CN108611417A - A kind of specific expressed collection of illustrative plates and analysis system for Diagnosis of Bladder - Google Patents
A kind of specific expressed collection of illustrative plates and analysis system for Diagnosis of Bladder 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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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
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses a kind of early stage carcinoma of urinary bladder urine miRNA specifically expressings spectrum, 423 5p of miR lowered by miR 126 5p of 22 3p, miR 375, miR and expression of up-regulated expression are constituted.The reverse transcriptase primer and detection primer of specifically expressing miRNAS in the early stage carcinoma of urinary bladder urine miRNA specifically expressings spectrum, as shown in SEQ ID NO 1~9.The present invention also provides a kind of carcinoma of urinary bladder miRNAS specifically expressings to compose testing and analysis system, which has higher diagnostic for early stage carcinoma of urinary bladder, is higher than traditional Urine exfoliated cells.
Description
Technical field
The present invention relates to technical field of molecular biological detection, and in particular to a kind of specific table for Diagnosis of Bladder
Up to collection of illustrative plates and analysis system.
Background technology
Carcinoma of urinary bladder is the most common malignant tumour of urinary system, and incidence and the death rate worldwide occupy uropoiesis
System tumor is the first, and has the tendency that rising in recent years, and about 75% patient is non-Myometrial involvement carcinoma of urinary bladder in diagnosis, is had
Still recurrence rate is also very high for very high survival rate, and remaining about 25% patient is exactly aggressive carcinoma of urinary bladder, there is very high death
Rate is required for thorough operative treatment mostly.Therefore, improve early stage carcinoma of urinary bladder recall rate for bladder cancer patients treatment and
Prognosis has very great meaning.Main method currently used for Diagnosis of Bladder has exfoliative cytology inspection, cystoscopy
It looks into and imageological examination etc..Although cystoscopy has higher detection efficiency, but it is invasive inspection, is not only taken
And very painful experience is brought to patient, therefore it is not suitable for use in generaI investigation.Although exfoliative cytology inspection has very high
Still its sensitivity is very low for specificity, especially for the tumour of low classification, and the influence of easy examined person's subjectivity.CT and ultrasound
It is not easy to find lesion smaller in bladder, there is certain limitation to the discovery of early stage carcinoma of urinary bladder.Therefore we need to find
Specificity and marker during sensitivity is all high and marker such as urine of convenient material drawing are used to find early stage carcinoma of urinary bladder, are
The diagnoses and treatment and prognosis of carcinoma of urinary bladder provide foundation.
MicroRNA (miRNAs) be it is a kind of be distributed widely in it is Eukaryotic highly conserved, non-coding albumen single-stranded
MicroRNA s, in post-transcriptional level by adjusting the expression of target gene to target mrna degradation or Translational repression to thin
The functions such as born of the same parents' differentiation, growth, metabolism and apoptosis regulate and control.Previous research confirms that miRNAs unconventionality expressions in tumour have
Increasing some a bit reduces and there is close contact with the generation of tumour, development, transfer and prognosis.Recent study discovery,
There is some miRNAs in urine is found not by miRNAs express spectras in comparative analysis tumor patient and control group urine
There is its specific miRNAs express spectra with tumour, new foundation is provided for the non-invasive early diagnosis of tumour.Therefore, really
Determine carcinoma of urinary bladder miRNAs specifically expressings spectrum, screen the miRNAs of differential expression, establish carcinoma of urinary bladder urine miRNAs diagnostic models,
Help to realize the early diagnosis to carcinoma of urinary bladder.
Invention content
Present invention is primarily intended to provide a kind of specific expressed collection of illustrative plates and analysis system for Diagnosis of Bladder.
To achieve the above object, the present invention uses following technical scheme：
One of the object of the invention provides a kind of carcinoma of urinary bladder miRNAS specifically expressings spectrum, by the miR-22-3p raised, miR-
375, miR-126-5p are constituted with the miR-423-5p that expression is lowered.
