CN105954389A - Diagnostic kit for bladder cancer and detection method based on human urine glycine, 3-phosphoglyceric acid and cytosine - Google Patents
Diagnostic kit for bladder cancer and detection method based on human urine glycine, 3-phosphoglyceric acid and cytosine Download PDFInfo
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- CN105954389A CN105954389A CN201610257966.2A CN201610257966A CN105954389A CN 105954389 A CN105954389 A CN 105954389A CN 201610257966 A CN201610257966 A CN 201610257966A CN 105954389 A CN105954389 A CN 105954389A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/025—Gas chromatography
Abstract
The invention relates to the technical field of medical biological detection, and specifically to the novel application of glycine, 3-phosphoglyceric acid and cytosine on preparing a diagnostic reagent or kit for bladder cancer. The contents of the glycine, 3-phosphoglyceric acid and cytosine in human urine can be detected by using the reagent and method in the invention. The invention further provides a method of detecting the contents of the glycine, 3-phosphoglyceric acid and cytosine in urine by using gas chromatography-mass spectrometric, and the method can be used for diagnosing bladder cancer. The kit and detection method of the invention are simple and reliable, have short period and high specificity, and are easy to promote clinically.
Description
Technical field
The present invention relates to technical field of biomedical detection, specifically, be based on human urine glycine, 3-
The bladder cancer diagnosis agent box of phosphoglyceric acid and cytosine and diagnostic method.
Background technology
Bladder cancer be China be also the tumor that urinary system sickness rate is the highest in world wide.Owing to bladder cancer has
There are high incidence, high relapse rate and the feature of high progression rates, therefore, can diagnose in clinical position in early days
New people of bladder tumor accurate evaluation and the postoperative high-risk bladder cancer patients of monitoring seem particularly necessary.Its diagnosis
Rely on Urine exfoliative cell and cystoscopy.Urine exfoliative cell sensitivity is low；Cystoscopy belongs to invasive operation,
The complication such as the damage of the pain of patient, urethral mucosa, hemorrhage, infection can be caused, and increase patient's
Misery and financial burden.Preferably tumor markers feature should be quick, effective, convenient, safety, warp
Ji, hurtless measure.
Metabolism group is subject the most emerging in systems biology, be one with research cell, tissue or device
The metabolite produced after official's biological metabolism is research purpose scientific domain.Owing to metabolite is metastable
Being present in body fluid, endogenous metabolism analysis of spectrum is more applied to the early diagnosis of tumor and grinds the most in recent years
Study carefully, such as carcinoma of prostate, hepatocarcinoma.In many malignant tumor, bladder cancer is preferably to use metabolism
Group learns the methods analyst urine endogenous metabolism spectrum malignant tumor as early diagnosis, because urine in bladder cavity
The directly tumor tissues at contact disease damage.It addition, the collection of urine is more convenient, simple and noinvasive.
Chinese patent literature CN102323351A mono-kind sets up bladder cancer patient urine specific metabolite spectrum
Method urine sample is carried out metabolism analysis of spectrum, data acquisition multivariate data statistical software founding mathematical models,
Relatively normal person and the difference of bladder cancer patients metabolite profile, so that it is determined that specific metabolic thing spectrum.This research
It is to use metabolite profile identification bladder cancer patients, operates more complicated.The potential of diagnosis is can be used as although giving
Biomarker, but diagnosis effect based on biomarker is not given, and this experiment is without external certificate.
Lin Yun footpath etc. establishes the urine sample metabolic components analysis side that a kind of derivatization combines with gas chromatography-mass spectrography
Method, is successfully applied to the urine metabolism group credit analysis of bladder cancer patients, finds 4 kinds of potential biomarkers:
Threonic acid THREONIC ACID., equisetic acid, hippuric acid and D-Glucose (Lin Yun footpath etc. GC-MS Analysis bladder
Cancer Urine in Patients metabolism group. analytical chemistry [J], 2008,36 (9), 1257-1260.).But be not given based on
The diagnosis effect of these 4 kinds of potential source biomolecule marks, and without confirmatory experiment.
