CN109055559A - A kind of specific primer detecting bladder cancer and kit and its detection method - Google Patents
A kind of specific primer detecting bladder cancer and kit and its detection method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
A kind of specific primer for detecting bladder cancer of the present invention and kit and its detection method, wherein specific primer includes β-GRS gene magnification primer and probe, NAG gene magnification primer and probe, HOXA13 gene magnification primer and probe, UBE2C gene magnification primer and probe；β-GRS gene magnification primer sequence is as shown in SEQ ID NO:1-2, and β-GRS gene probe is as shown in SEQ ID NO:3；NAG gene magnification primer sequence is as shown in SEQ ID NO:4-5, and NAG gene probe is as shown in SEQ ID NO:6；HOXA13 gene magnification primer sequence is as shown in SEQ ID NO:7-8, and HOXA13 gene probe is as shown in SEQ ID NO:9；UBE2C gene magnification primer sequence is as shown in SEQ ID NO:10-11, and UBE2C gene probe is as shown in SEQ ID NO:12.Have many advantages, such as that detection speed is fast, sensibility is high and specific good using kit prepared by above-mentioned primer and probe；This method does not depend on cystoscopy simultaneously, carries out fluorogenic quantitative detection directly against marker, easy to operate, therefore is suitble to the popularization and application in clinical examination work.
Description
Technical field
The invention belongs to nucleic acid in vitro augmentation detection fields, and in particular to it is a kind of detect bladder cancer specific primer and examination
Agent box and its detection method.
Background technique
Bladder cancer is one of five kinds of most common malignant tumours in the whole world.2010 bladder cancer cases for being only in the U.S. expect
It is 67,160, it is contemplated that dead 13,750 (A.Jemal etc., Cancer Statistics, 2010CA:A Cancer
Journal for Clinicians 60,277-300,2010).When early detection then, the survival rate in 5 years is about
94%, therefore timely intervention can significantly improve the probability of patient's survival.Currently, the tumor of bladder for being more than 80% is non-infiltration
The papilloma (pTa or pT1) of property, but remainder shows muscle during diagnosis and infiltrates and have more less advantageous pre-
Afterwards.Although muscle invasive disease needs radical surgery, non-muscle invasive tumor is being with or without Bladder intravesical instillation
In the case of, it can more conservatively be treated by transurethral resection tumour；However, 70% or more the trouble with early stage disease
Person can the first two years recurrence after diagnosis, this makes bladder cancer become one of most common cancer.If be not treated in time, these
Initial non-infiltration venereal disease change can develop into muscle wellability lesion (F.Millan-Rodriguez etc., J.Urol.164,680-
84,2000).The rebound phenomenon of bladder cancer means that patient needs at least annual stringent monitoring.For the quality of life
With positive clinical effectiveness, palindromia and tentative diagnosis no less important are found in time.
It is cut currently, the most reliable method for bladder cancer detection is combined for cystoscopy with the living tissue of damaged part
Piece inspection.But the technology is time-consuming, be wellability and its sensibility is only about 90%, it means that about 10% cancer patient
It cannot be detected with these methods.Urinary cytology method in shallow method can detect flaking by microscope
Malignant cell is current preferred method.Although the specificity of cytology is about 95%, to the sensitivity of low-grade trauma
Property is very low, is influenced highly dependent upon sample quality and by the difference in height between observer.
It has attempted to detect the genetic marker in bladder living tissue specimen in the prior art.Most common method is
Microarray analysis and is obtained from patient by containing the array with the oligonucleotides of the partial complementarity of the genetic marker of deduction in method
MRNA the or cDNA sample of sample is contacted.With these methods, some recent reports have identified the bladder of a variety of suppositions
Cancer marker.But array technique is in contrast non-quantitation and variation is larger.