The two of the object of the invention provide one group of primer for detecting carcinoma of urinary bladder miRNAS specifically expressings spectrum described above,
Including following primer：
(1) sequence of miR-22-3p reverse transcriptions forward primer such as SEQ ID NO：Shown in 1, miR-22-3p detections are positive
The sequence of primer such as SEQ ID NO：Shown in 2；
(2) sequence of the reverse transcription forward primer of miR-375 such as SEQ ID NO：Shown in 3, miR-375 detections forward direction is drawn
The sequence of object such as SEQ ID NO：Shown in 4；
(3) sequence of the reverse transcription forward primer of miR-126-5p such as SEQ ID NO：Shown in 5, the inspection of miR-126-5p
Survey the sequence such as SEQ ID NO of forward primer：Shown in 6；
(4) sequence of the reverse transcription forward primer of miR-423-5p such as SEQ ID NO：Shown in 7, the inspection of miR-423-5p
Survey the sequence such as SEQ ID NO of forward primer：Shown in 8；
The general inverse detection primer of (5) four kinds of miRNAs such as SEQ ID NO：Shown in 9.
The three of the object of the invention provide more than one described primers in preparing detection carcinoma of urinary bladder reagent and/or kit
Application.
The four of the object of the invention provide a kind of kit or biochip of detection carcinoma of urinary bladder, including SEQ ID NO：1~
SEQ ID NO：9 primers.
The five of the object of the invention provide above-described kit or biochip in preparing detection carcinoma of urinary bladder reagent
Using.
The six of the object of the invention provide more than one carcinoma of urinary bladder miRNAS specifically expressings spectrum testing and analysis systems, packet
Include quantitative detection module, input module, analysis module and output module.
Further, the quantitative detection module includes；It is described to determine detection module using in primer detection urine described above
MiR-22-3p, miR-375, miR-126-5p and miR-423-5p expression quantity, using 2-△△CtMethod is analyzed respectively, is obtained
MiR-22-3p expresses numerical quantity A22, miR-375 and expresses numerical quantity A375, miR-126-5p expression numerical quantity A126 and miR-
423-5p expresses numerical quantity A423.
Further, the analysis module includes that logit models calculate and ROC curve analysis；The logit models are：
Logit (P=BC)=1.0274-0.2828*A22-0.1722*A375-0.1146*A126+0.2907*A423；
The A22 is that miR-22-3p expresses numerical quantity, and A375 is that miR-375 expresses numerical quantity, and A126 is miR-126-5p expression quantity
Numerical value and A423 are that miR-423-5p expresses numerical quantity.
The seven of the object of the invention provide the analysis system operation method described in more than one, include the following steps：
(1) using quantitative detection module, miR-22-3p expression numerical quantities A22, miR-375 is obtained and expresses numerical quantity A375,
MiR-126-5p expresses numerical quantity A126 and miR-423-5p expression numerical quantity A423；
(2) A22, A375, A126 and A423 numerical value obtained by above-mentioned steps (1) are input into input module；
(3) analysis module carries out operational analysis；
(4) output module output 1 or 0；Wherein 0 represents carcinoma of urinary bladder detection feminine gender；1 represents the carcinoma of urinary bladder detection positive.
The present invention is primarily based on non-invasive bladder cancer patient, aggressive bladder cancer patients and the health of routine clinical collection
The Technology Ways of collator's urine specimen screening differential expression miRNAs, using " libraries miRNAs cDNA+library expands in advance+
The research strategy of Miseq deep sequencings " has carried out miRNAs express spectras in three groups of urine specimens to compare analysis, and screening altogether goes out
13 differential expression miRNAs, sequence are shown in Table 1.Then reverse transcription-real time fluorescence quantifying PCR method of SYBR Green is used,
Gene expression abundances of the above-mentioned 13 differential expression miRNAs in bladder cancer patients and control urine is verified, it is final to determine
With carcinoma of urinary bladder relevant 4 miRNAs, include the miR-22-3p of up-regulated expression, miR-375, miR-126-5p and lowers
miR-423-5p.And autonomous Design its reverse transcription primer and Direct/Reverse detection primer, specifically it is shown in Table 2.In turn, one is provided
Kind carcinoma of urinary bladder miRNAS specifically expressings compose testing and analysis system, including quantitative detection module, input module, analysis module and output
Module, the analysis module provide following calculation formula using multiple logistic regression method：
Logit (P=BC)=1.0274-0.2828*A22-0.1722*A375-0.1146*A126+0.2907*A423；
The A22 is that miR-22-3p expresses numerical quantity, and A375 is that miR-375 expresses numerical quantity, and A126 is miR-126-5p expression quantity
Numerical value and A423 are that miR-423-5p expresses numerical quantity.