The problem existed based on above research, the present invention uses the selection ion of gas chromatography-mass spectrography to sweep
Retouching method, the method has reproducible, highly sensitive, and the simple advantage of Data Matching.Use from sending out
Now arrive the diagnosis marker that the Policy Filtering of checking is potential, successfully filter out based on human urine glycine, 3-
The bladder cancer combined diagnosis mark of phosphoglyceric acid and cytosine, the diagnostic sensitivity of this composite marker thing and
Specificity is all good, and the test kit of this combined diagnosis mark yet there are no report.
Summary of the invention
It is an object of the invention to low for Diagnosis of Bladder sensitivity or there is the problems such as traumatic, it is provided that be a kind of
The application in Diagnosis of Bladder of the small molecule metabolites of combination, provides simultaneously and can be used for the little of combinations thereof
The analyzing detecting method of molecule metabolites.
For achieving the above object, the technical solution used in the present invention is as follows:
(1) metabonomic technology of gas chromatography-mass spectrography is used, to normal person and bladder cancer patients
Urine carry out metabolic profiling analysis, metabolite is screened, screening conditions include P value, believable put
Letter is interval, change multiplying power and AUC etc., retains the difference metabolite meeting condition.
(2) to the difference metabolite retained in (1), use Software of Data Statistics SPSS, pass through binary
Logistic regression analysis method, returns as composite marker thing variable, employing ROC (receiver operating
Characteristic) curve evaluates susceptiveness and the specificity of composite marker thing.
(3) use of composite marker thing: glycine, 3-phoshoglyceric acid and born of the same parents are phonetic in bladder cancer patients
The content of pyridine is lowered.Use Software of Data Statistics SPSS, by binary logical regression analysis method, by sweet
It is composite marker thing variable X that propylhomoserin, 3-phoshoglyceric acid and cytosine return, and dualistic logistic regression equation is:
X=1.3333-8.891* glycine * 10-8-4.8113* phosphoglyceric acid * 10-5-5.62* cytosine * 10-5,
P=1/ (1+e-X)
Wherein P is the probability of bladder cancer, when P value is more than 0.6245, is diagnosed as bladder cancer.
(4) apply another batch of independent sample that the composite marker thing found in (3) is verified, really
Determining glycine, 3-phoshoglyceric acid and cytosine can be as the composite marker thing of auxiliary diagnosing bladder cancer；
(5) device included by diagnostic system: chromatographic column is Agilent DB-5 MS chromatographic column (30
m×0.25mm×0.25μm)；Detecting instrument is gas chromatograph-mass spectrometer (GC-MS).
(6) determine that test kit forms:
A. urine sample pre-treatment reagent includes: A liquid, 15mg/mL urase；B liquid, methanol；C liquid, molten
Molten in the methoxamine that concentration is 20mg/mL of pyridine；D liquid, commercially available N-methyl-N (TMS)
Trifluoroacetamide.Wherein, the purpose of A liquid is to remove the carbamide in urine, because urea content is the highest, seriously
Have impact on the analysis detection of other metabolite；B liquid is to remove urease activity, and extracts the metabolism in urine
Thing；C liquid is that the metabolite containing carbonyl is carried out oximation reaction, to prevent its open loop closed loop；D liquid is to generation
Thank to thing and carry out Silylation, to increase the volatility of metabolite, beneficially gas chromatography-mass spectrum detection.
B. instrumental conditions:
Described GC conditions: urine sample metabolic profiling analysis gas phase condition (Ye, the G. of list of references report；
Zhu,B.；Yao,Z.；Yin,P.；Lu,X.；Kong,H.；Fan,F.；Jiao,B.；Xu,G.,Analysis of
urinary metabolic signatures of early hepatocellular carcinoma recurrence after
surgical removal using gas chromatography-mass spectrometry.J Proteome Res
2012,11 (8), 4361-72.), specific as follows: DB-5 MS chromatographic column (30m × 0.25mm × 0.25 μm,
Agilent Technologies,USA)；Column temperature uses temperature programming, and 70 DEG C keep 3min, with 4 DEG C/min
Speed rise to 220 DEG C, then rise to 300 DEG C with the speed of 8 DEG C/min, keep 10min.Injector temperature
300 DEG C, sampling volume 1 μ L, split ratio 1:10.With helium (99.9995%, China) as carrier gas,
In post, flow velocity is 1.19ml/min, keeps constant speed mode.
