
T cells after wild-type plasmid transfection
NF-H 2 O
nuclease-free water
wherein the sequences and concentration of each primer pair and probe in Primer&Probe A are as follows:
the forward primer, reverse primer and probe for the FGFR3 point mutation are the sequences set forth in SEQ ID NOs: 10, 11 and 12, and the concentrations are 25 μM, 25 μM and 15 μM respectively;
the forward primer, reverse primer and probe for the TERT point mutation are the sequences set forth in SEQ ID NOs: 16, 17 and 18, and the concentrations are 25 μM, 25 μM and 10 μM respectively;
the forward primer, reverse primer and probe for the PLEKHSI point mutation are the sequences set forth in SEQ ID NOs: 22, 23 and 24, and the concentrations are 25 μM, 25 μM and 8 μM respectively; and
the forward primer, reverse primer and probe for the internal reference gene are the sequences set forth in SEQ ID NOs: 41-43 and the concentrations are 2.5 μM, 2.5μM and 5 μM respectively; and
wherein the sequences and concentration of each primer pair and probe in Primer&Probe B are as follows:
the forward primer, reverse primer and probe for the OTX1 methylation are the sequences set forth in SEQ ID NOs: 25, 29 and 30, and the concentrations are 25 μM, 25 μM and 15 μM respectively;
the forward primer, reverse primer and probe for the NID2 methylation are the sequences set forth in SEQ ID NOs: 31, 34 and 35, and the concentrations are 25 μM, 25 μM and 15 μM respectively;
the forward primer, reverse primer and probe for the NRN1 methylation are the sequences set forth in SEQ ID NOs: 36, 38 and 37, and the concentrations are 25 μM, 25μM and 15 μM respectively; and
the forward primer, reverse primer and probe for the internal reference gene are the sequences set forth in SEQ ID NOs: 41-43, and the concentrations are 2.5 μM, 2.5 μM and 5 μM, respectively.