To can indicate that blood existing for bladder cancer or urine markers carry out fluorogenic quantitative detection and provide for improving this
A kind of potential method of disease detection.Special cancer marker may also provide for monitoring disease process and guarantee to be monitored outer
The method of the effect of section, radiation and chemotherapy treatment.Such as by liver detoxification in conjunction with glucuronic acid, the carcinogen of inactivation can
β-GRS cracking reduction, discharges carcinogenic movable part again in being urinated, and causes bladder cancer, and therefore, β-GRS increases mark in urine
Will has the trend that bladder cancer occurs；The pathological grading of NAG activity and tumor of bladder has certain relationship, and tumor of bladder classification is got over
Height, growth is faster, and outer peripheral portion blood supply is poorer, easier necrosis, therefore NAG is also corresponding higher in urinating；HOXA13, UBE2C can
In bladder cancer tissues and other tumor tissues height or constantly accumulate, to HOXA13, UBE2C in urine carry out detection and
The sensibility and specificity of bladder cancer detection quantitatively can be improved.But in the prior art for bladder cancer, still remaining can
The problems such as marker sensibility and specificity is insufficient, fluorogenic quantitative detection primer specificity is poor, quantitative detection sensitivity is low.
Summary of the invention
For technological gap in the prior art, the object of the present invention is to provide a kind of specific primers for detecting bladder cancer
The cystoscopy not being easily accepted by with kit and its detection method, this method without relying on expensive, cumbersome and usual patient, directly
It connects and carries out fluorogenic quantitative detection, detection sensitivity height, high specificity for urine or tissue markers.
In order to achieve the above object, the present invention is achieved through the following technical solutions ground:
The first purpose of this invention is to propose one group of specific primer and probe for being used to detect bladder cancer, including β-
GRS gene magnification primer and probe, NAG gene magnification primer and probe, HOXA13 gene magnification primer and probe, UBE2C base
Gene-amplification primer and probe；β-GRS gene magnification primer the sequence is as shown in SEQ ID NO:1-2, and β-GRS gene probe is such as
Shown in SEQ ID NO:3；The NAG gene magnification primer sequence is as shown in SEQ ID NO:4-5, NAG gene probe such as SEQ
Shown in ID NO:6；The HOXA13 gene magnification primer sequence is as shown in SEQ ID NO:7-8, HOXA13 gene probe such as SEQ
Shown in ID NO:9；The UBE2C gene magnification primer sequence is as shown in SEQ ID NO:10-11, UBE2C gene probe such as SEQ
Shown in ID NO:12.
It further, further include reference gene primer pair and TaqMan probe, the reference gene primer sequence such as SEQ
Shown in ID NO:13-14, the TaqMan probe sequence of the reference gene is as shown in SEQ ID NO:15.
Second object of the present invention is to propose a kind of kit for detecting bladder cancer, and the kit includes for examining
The specific primer and probe for surveying bladder cancer, specifically include β-GRS gene magnification primer and probe, NAG gene magnification primer and
Probe, HOXA13 gene magnification primer and probe, UBE2C gene magnification primer and probe；β-GRS gene magnification primer the sequence
Column are as shown in SEQ ID NO:1-2, and β-GRS gene probe is as shown in SEQ ID NO:3；The NAG gene magnification primer sequence
As shown in SEQ ID NO:4-5, NAG gene probe is as shown in SEQ ID NO:6；The HOXA13 gene magnification primer sequence is such as
Shown in SEQ ID NO:7-8, HOXA13 gene probe is as shown in SEQ ID NO:9；The UBE2C gene magnification primer sequence is such as
Shown in SEQ ID NO:10-11, UBE2C gene probe is as shown in SEQ ID NO:12.
Further, the kit further includes reference gene primer pair and TaqMan probe, the reference gene primer
Sequence is as shown in SEQ ID NO:13-14, and the TaqMan probe sequence of the reference gene is as shown in SEQ ID NO:15.
Further, the kit further includes external control gene primer and probe, the external control gene primer sequence such as SEQ
Shown in ID NO:16-17, the external control gene order such as SEQ ID NO:18.