Described Mass Spectrometry Conditions: EI source, 70eV, transmission line and ion source temperature are 280 DEG C and 230 DEG C respectively.
Using full scan type collection data (m/z 33-600) or choice ion pattern to gather data, scanning speed is
0.25s/scan.Detector voltage is set as that for 1.09kV, the solvent mute time is 5min, and initial data is adopted
Integrate the time as 5min.
Process according to the data of urine sample full scan type collection, select the characteristic ion of metabolite, right
Sample carries out selecting ion scan to gather data, highly sensitive, reproducible, can improve the quality of data.Its
Middle glycine, cytosine, the characteristic ion of 3-phoshoglyceric acid are m/z 174,254,357 respectively.Entirely
Scan pattern and select the total ion current figure of ion scan pattern and glycine, 3-phoshoglyceric acid and cytosine
Peak, as shown in Fig. 1 (a-h).
During the detection of described gas chromatography-mass spectrum, the data according to full scan pattern are qualitative to metabolite, by matter
In spectrogram and NIST11 mass spectral database, glycine, cytosine, the standard mass spectrum of 3-phoshoglyceric acid compare
Right, matching degree reaches to be considered when more than 70% glycine, cytosine, 3-phoshoglyceric acid, and uses
Standard substance are verified.
(7) the application effect of the urine sample test sample present invention is used.Be respectively adopted two batches normal person and
The urine sample sample of bladder cancer patients, wherein bladder cancer comprise bladder transitional cell carcinoma, sarcoma of bladder, bladder scale cancer,
The malignant tumor such as adenocarcinoma of bladder, Bladder metastasis tumor.Use composite marker thing normal person can obtain with bladder cancer patients
Differentiation to good: at discovery phase, its AUC is respectively 88.0%, and sensitivity 78.1% is special
Degree 96.2%, as shown in Figure 2；At large quantities of sample individual authentication stages, its AUC=0.705, sensitivity is
62.0%, specificity is 72.5%.
For above technical scheme, a first aspect of the present invention, it is provided that glycine, 3-phoshoglyceric acid and born of the same parents
Pyrimidine is as diagnosis marker application in preparing bladder cancer diagnosis agent or test kit.
In described diagnostic reagent or test kit detection urine, glycine, 3-phoshoglyceric acid and cytosine contains
Amount.Following weight, Y=1.3333-8.891* glycine * 10 is given to testing result-8-4.8113* phosphoglycerol
Acid * 10-5-5.62* cytosine * 10-5, by the Y value diagnosing bladder cancer obtained.When Y value is more than 0.6245
Time, it is diagnosed as bladder cancer.
Described diagnostic reagent or test kit are for using glycine, 3-phosphoric acid in gas chromatography-mass spectrum detection urine
The combination of the reagent of the content of glyceric acid and cytosine.
In described employing gas chromatography-mass spectrum detection urine, glycine, 3-phoshoglyceric acid and cytosine contains
The reagent of amount includes: A liquid, 15mg/ml urase；B liquid, commercial methanol；C liquid, is dissolved in the dense of pyridine
Degree is molten for the methoxamine of 20mg/mL；D liquid, commercially available methyl-trimethyl-silyl trifluoroacetamide.
Described bladder cancer is bladder transitional cell carcinoma, sarcoma of bladder, bladder scale cancer, adenocarcinoma of bladder, Bladder metastasis
The malignant tumor such as tumor.