The advantageous effect that the present invention obtains：
The present invention is constituted with the miR-423-5p that the miR-22-3p raised, miR-375, miR-126-5p and expression are lowered
The specific expressed collection of illustrative plates for detecting carcinoma of urinary bladder establishes testing and analysis system for detecting carcinoma of urinary bladder, is to Diagnosis of Bladder AUC
0.922, sensibility 85.9%, specificity is 84.4%, higher than traditional Urine exfoliated cells inspection (AUC 0.617,
Sensibility is 26.6%, 96.9%) specificity is.
Description of the drawings
Fig. 1：Screening stage：Expression of four kinds of miRNAs in carcinoma of urinary bladder group and control group；
Fig. 2：Screening stage：Diagnosis ROC curves of four kinds of miRNAs to carcinoma of urinary bladder；
Fig. 3：Screening stage：Diagnosis ROC curve of the diagnostic model (panel) of foundation to carcinoma of urinary bladder；
Fig. 4：Qualify Phase：Expression of four kinds of miRNAs in carcinoma of urinary bladder group and control group；
Fig. 5：Qualify Phase：Diagnosis ROC curves of four kinds of miRNAs to carcinoma of urinary bladder；
Fig. 6：Qualify Phase：The diagnosis ROC of the diagnostic model (panel) of foundation and Urine exfoliated cells to carcinoma of urinary bladder
Curve；
Fig. 7：Qualify Phase：Diagnosis ROC curve of the diagnostic model (panel) of foundation to Staging Bladder；
Abscissa in the above figure：Specificity, ordinate：Sensibility.
Specific implementation mode
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another
It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific implementation mode, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation and/or combination thereof.
One of the object of the invention provides a kind of carcinoma of urinary bladder miRNAS specifically expressings spectrum, by the miR-22-3p raised, miR-
375, miR-126-5p are constituted with the miR-423-5p that expression is lowered.
Further, the miRNAS comes from urine.
The two of the object of the invention provide one group of primer for detecting carcinoma of urinary bladder miRNAS specifically expressings spectrum described above,
Including following primer：
(1) sequence of miR-22-3p reverse transcriptions forward primer such as SEQ ID NO：Shown in 1, miR-22-3p detections are positive
The sequence of primer such as SEQ ID NO：Shown in 2；
(2) sequence of the reverse transcription forward primer of miR-375 such as SEQ ID NO：Shown in 3, miR-375 detections forward direction is drawn
The sequence of object such as SEQ ID NO：Shown in 4；
(3) sequence of the reverse transcription forward primer of miR-126-5p such as SEQ ID NO：Shown in 5, the inspection of miR-126-5p
Survey the sequence such as SEQ ID NO of forward primer：Shown in 6；
(4) sequence of the reverse transcription forward primer of miR-423-5p such as SEQ ID NO：Shown in 7, the inspection of miR-423-5p
Survey the sequence such as SEQ ID NO of forward primer：Shown in 8；
The general inverse detection primer of (5) four kinds of miRNAs such as SEQ ID NO：Shown in 9.
The three of the object of the invention provide more than one described primers in preparing detection carcinoma of urinary bladder reagent and/or kit
Application.
The four of the object of the invention provide a kind of kit or biochip of detection carcinoma of urinary bladder, including SEQ ID NO：1~
SEQ ID NO：9 primers.
The five of the object of the invention provide above-described kit or biochip in preparing detection carcinoma of urinary bladder reagent
Using.
The six of the object of the invention provide more than one carcinoma of urinary bladder miRNAS specifically expressings spectrum testing and analysis systems, packet
Include quantitative detection module, input module, analysis module and output module.