Further, the kit further includes PCR MIX reaction solution and deionized water.
Further, the PCRMIX reaction solution includes Taq archaeal dna polymerase, buffer, dNTPs and MgCl2；It is described
The concentration of Taq archaeal dna polymerase is 5U/ μ l；The dNTPs includes dATP, dGTP, dCTP, dTTP, their concentration is 2.5
μM；The MgCl2Concentration be 250mM.
Third object of the present invention is to propose that a kind of detection method of bladder cancer, this method are wanted in 3-7 using right
The expression of β-GRS, at least one marker of NAG, HOXA13, UBE2C in described in any item kit detection tissues or urine
Amount, includes the following steps:
(1) reaction solution I, reaction solution II, reaction solution III, reaction solution IV, reaction solution V are prepared, the reaction solution I includes PCR
Buffer, archaeal dna polymerase, primer mixture SEQ ID NO:1-2, probe SEQ ID NO:3, internal reference mixture SEQ ID NO:
13-15；The reaction solution II includes PCR buffer, archaeal dna polymerase, primer mixture SEQ ID NO:4-6, probe SEQ ID
NO:6, internal reference mixture SEQ ID NO:13-15；The reaction solution III includes PCR buffer, archaeal dna polymerase, primer mixture
SEQ ID NO:7-8, probe SEQ ID NO:9, internal reference mixture SEQ ID NO:13-15；The reaction solution IV is slow including PCR
Fliud flushing, archaeal dna polymerase, primer mixture SEQ ID NO:10-11, probe SEQ ID NO:12, internal reference mixture SEQ ID
NO:13-15；The reaction solution V includes PCR buffer, archaeal dna polymerase, external control mixture SEQ ID NO:16-18；
(2) extraction purification sample to be tested DNA, and concentration and purity testing are carried out to the DNA of extraction；The sample to be tested is
Bladder cast-off cells in urine；
(4) DNA sample of known concentration in step (2) is diluted to 2~20ng/ μ l with TE；
(4) 2 μ l steps are respectively added into reaction solution I, reaction solution II, reaction solution III, reaction solution IV, reaction solution V respectively
(3) spare sample DNA has been diluted in as template；
(5) PCR program, real-time fluorescence PCR detection are set；
(5) Analysis of test results.
Further, the final concentration of each primer is 100-400nm in reaction solution in the step (1), each
The final concentration of probe is 100-400nm.
Further, fluorescence detection channel selects FAM, VIC sense channel in the step (5), wherein mutation and external control
The channel channel selecting FAM, internal reference select the channel VIC.
Compared with prior art, the advantages of the present invention are as follows:
(1) technical solution of the present invention is by the high primer and fluorescence probe of design specificity, be reconfigured to it is easy to use,
The reliable kit of testing result designs scientific and reasonable PCR reaction system, so that the present invention has, detection speed is fast, examines
Survey the advantages that sensibility is high and specificity is good；The wing that this method is not easily accepted by without relying on expensive, cumbersome and usual patient simultaneously
Guang spectroscopy carries out fluorogenic quantitative detection directly against urine or tissue markers, easy to operate；Therefore this method is particularly suitable for
The popularization and application in clinical examination work.
(2) the external control reaction solution that the present invention designs can accurately reflect template quality to be measured, prevent because template quantity is insufficient or suppression
False negative result caused by preparation is too strong, can also monitor the false positive that may cause because template quantity is excessively high, while in abrupt climatic change
It is added to internal control primer probe in reaction solution, effectively false negative can be avoided to occur by the amplification of internal reference, finally ensure reagent
Box testing result is accurate.
Specific embodiment
Applicant is in conjunction with specific embodiments described in further details technical solution of the present invention below, so that this field
Technical staff may be better understood the present invention and can be practiced, but range is claimed not as the present invention in illustrated embodiment
Restriction.