A second aspect of the present invention, it is provided that a kind of bladder cancer diagnosis agent or test kit, described diagnostic reagent
Or test kit includes detecting the reagent of the content of glycine, 3-phoshoglyceric acid and cytosine in urine.
Described diagnostic reagent or test kit include: A liquid, 15mg/ml urase；B liquid, commercial methanol；
C liquid, the methoxamine that concentration is 20mg/mL being dissolved in pyridine is molten；D liquid, commercially available methyl-trimethyl-first silicon
Alkyl trifluoroacetamide.
A third aspect of the present invention, it is provided that in detection urine, glycine, 3-phoshoglyceric acid and cytosine contains
The application in preparing bladder cancer diagnosis agent box of the reagent of amount.
In described detection urine, the reagent of the content of glycine, 3-phoshoglyceric acid and cytosine is for using gas
The reagent of the content of glycine, 3-phoshoglyceric acid and cytosine in phase chromatography-mass spectroscopy detection urine.Described
Reagent includes: A liquid, 15mg/ml urase；B liquid, commercial methanol；C liquid, the concentration being dissolved in pyridine is
The methoxamine of 20mg/mL is molten；D liquid, commercially available methyl-trimethyl-silyl trifluoroacetamide.
A fourth aspect of the present invention, it is provided that glycine, 3-phoshoglyceric acid and cytosine content in a kind of urine
Detection method, comprise the following steps: by urine sample after above-mentioned A, B, C, D liquid processes successively,
With glycine, 3-phoshoglyceric acid and the content of cytosine in gas chromatography-mass spectrum detection urine sample.
During the detection of described gas chromatography-mass spectrum, survey urine specimen difference metabolite charge-mass ratio and peak index, will
In charge-mass ratio and NIST05 mass spectral database, Standard Metabolism thing glycine, cytosine, 3-phoshoglyceric acid compare
Relatively, matching degree reaches to be considered when more than 70% glycine, cytosine, 3-phoshoglyceric acid.With correspondence
Peak index draw content.
Described GC conditions: DB-5 MS chromatographic column (30m × 0.25mm × 0.25 μm, Agilent
Technologies,USA)；Column temperature uses temperature programming, and 70 DEG C keep 3min, with the speed of 4 DEG C/min
Rise to 220 DEG C, then rise to 300 DEG C with the speed of 8 DEG C/min, keep 10min.Injector temperature 300 DEG C,
Sampling volume 1 μ L, split ratio 1:10.With helium (99.9995%, China) as carrier gas, flow velocity in post
For 1.19ml/min, keep constant speed mode.
Described Mass Spectrometry Conditions: EI source, 70eV, transmission line and ion source temperature are 280 DEG C and 230 DEG C respectively.
Use full scan pattern (m/z 33-600) or choice ion pattern gather data (glycine, cytosine,
The characteristic ion of 3-phoshoglyceric acid is m/z 174,254,357 respectively), scanning speed is 0.25s/scan.
Detector voltage is set as that 1.09kV, solvent mute time are 5min, and initial data acquisition time is 5min.
A fifth aspect of the present invention, it is provided that one utilizes above-mentioned diagnostic reagent or diagnostic kit detection bladder cancer
Method, comprise the following steps: by urine sample after above-mentioned A, B, C, D liquid processes successively, use
Glycine, 3-phoshoglyceric acid and the content of cytosine in gas chromatography-mass spectrum detection urine sample, to detection
Result gives following weight, Y=1.3333-8.891* glycine * 10-8-4.8113* phosphoglyceric acid
*10-5-5.62* cytosine * 10-5, by the Y value diagnosing bladder cancer obtained.When Y value is more than 0.6245,
It is diagnosed as bladder cancer.