Further, the quantitative detection module includes；It is described to determine detection module using in primer detection urine described above
MiR-22-3p, miR-375, miR-126-5p and miR-423-5p expression quantity, using 2-△△CtMethod is analyzed respectively, is obtained
MiR-22-3p expresses numerical quantity A22, miR-375 and expresses numerical quantity A375, miR-126-5p expression numerical quantity A126 and miR-
423-5p expresses numerical quantity A423.
Further, the analysis module includes that logit models calculate and ROC curve analysis；The logit models are：
Logit (P=BC)=1.0274-0.2828*A22-0.1722*A375-0.1146*A126+0.2907*A423；
The A22 is that miR-22-3p expresses numerical quantity, and A375 is that miR-375 expresses numerical quantity, and A126 is miR-126-5p expression quantity
Numerical value and A423 are that miR-423-5p expresses numerical quantity.
The seven of the object of the invention provide the analysis system operation method described in more than one, include the following steps：
(1) using quantitative detection module, miR-22-3p expression numerical quantities A22, miR-375 is obtained and expresses numerical quantity A375,
MiR-126-5p expresses numerical quantity A126 and miR-423-5p expression numerical quantity A423；
(2) A22, A375, A126 and A423 numerical value obtained by above-mentioned steps (1) are input into input module；
(3) analysis module carries out operational analysis；
(4) output module output 1 or 0；Wherein 0 represents carcinoma of urinary bladder detection feminine gender；1 represents the carcinoma of urinary bladder detection positive.
In order to enable those skilled in the art can clearly understand technical scheme of the present invention, below with reference to tool
The embodiment of the body technical solution that the present invention will be described in detail.
Embodiment one
1, research object
Collator of the urine specimen from bladder cancer patients and eliminating excreting excrement system tumour, materials pass through the same of subject
Meaning.All bladder cancer patients are all from the inpatient of Shandong hospital Urology Surgery, are all the patients receiving treatment that is admitted to hospital for the first time
And do not received any drug and operative treatment before, it is by stages postoperative pathological examination inspection or bladder with histological type
What spectroscopy confirmed.Include the urine specimen of 86 bladder cancer patients and 86 normal healthy controls in screening stage, in verification rank
Section includes the urine specimen of 64 bladder cancer patients and 64 normal healthy controls.These urine specimens are all not receive any hand
It is obtained before art or drug therapy.
2, collection of specimens
5ml urines are acquired, 4000 leave the heart 10 minutes under the conditions of having acquired in 2 hours at 4 DEG C, then in 4 DEG C of condition
Lower 12000 leave the extra cell fragment of heart removal in 15 minutes, supernatant are stored in -80 DEG C of refrigerator.
3, library construction and Miseq sequencings
Select miRCURYTM RNA Isolation kits to RNA in bladder cancer patients and collator's urine supernatant into
Row extraction.After RNA is concentrated, built using NEBNextMultiplex Small RNA Library Prep Set kits
Vertical cDNA library.Concentration in library and fragment length are completed using Agilent 2100Bioanalyzer detection structures, are used in combination
KAPA bis- generations sequencing library quantification kits detect molecular concentration in library.It finally carries out machine sequencing and carries out bioinformatics
Analysis.
4、RT-qPCR
Use One StepMiRNA cDNA Synthesis Kit Reverse Transcriptase kits (Takara
Company) for the above-mentioned mRNA of reverse transcription at cDNA, reverse transcription system 20ul includes 10 μ l of 2 × miRNA Reaction Buffer
3 μ l serum that of Mix2 μ l of miRNA Primescript RT Enzyme Mix, 2 0.1% BSA and of μ l of
3 μ l serum buffer of mixed with, are incubated 60 minutes 60 minutes, 85 DEG C of 5S, 4 DEG C under conditions of 37 DEG C, acquisition
CDNA 10000 leaves the heart 10 minutes again, then carries out PCR reactions, runs on 7500 real-time fluorescence quantitative PCR analyzers of ABI
(ABI companies).