Embodiment 1:
The invention proposes one group for detecting the specific primer and probe of bladder cancer, including β-GRS gene amplification primer
Object and probe, NAG gene magnification primer and probe, HOXA13 gene magnification primer and probe, UBE2C gene magnification primer and spy
Needle, reference gene primer pair and TaqMan probe；β-GRS gene magnification primer sequence is as shown in SEQ ID NO:1-2, β-GRS
Gene probe is as shown in SEQ ID NO:3；NAG gene magnification primer sequence is as shown in SEQ ID NO:4-5, NAG gene probe
As shown in SEQ ID NO:6；HOXA13 gene magnification primer sequence is as shown in SEQ ID NO:7-8, and HOXA13 gene probe is such as
Shown in SEQ ID NO:9；UBE2C gene magnification primer sequence is as shown in SEQ ID NO:10-11, UBE2C gene probe such as SEQ
Shown in ID NO:12；Reference gene primer sequence is as shown in SEQ ID NO:13-14, reference gene probe sequence such as SEQ ID
Shown in NO:15.Wherein detection probe is by fluorescent marker, and 5 ' ends are marked with reporter group, and 3 ' ends are marked with non-glimmering
Optical quenching group, the detection probe in the present embodiment are as follows:
1. β-GRS detection probe SEQ ID NO:3
5`-FAM-aggagataaatggaaacaa-MGB-3`
2. NAG detection probe SEQ ID NO:6
5`-FAM-caggctgagttttggtc-MGB-3`
3. HOXA13 detection probe SEQ ID NO:9
5`-FAM-taaaggtaacaacaaaggtatatt-MGB-3`
4. UBE2C detection probe SEQ ID NO:12
5`-FAM-atctacttggagcctgcaccattggag-BHQ1-3`
5. reference gene detection probe SEQ ID NO15:
5`-FAM-tacccattctctgcttgacagtcctgc-BHQ1-3`
The present invention also proposes that a kind of kit for detecting bladder cancer, the kit include above-mentioned one group for detecting bladder
The specific primer and probe of cancer, while further including external control gene primer and probe, PCR MIX reaction solution and deionized water,
Middle external control gene primer sequence is as shown in SEQ ID NO:16-17, external control genetic test probe sequence such as SEQ ID NO:18,
For middle detection probe by fluorescent marker, 5 ' ends are marked with reporter group, and 3 ' ends are marked with non-fluorescence quenching group, this implementation
Detection probe in example is as follows:
SEQ ID NO:18 5`-FAM-agacttaatagtccgcgtgggcgacg-BHQ1-3`
Embodiment 2: being detection sample, the foundation of bladder cancer detection method with urine
It is normal to bladder cast-off cells in 15 transitional cell bladder carcinomas and 2 tested using the kit in embodiment 1
Bladder cast-off cells detect in person's urine, which has obtained knowing and agreeing to for measured.This 15 patients pass through
Clinical cystoscope (goldstandard) method is diagnosed, and wherein non-infiltration bladder cancer patients 7 (FJ-01~FJ-7), wellability
Bladder cancer patients 8 (J-01~J-8).Normal subjects' number is E-01~E-02, and the specific detection method is as follows:
1. preparing reaction solution
Reaction solution I, reaction solution II, reaction solution III, reaction solution IV, reaction solution V are prepared, wherein reaction solution I is slow including PCR
Fliud flushing, archaeal dna polymerase, primer mixture SEQ ID NO:1-2, probe SEQ ID NO:3, internal reference mixture SEQ ID NO:
13-15；Reaction solution II includes PCR buffer, archaeal dna polymerase, primer mixture SEQ ID NO:4-6, probe SEQ ID NO:
6, internal reference mixture SEQ ID NO:13-15；Reaction solution III includes PCR buffer, archaeal dna polymerase, primer mixture SEQ ID
NO:7-8, probe SEQ ID NO:9, internal reference mixture SEQ ID NO:13-15；Reaction solution IV is poly- including PCR buffer, DNA
Synthase, primer mixture SEQ ID NO:10-11, probe SEQ ID NO:12, internal reference mixture SEQ ID NO:13-15；Instead
Answering liquid V includes PCR buffer, archaeal dna polymerase, external control mixture SEQ ID NO:16-18.Negative quality-control product is 18,000,000 or more
Nuclease-free water.