The invention has the advantages that:
1, present invention employs substantial amounts of bladder cancer clinic urine specimen, and use from being found to grinding of checking
Studying carefully strategy, the research for the present invention provides sound assurance；
2, the present invention is used basic detection method to be quantitative gas chromatography-mass-spectrometric technique, and this technology is existing
It is the detection method of high sensitivity, high accuracy for Laboratory Diagnosis has been found to, uses and select ion to sweep
Retouch and further increase sensitivity, improve repeatability, be beneficial to find the important metabolite of low content.And
And the present invention uses (the automated mass spectral of AMDIS software in this technology
Deconvolution and identification system, automatic deconvolution software) and ChromaTOF software
And similarity searching software (NIST Mass Spectral Search Program Version 2.0), can be accurate
Glycine, 3-phoshoglyceric acid and cytosine in various samples are done qualitative analysis by ground；Select ion scan mould
Formula detection glycine, 3-phoshoglyceric acid and cytosine, be substantially the quantitative skill of a kind of urine metabolism group
Art detect, have easy and simple to handle, detect sensitive, specificity good, repeated high；
3, composite marker thing involved in the present invention comprises glycine, 3-phoshoglyceric acid and cytosine,
Discovery phase, its AUC is respectively 88.0%, sensitivity 78.1%, specificity 95.2%；At big lot sample
The product individual authentication stage, its AUC=0.705, sensitivity is 62.0%, and specificity is 72.5%, compared to
Urine exfoliated cells detects, and its clinical reference value and credibility are higher.So, the detection examination of the present invention
Agent box, can improve recall rate and the accuracy rate of bladder cancer, and the clinical treatment for bladder cancer has great
Meaning；
4, the invention provides a kind of bladder cancer detection kit based on quantitative gas chromatography-mass spectrum,
Can be strong by detecting the peak that in patient's urina sanguinis, the content of glycine, 3-phoshoglyceric acid and cytosine is formed
Bladder cancer is made diagnosis by degree.In vitro sample is used to detect, detection method is easy especially, the cycle is short,
Highly sensitive, it is effectively supplementing of existing detectable.
Accompanying drawing explanation
The spectrogram of Fig. 1 urine sample sample.The total ion current figure of (a) full scan pattern；B () selects ion scan mould
The total ion current figure of formula；(c-h) glycine, 3-phoshoglyceric acid and cytosine are respectively in full scan pattern and choosing
Select the quasi-molecular ions of ion mode.
Fig. 2 discovery phase (Training Phase) diagnostic cast；Caption: Sensitivity-sensitivity,
Specificity-specificity, Criterion (cut-off value)-dividing value.
Fig. 3 Qualify Phase (Test Phase) diagnostic cast；Caption: Sensitivity-sensitivity, Specificity-
Specificity.
Detailed description of the invention
The detailed description of the invention provided the present invention below in conjunction with embodiment elaborates.
Embodiment 1:
Take 100 μ L urines with 1:1 ratio add concentration for this test kit A liquid (15mg/mL urase), use
Vortex mixed instrument concussion mixing 10 seconds, enzymolysis 15 minutes in water-bath at 37 DEG C.1:4 in above-mentioned mixed liquor
Add this test kit B liquid (methanol), with vortex mixed instrument concussion mixing 30 seconds, in high speed centrifuge from
The heart (14000rpm, 15min, 4 DEG C), Aspirate supernatant lyophilizing.In lyophilizing sample, add 50 μ L originally
Test kit C liquid (is dissolved in the methoxamine solution that concentration is 20mg/mL of pyridine), and the concussion of vortex mixed instrument is mixed
Even 1 minute, more ultrasonic 15 minutes of room temperature, be positioned over water-bath 90 minutes at 37 DEG C.It is subsequently adding this reagent
Box 40 μ L D liquid (N-methyl-N-(TMS) trifluoroacetamide), the concussion mixing of vortex mixed instrument
10 seconds, water-bath 60 minutes at 37 DEG C.High speed centrifuge centrifugal (14000rpm, 10min, 4 DEG C),
Take supernatant sample introduction analysis.