PCR reaction systems (25 μ l)：
Template DNA：2ul
SYBR Premix Ex TaqⅡ：12.5μl
DyeⅡ：0.5μl
Sense primer (10 μM)：1μl
Downstream primer (10 μM)：1μl
Sterile water：8μl
Reaction condition is：95 DEG C of 30 seconds → 1 cycles；(95 DEG C 5 seconds, 57 DEG C 34 seconds) → 45 cycles；Addition dissolving is bent
Line.
Each sample is repeated three times, and miRNA expression quantity uses 2-△△CtIt is analyzed.
5, data processing and statistical analysis
The concentration difference for comparing miRNAS between non-matching group with Mann-Whitney U tests, uses he
Wilcoxon test more perioperatively match the expression quantity of miRNAS in urine specimen, are determined with COX recurrence independent
Predictive factor is drawn survivorship curve with Kaplan-Meier, is commented diagnostic with ROC curve and area under the curve AUC
Estimate, others are analyzed with 17.0 softwares of SPSS.
6, testing result
1) through Miseq sequencing analysis, we pick 23 miRNAs in carcinoma of urinary bladder group and healthy control group, 14
Up-regulation, 9 are lowered, and sequence is as shown in table 1.
The difference miRNAs sequences sifted out are sequenced in table 1
2) it is verified with RT-qPCR, with p<0.05 finally determines 4 in carcinoma of urinary bladder and normal healthy controls for inclusion criteria
The statistically significant miRNAs of differential expression in group, miR-22-3p, miR-375, miR-126-5p up-regulated expressions, miR-
423-5p expression is lowered, and detection primer is autonomous Design, primer (such as SEQ ID NO as shown in table 2：Shown in 1~9).
2 primer sequence of table
3) as can be seen from Figure 1 come, screening stage, miR-22-3p, miR-375, miR-126-5p expression is apparent to rise
Height, miR-423-5p expression are substantially reduced.The ROC curve of this 4 miRNAs diagnosing bladder cancers is established respectively, as shown in Fig. 2,
The AUC of miR-22-3p, miR-375, miR-126-5p, miR-423-5p are respectively 0.810,0.797,0.731 and 0.769.
4) the statistically significant miRNAs of this 4 differential expressions is substituted into Multivariate logistic regression analysis, and built
Vertical Combining diagnosis model.
Calculation formula is：
Logit (P=BC)=1.0274-0.2828*A22-0.1722*A375-0.1146*A126+0.2907*A423；
The A22 is that miR-22-3p expresses numerical quantity, and A375 is that miR-375 expresses numerical quantity, and A126 is miR-126-5p expression quantity
Numerical value and A423 are that miR-423-5p expresses numerical quantity, and ROC curve is shown in Fig. 3, AUC 0.913.
5) Qualify Phase, miR-22-3p, miR-375, miR-126-5p expression is significantly raised, and miR-423-5p expression is bright
Fig. 4 is shown in aobvious reduction.The ROC curve of this 4 miRNAs diagnosing bladder cancers is established respectively, as shown in figure 5, miR-22-3p, miR-
The AUC of 375, miR-126-5p, miR-423-5p are respectively 0.819,0.742,0.692 and 0.703.
6) it in Qualify Phase, is analyzed through ROC curve, this model is 0.922 to Diagnosis of Bladder AUC, and sensibility is
85.9%, specificity is 84.4%, checks that (AUC 0.617, sensibility are higher than traditional Urine exfoliated cells
26.6%, specificity is 96.9%, as shown in Figure 6.
7) the analyzing by stages to bladder cancer patients using the diagnostic model includes Ta, T1, T2-T4, diagnosis by stages
The AUC of Ta, T1, T2-T4 patient are respectively 0.886,0.903 and 0.962, as shown in Figure 7.Illustrate that the model can be realized to wing
Guang cancer early diagnoses.
SEQUENCE LISTING
<110>Shandong University The Second Hospital
<120>A kind of specific expressed collection of illustrative plates and analysis system for Diagnosis of Bladder
<130>
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 44
<212> DNA
<213>Artificial sequence
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<213>Artificial sequence
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acactccagc tgggaagctg ccagttgaag 30
<210> 3
<211> 44
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<213>Artificial sequence
<400> 3
ctcaactggt gtcgtggagt cggcaattca gttgagtcac gcga 44
<210> 4
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<213>Artificial sequence
<400> 4
acactccagc tgggtttgtt cgttcggctc 30
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<213>Artificial sequence
<400> 5
ctcaactggt gtcgtggagt cggcaattca gttgagcgcg tacc 44
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tggtgtcgtg gagtcg 16
Claims (10)
1. a kind of carcinoma of urinary bladder miRNAS specifically expressings spectrum, which is characterized in that by the miR-22-3p raised, miR-375, miR-
The miR-423-5p that 126-5p and expression are lowered is constituted.