Wherein the system of PCR buffer includes: 55 × Buffer of μ L (TrispH8.0, KCl), 0.25 in the first reaction
μLMgCl2(250mM), 2 μ L dNTP Mix (10mM), 12.55 μ L deionized waters；Primer, spy in adding in each reaction solution
(10 μM) of needle mixed liquor are 3 μ L, each 1 μ L of upstream and downstream primer and probe, 0.2 μ L of chemical modification hot start Taq polymerase (5U/ μ l).
In the present embodiment be 15 samples to be tested, therefore need to prepare 15 groups of reaction solutions (every group of reaction solution include reaction
Liquid I, reaction solution II, reaction solution III, reaction solution IV, reaction solution V)
2. extraction purification sample to be tested DNA, and concentration and purity testing are carried out to the DNA of extraction
(1) urine specimen acquisition and centrifugation: acquiring the midstream urine 50mL in each patient's urina sanguinis, average to two 50mL
Centrifuge tube, immediately with 2000g centrifugation 20 minutes, remove supernatant.
(2) urine precipitating extract DNA: according to DNeasy Blood&Tissue Kit (producer: Qiagen, article No.:
69504) kit specification extracts urine and precipitates DNA, Qubit quantitative detection DNA concentration.DNA concentration need to be more than or equal to
2.5ng/ μ L, as a result as follows:
Table 1
| Catalogue number(Cat.No.) | Concentration (ng/ μ l) | 260/280 |
| FJ-01 | 84.5 | 1.96 |
| FJ-02 | 75.0 | 1.82 |
| FJ-03 | 42.6 | 1.72 |
| FJ-04 | 92.2 | 1.80 |
| FJ-05 | 55.4 | 1.75 |
| FJ-06 | 82.5 | 1.87 |
| FJ-07 | 75.0 | 1.82 |
| J-01 | 40.6 | 1.65 |
| J-02 | 92.2 | 1.80 |
| J-03 | 65.4 | 1.73 |
| J-04 | 87.5 | 1.85 |
| J-05 | 63.0 | 1.74 |
| J-06 | 42.6 | 1.72 |
| J-07 | 91.2 | 1.68 |
| J-08 | 68.4 | 1.75 |
| E-01 | 63.2 | 1.85 |
| E-02 | 92.7 | 1.72 |
3. the DNA sample of known concentration is diluted to 2~20ng/ μ L with TE
4. 2 μ L are respectively added into reaction solution I, reaction solution II, reaction solution III, reaction solution IV, reaction solution V respectively to have diluted
Spare sample DNA carries out PCR amplification as template.
5. PCR program, real-time fluorescence PCR detection is arranged
Fluorescence detection channel selects FAM, VIC/HEX sense channel, wherein mutation and the channel external control channel selecting FAM, interior
The channel VIC is selected in participation in the election.
PCR program are as follows: 52 DEG C of 30min, (95 DEG C of 15s, 60 DEG C of 40s) × 45cycles, 60 DEG C are collected simultaneously 2 kinds of fluorescence letters
Number.
6. Analysis of test results
(1) criterion
1. negative quality-control product: the channel FAM, the channel VIC are straight line or slight oblique line without amplification curve or amplification curve,
Without obvious Exponential growth stage, value >=51 Ct.
2. external control reaction solution: FAM channel C t value 36≤Ct≤45, sample DNA additional amount are moderate；FAM channel C t value > 45,
Sample genomic dna extraction is second-rate, and only the higher sample of mutant DNA content, which can detecte out, is mutated, mutant DNA content
Sample is extracted in lower sample suggestion again, increases genomic DNA additional amount；FAM channel C t value < 36, sample additional amount mistake
It is high, it is proposed that be detected again after dilution.