The condition of gas chromatograph-mass spectrometer (GC-MS) is as follows:
Chromatographic condition: DB-5 MS chromatographic column (30m × 0.25mm × 0.25 μm, Agilent
Technologies,USA)；Column temperature uses temperature programming, and 70 DEG C keep 3min, with the speed of 4 DEG C/min
Rise to 220 DEG C, then rise to 300 DEG C with the speed of 8 DEG C/min, keep 10min.Injector temperature 300 DEG C,
Sampling volume 1 μ L, split ratio 1:10.With helium (99.9995%, China) as carrier gas, flow velocity in post
For 1.19mL/min, keep constant speed mode.
Mass Spectrometry Conditions: EI source, 70eV, transmission line and ion source temperature are 280 DEG C and 230 DEG C respectively.Electricity
Sub-bombarding energy is 70eV, and scanning speed is 0.25s/scan.Detector voltage is set as 1.09kV, solvent
Mute time is 5 minutes, and initial data acquisition time is 5 minutes.
To urine sample sample use full scan pattern analysis, the m/z of scanning of the mass spectrum scope at 33-600, total ion
Flow graph is shown in Fig. 1 (a).Use automatic deconvolution AMDIS software and ChromaTOF software to spectrogram at
Reason, by Standard Metabolism thing glycine, cytosine, 3-phoshoglyceric acid mass spectrum in mass spectrum and NIST11 storehouse
Figure is compared, and matching degree reaches to be considered when more than 70% glycine, cytosine, 3-phoshoglyceric acid,
Finally use standard substance that it is verified.And filter out the characteristic ion of metabolite, set up and select ion to sweep
Retouch method, total ion current figure is shown in Fig. 1 (b), and the method is highly sensitive, reproducible, beneficially low content
Metabolite.Wherein glycine, cytosine, the characteristic ion of 3-phoshoglyceric acid be respectively m/z 174,254,
357, when full scan pattern analysis, cytosine and 3-phoshoglyceric acid peak are below quantitative limit (signal to noise ratio are little
In 10), detection difficult, and use choice ion pattern analysis so that it is sensitivity has been respectively increased 19.0,
10.9,18.9 times, thus realize accurate quantitative analysis, peak shape is also greatly improved, and is specifically shown in Fig. 1 (c-h).
Embodiment 2:
Before urine sample sample collection gathers, include volunteer in and sign Informed Consent Form.
Leave and take bladder cancer patients (32 example) and the urina sanguinis of normal person's (21 example), take 100 μ L urines with 1:1
It is this test kit A liquid (15mg/mL urase) that ratio adds concentration, shakes mixing 10 with vortex mixed instrument
Second, enzymolysis 15 minutes in water-bath at 37 DEG C.In above-mentioned mixed liquor, 1:4 adds this test kit B liquid (first
Alcohol), with vortex mixed instrument concussion mixing 30 seconds, centrifugal in high speed centrifuge (14000rpm, 15min,
4 DEG C), Aspirate supernatant lyophilizing.In lyophilizing sample, add 50 μ L this test kit C liquid and (be dissolved in pyridine
The methoxamine solution that concentration is 20mg/mL), vortex mixed instrument concussion mixing 1 minute, then room temperature is ultrasonic
15 minutes, it is positioned over water-bath 90 minutes at 37 DEG C.It is subsequently adding this test kit 40 μ L D liquid (N-methyl
-N-(TMS) trifluoroacetamide), vortex mixed instrument concussion mixing 10 seconds, water-bath at 37 DEG C
60 minutes.High speed centrifuge centrifugal (14000rpm, 10min, 4 DEG C), takes supernatant sample introduction analysis.
The condition of gas chromatograph-mass spectrometer (GC-MS) is as follows:
Chromatographic condition: DB-5 MS chromatographic column (30m × 0.25mm × 0.25 μm, Agilent
Technologies,USA)；Column temperature uses temperature programming, and 70 DEG C keep 3min, with the speed of 4 DEG C/min
Rise to 220 DEG C, then rise to 300 DEG C with the speed of 8 DEG C/min, keep 10min.Injector temperature 300 DEG C,
Sampling volume 1 μ L, split ratio 1:10.With helium (99.9995%, China) as carrier gas, flow velocity in post
For 1.19mL/min, keep constant speed mode.