2. the specific expressed spectrums of carcinoma of urinary bladder miRNAS as described in claim 1, which is characterized in that the miRNAS is from urine
Liquid.
3. one group of primer for requiring 1 carcinoma of urinary bladder miRNAS specifically expressings spectrum for test right, which is characterized in that including with
Lower primer：
(1) sequence of miR-22-3p reverse transcriptions forward primer such as SEQ ID NO：Shown in 1, miR-22-3p detects forward primer
Sequence such as SEQ ID NO：Shown in 2；
(2) sequence of the reverse transcription forward primer of miR-375 such as SEQ ID NO：Shown in 3, miR-375 detects the sequence of forward primer
Row such as SEQ ID NO：Shown in 4；
(3) sequence of the reverse transcription forward primer of miR-126-5p such as SEQ ID NO：Shown in 5, the detection of miR-126-5p is positive
The sequence of primer such as SEQ ID NO：Shown in 6；
(4) sequence of the reverse transcription forward primer of miR-423-5p such as SEQ ID NO：Shown in 7, the detection of miR-423-5p is positive
The sequence of primer such as SEQ ID NO：Shown in 8；
The general inverse detection primer of (5) four kinds of miRNAs such as SEQ ID NO：Shown in 9.
4. application of the primer as claimed in claim 3 in preparing detection carcinoma of urinary bladder reagent and/or kit.
5. a kind of kit or biochip of detection carcinoma of urinary bladder, which is characterized in that including SEQ ID NO：1~SEQ ID NO：
9 primers.
6. kit according to claim 5 or biochip are preparing the application in detecting carcinoma of urinary bladder reagent.
7. a kind of carcinoma of urinary bladder miRNAS specifically expressings as described in claim 1 compose testing and analysis system, it is characterised in that：Including fixed
Measure detection module, input module, analysis module and output module.
8. analysis system as claimed in claim 7, which is characterized in that the quantitative detection module includes；The regular inspection surveys mould
Block is expressed using miR-22-3p, miR-375, miR-126-5p and miR-423-5p in primer detection urine described in claim 3
Amount, using 2-△△CtMethod is analyzed respectively, is obtained miR-22-3p expression numerical quantities A22, miR-375 and is expressed numerical quantity A375,
MiR-126-5p expresses numerical quantity A126 and miR-423-5p expression numerical quantity A423.
9. analysis system as claimed in claim 7, which is characterized in that the analysis module includes the calculating of logit models and ROC
Tracing analysis；The logit models are：
Logit (P=BC)=1.0274-0.2828*A22-0.1722*A375-0.1146*A126+0.2907*A423；It is described
A22 be miR-22-3p express numerical quantity, A375 be miR-375 express numerical quantity, A126 be miR-126-5p express numerical quantity and
A423 is that miR-423-5p expresses numerical quantity.
10. analysis system operation method as claimed in claim 7, which is characterized in that include the following steps：
(1) using quantitative detection module, miR-22-3p expression numerical quantities A22, miR-375 is obtained and expresses numerical quantity A375, miR-
126-5p expresses numerical quantity A126 and miR-423-5p expression numerical quantity A423；
(2) A22, A375, A126 and A423 numerical value obtained by above-mentioned steps (1) are input into input module；
(3) analysis module carries out operational analysis；
(4) output module output 1 or 0；Wherein 0 represents carcinoma of urinary bladder detection feminine gender；1 represents the carcinoma of urinary bladder detection positive.
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Cited By (1)
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| CN110541030A (en) * | 2019-08-22 | 2019-12-06 | 广州医科大学附属肿瘤医院 | bladder cancer detection kit and application thereof |
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