3. sample mutation type criterion
Table 2
(2) amplification is shown:
The channel negative quality-control product FAM, VIC without amplification curve, external control reaction solution FAM channel C t value 36≤Ct≤
Between 45, illustrates that sample Quality Control is qualified, pattern detection result can be determined according to criterion, in 17 samples, E-01-
02 without amplification curve, and showing the sample, there is no markers relevant to bladder cancer；4 labels of FJ-01-07, J-01-08
Quality testing sample has amplification curve, and specific testing result is as shown in the table:
Table 3
| Catalogue number(Cat.No.) | β-GRS | NAG | HOXA13 | UBE2C |
| FJ-01 | 37.9 | 45.2 | 46.3 | 38.9 |
| FJ-02 | 38.6 | 41.3 | 45.2 | 39.7 |
| FJ-03 | 37.6 | 45.6 | 44.9 | 39.6 |
| FJ-04 | 36.9 | 45.2 | 48.6 | 41.3 |
| FJ-05 | 45.6 | 44.3 | 48.6 | 44.6 |
| FJ-06 | 44.9 | 48.9 | 45.2 | 44.5 |
| FJ-07 | 49.5 | 44.8 | 44.3 | 41.9 |
| J-01 | 50.1 | 45.6 | 48.5 | 45.6 |
| J-02 | 48.6 | 44.1 | 45.6 | 44.3 |
| J-03 | 44.6 | 41.3 | 44.9 | 39.5 |
| J-04 | 41.3 | 40.2 | 48.7 | 38.7 |
| J-05 | 42.6 | 40.9 | 45.6 | 41.2 |
| J-06 | 44.2 | 50.2 | 45.0 | 40.9 |
| J-07 | 38.9 | 41.6 | 44.6 | 42.3 |
| J-08 | 39.7 | 48.9 | 48.7 | 50.2 |
It can be seen that in 15 transitional cell bladder carcinomas that 4 kinds of markers are equal in bladder cast-off cells from the data in upper table
In the presence of consistent with legitimate reading.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Sequence table
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Claims (10)
1. one group for detecting the specific primer and probe of bladder cancer, which is characterized in that including β-GRS gene magnification primer and
Probe, NAG gene magnification primer and probe, HOXA13 gene magnification primer and probe, UBE2C gene magnification primer and probe；
β-GRS gene magnification primer the sequence is as shown in SEQ ID NO:1-2, and β-GRS gene probe is as shown in SEQ ID NO:3；
The NAG gene magnification primer sequence is as shown in SEQ ID NO:4-5, and NAG gene probe is as shown in SEQ ID NO:6；It is described
HOXA13 gene magnification primer sequence is as shown in SEQ ID NO:7-8, and HOXA13 gene probe is as shown in SEQ ID NO:9；Institute
UBE2C gene magnification primer sequence is stated as shown in SEQ ID NO:10-11, UBE2C gene probe is as shown in SEQ ID NO:12.
2. one group according to claim 1 for detecting the specific primer and probe of bladder cancer, which is characterized in that also wrap
Reference gene primer pair and TaqMan probe are included, the reference gene primer sequence is described interior as shown in SEQ ID NO:13-14
Join the TaqMan probe sequence of gene as shown in SEQ ID NO:15.
3. a kind of kit for detecting bladder cancer, which is characterized in that the kit includes the specificity for detecting bladder cancer
Primer and probe, specifically includes β-GRS gene magnification primer and probe, NAG gene magnification primer and probe, HOXA13 gene expand
Increase primer and probe, UBE2C gene magnification primer and probe；β-GRS gene magnification primer the sequence such as SEQ ID NO:1-2
Shown, β-GRS gene probe is as shown in SEQ ID NO:3；The NAG gene magnification primer sequence such as SEQ ID NO:4-5 institute
Show, NAG gene probe is as shown in SEQ ID NO:6；The HOXA13 gene magnification primer sequence such as SEQ ID NO:7-8 institute
Show, HOXA13 gene probe is as shown in SEQ ID NO:9；The UBE2C gene magnification primer sequence such as SEQ ID NO:10-11
Shown, UBE2C gene probe is as shown in SEQ ID NO:12.