Mass Spectrometry Conditions: EI source, 70eV, transmission line and ion source temperature are 280 DEG C and 230 DEG C respectively.Electricity
Sub-bombarding energy is 70eV, uses choice ion pattern scanning (glycine, cytosine, 3-phoshoglyceric acid
Characteristic ion be m/z 174,254,357 respectively), scanning speed is 0.25s/scan.Detector voltage
Being set as that 1.09kV, solvent mute time are 5 minutes, initial data acquisition time is 5 minutes.
Process urine sample with this test kit and analyzed by gas chromatograph-mass spectrometer (GC-MS), face total to initial data
Carry out univariate analysis after long-pending normalization, find 32 example bladder cancer patients and the urine of 21 example Healthy Peoples are total to
With the presence of 101 metabolite significant differences, after rejecting the metabolite with passive confidence interval, remain 75
Individual difference metabolite (p < 0.05)；Further screening change multiplying power more than 1.4 or is less than 0.714, has
34 difference metabolite；Using statistical analysis software SPSS again, the AUC screening single metabolite is big
In 0.7, have 22 difference metabolite；Finally 22 the difference metabolite retained are carried out binary to patrol
Collecting regression analysis, finishing screen selects glycine, cytosine, the variable X value of 3-phoshoglyceric acid combination, returns
Return equation as follows:
X=1.3333-8.891* glycine * 10-8-4.8113* phosphoglyceric acid * 10-5-5.62* cytosine * 10-5
P=1/ (1+e-X)
Wherein P is the probability of bladder cancer, when P value is more than 0.6245, is diagnosed as bladder cancer.This combination
Mark may be used for distinguishing bladder cancer patients group and healthy population, and diagnosis effect is shown in Table 1.
Table 1
| Cut-off value (Training Phase) | ＞ 0.6245 |
| Sensitivity (%) | 78.1 |
| Specificity (%) | 95.2 |
| Positive predictive value % | 96.2 |
| Negative predictive value % | 74.1 |
| AUC (area under curve) | 0.88 |
For verifying above-mentioned application, the present invention have collected the urine of 79 example bladder cancer patients and 51 example Healthy Peoples again
Liquid, through sample preprocessing, by this kit measurement glycine, cytosine and glycerol 3-phosphate acid content
After, by aforesaid computational methods, the effect of checking P value diagnosing bladder cancer.Result prompting uses the present invention
Test kit and application process, the composite marker thing diagnosis bladder of glycine, cytosine and 3-phoshoglyceric acid
The diagnosis curve AUC=0.705 of cancer, sensitivity is 62.0%, and specificity is 72.5%, positive predictive value 77.8%
With negative predictive value 55.2%, specifically it is shown in Table 2, hence it is evident that be better than existing urine sediment Diagnosis of Bladder
Method.
Table 2
| Cut-off value (Test Phase) | ＞ 0.6245 |
| Sensitivity (%) | 62.0 |
| Specificity (%) | 72.5 |
| Positive predictive value % | 77.8 |
| Negative predictive value % | 55.2 |
| AUC (area under curve) | 0.705 |
Embodiment 3
59 years old male of one Healthy People, the Y value that the reagent of the employing present invention and detection method obtain is
0.3974, less than 0.6245；76 years old male of one hematuria patient, uses reagent and the detection method of the present invention
The Y value obtained is 1.1184, suspects for bladder cancer, and row cystoscopy is also demonstrate,proved after doing histopathologic examination
Actually Urothelial Carcinoma of Bladder, the checking obtained with Y value is consistent.
Below preferred embodiment to the invention is illustrated, but the invention does not limit
In described embodiment, those of ordinary skill in the art also may be used on the premise of the invention spirit
Making modification or the replacement of all equivalents, the modification of these equivalents or replacement are all contained in the application right and want
In seeking limited range.
Claims (10)
1. glycine, 3-phoshoglyceric acid and cytosine answering in preparing bladder cancer diagnosis agent or test kit
With.