4. a kind of kit for detecting bladder cancer according to claim 3, which is characterized in that the kit further includes interior
Join gene primer to and TaqMan probe, the reference gene primer sequence is as shown in SEQ ID NO:13-14, the internal reference base
The TaqMan probe sequence of cause is as shown in SEQ ID NO:15.
5. a kind of kit for detecting bladder cancer according to claim 3, which is characterized in that the kit further includes outer
Gene primer and probe are controlled, the external control gene primer sequence is as shown in SEQ ID NO:16-17, and the external control gene order is such as
SEQ ID NO:18。
6. a kind of kit for detecting bladder cancer according to claim 3, which is characterized in that the kit further includes
PCR MIX reaction solution and deionized water.
7. a kind of kit for detecting bladder cancer according to claim 6, which is characterized in that the PCR MIX reaction solution
Including Taq archaeal dna polymerase, buffer, dNTPs and MgCl2；The concentration of the Taq archaeal dna polymerase is 5U/ μ l；It is described
DNTPs includes dATP, dGTP, dCTP, dTTP, their concentration is 2.5 μM；The MgCl2Concentration be 250mM.
8. a kind of detection method of bladder cancer, which is characterized in that this method wants kit described in any one of 3-7 using right
The expression quantity for detecting β-GRS, at least one marker of NAG, HOXA13, UBE2C in tissue or urine, includes the following steps:
(1) reaction solution I, reaction solution II, reaction solution III, reaction solution IV, reaction solution V are prepared, the reaction solution I is buffered including PCR
Liquid, archaeal dna polymerase, primer mixture SEQ ID NO:1-2, probe SEQ ID NO:3, internal reference mixture SEQ ID NO:13-
15；The reaction solution II includes PCR buffer, archaeal dna polymerase, primer mixture SEQ ID NO:4-6, probe SEQ ID NO:
6, internal reference mixture SEQ ID NO:13-15；The reaction solution III includes PCR buffer, archaeal dna polymerase, primer mixture SEQ
ID NO:7-8, probe SEQ ID NO:9, internal reference mixture SEQ ID NO:13-15；The reaction solution IV is buffered including PCR
Liquid, archaeal dna polymerase, primer mixture SEQ ID NO:10-11, probe SEQ ID NO:12, internal reference mixture SEQ ID NO:
13-15；The reaction solution V includes PCR buffer, archaeal dna polymerase, external control mixture SEQ ID NO:16-18；
(2) extraction purification sample to be tested DNA, and concentration and purity testing are carried out to the DNA of extraction；The sample to be tested is urine
Middle bladder cast-off cells；
(3) DNA sample of known concentration in step (2) is diluted to 2~20ng/ μ l with TE；
(4) it is respectively added into reaction solution I, reaction solution II, reaction solution III, reaction solution IV, reaction solution V in 2 μ l steps (3) respectively
Spare sample DNA has been diluted as template；
(5) PCR program, real-time fluorescence PCR detection are set；
(6) Analysis of test results.
9. a kind of detection method of bladder cancer according to claim 8, which is characterized in that reaction solution in the step (1)
In the final concentration of each primer be 100-400nm, the final concentration of each probe is 100-400nm.
10. a kind of detection method of bladder cancer according to claim 8, which is characterized in that fluorescence is examined in the step (5)
Channel selecting FAM, VIC sense channel is surveyed, wherein mutation and the channel external control channel selecting FAM, internal reference selects the channel VIC.
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| CN111154879A (en) * | 2020-03-06 | 2020-05-15 | 北京恩泽康泰生物科技有限公司 | Biomarker and kit for bladder cancer diagnosis or recurrence monitoring |
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Application publication date: 20181221 |