Glycine the most according to claim 1,3-phoshoglyceric acid and cytosine are examined in preparation bladder cancer
Application in disconnected reagent or test kit, it is characterised in that in described diagnostic reagent or test kit detection urine
The content of glycine, 3-phoshoglyceric acid and cytosine.
Glycine the most according to claim 1,3-phoshoglyceric acid and cytosine are examined in preparation bladder cancer
Application in disconnected reagent or test kit, it is characterised in that described diagnostic reagent or test kit are for using gas phase
The combination of the reagent of the content of glycine, 3-phoshoglyceric acid and cytosine in chromatography-mass spectroscopy detection urine.
Glycine the most according to claim 3,3-phoshoglyceric acid and cytosine are examined in preparation bladder cancer
Application in disconnected reagent or test kit, it is characterised in that described employing gas chromatography-mass spectrum detection urine
The reagent of the content of middle glycine, 3-phoshoglyceric acid and cytosine includes: A liquid, 15mg/mL urase；
B liquid, methanol；C liquid, the methoxamine that concentration is 20mg/mL being dissolved in pyridine is molten；D liquid, N-methyl-N (three
Methyl-monosilane base) trifluoroacetamide.
5. preparing wing according to claim 1-4 arbitrary described glycine, 3-phoshoglyceric acid and cytosine
Application in Guang cancer diagnostic reagent or test kit, it is characterised in that described bladder cancer be bladder transitional cell carcinoma,
The malignant tumor such as sarcoma of bladder, bladder scale cancer, adenocarcinoma of bladder, Bladder metastasis tumor.
6. a bladder cancer diagnosis agent or test kit, it is characterised in that described diagnostic reagent or test kit
Including the reagent of the content of glycine, 3-phoshoglyceric acid and cytosine in detection urine.
A kind of bladder cancer diagnosis agent the most according to claim 6 or test kit, it is characterised in that institute
Diagnostic reagent or the test kit stated include: A liquid, 15mg/mL urase；B liquid, methanol；C liquid, is dissolved in
The concentration of pyridine is that the methoxamine of 20mg/mL is molten；D liquid, N-methyl-N (TMS) trifluoroacetyl
Amine.
8. in detection urine, bladder cancer prepared by the reagent of the content of glycine, 3-phoshoglyceric acid and cytosine
Application in diagnostic kit.
9. a detection method for glycine, 3-phoshoglyceric acid and cytosine content in urine, including following
Step: by urine sample after A, B, C, D liquid processes successively, detect urine with gas chromatography-mass spectrum
The content of glycine, 3-phoshoglyceric acid and cytosine in sample；
Wherein, A liquid, 15mg/mL urase；B liquid, methanol；C liquid, the concentration being dissolved in pyridine is 20mg/mL
Methoxamine molten；D liquid, N-methyl-N-(TMS) trifluoroacetamide.
Glycine, 3-phoshoglyceric acid and cytosine in a kind of urine the most according to claim 9
The detection method of content, it is characterised in that described GC conditions: DB-5MS chromatographic column；Column temperature
Using temperature programming, 70 DEG C keep 3min, rise to 220 DEG C with the speed of 4 DEG C/min, then with 8 DEG C/min
Speed rise to 300 DEG C, keep 10min；Injector temperature 300 DEG C, sampling volume 1 μ L, split ratio
1:10；Using helium as carrier gas, in post, flow velocity is 1.19ml/min, keeps constant speed mode；
Described Mass Spectrometry Conditions: EI source, 70eV, transmission line and ion source temperature are 280 DEG C and 230 DEG C respectively.
Use full scan mode m/z 33-600, or choice ion pattern gathers data, glycine, cytosine, 3-
The characteristic ion of phosphoglyceric acid is m/z 174,254,357 respectively, and scanning speed is 0.25s/scan；Inspection
Surveying device voltage and be set as that 1.09kV, solvent mute time are 5min, initial data acquisition time is 5min.
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