CN106485082B - A kind of method for building up of the OPLS-DA diagnostic model based on refining metabolism group and its application - Google Patents
A kind of method for building up of the OPLS-DA diagnostic model based on refining metabolism group and its application Download PDFInfo
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Abstract
The invention discloses a kind of method for building up of OPLS-DA diagnostic model based on refining metabolism group and its application, include the following steps: to screen modeling object and sample acquisition；Fabrication quality control sample；Sample extraction；Sample derivatization；Chromatography-mass spectral analysis；The use of Quality control samples；The identification of compound；OPLS-DA modeling.OPLS-DA diagnostic model of this kind based on refining metabolism group, the OPLS-DA model that the model is better than using the parameters such as semen volume, sperm concentration, sperm motility rate, sperm motility and the sperm motility mode in seminal parameters to establish to the differentiation effect of UMI, the former sensitivity and specificity is respectively 98% and 95%, and the sensitivity and specificity of the latter is only 67% and 73%.
Description
Technical field
The present invention relates to male infertility infertility field of biotechnology, and in particular to a kind of based on refining metabolism group
The method for building up of OPLS-DA diagnostic model and its application.
Background technique
The male sterility (Unexplained Male Infertility, UMI) of unknown cause refers to Sperm routine analysis
As a result in the normal range, and the male sterility phenomenon after body and cryptorrhea is excluded.In addition to erection problem and sexual intercourse
Outside factor, immunogene because and sperm function obstacle may also will lead to such situation.The disease of the male sterility of unknown cause
Because including low level leukocyte sperm disease and mitochondria DNA polymerase gene pleiomorphism etc..Infertile is that the whole world is generally deposited
Between husband and wife the problem of, can not be pregnant after being clinically defined as regular sexual intercourse in a couple 1 year, it is estimated that have 4-17%
Man and wife can go to a doctor, to correct their infertile, but generally, it is considered that more infertile Mr. and Mrs will not go diagnosis and treatment.It adjusts
Display is looked into, male factor is main or single factor the 20-50% or so for accounting for infertile reason.
Currently, Sperm routine analysis, which is still that male factor is infertile, mainly to be commented in addition to detailed medical history and comprehensive physical examination
Valence tool, this way are based on seminal parameters such as sperm concentration, and motility has proved to be the significant related fact to form.
Have the characteristics that widely used in Infertility male preliminary assessment, the normal reference value of seminal parameters is tested in the semen analysis of Noninvasive
The standard of range can change according to the revision and variation of World Health Organization's laboratory manual.2010, the World Health Organization
(WHO) new reference value is established, the reference value of current human seminal fluid's parameter is reported than in the past to be substantially reduced.From eight countries
About 2000 people and companion's life-time were less than 12 months and the male individual of companion's pregnancy is chosen as providing seminal parameters ginseng
The sample of examination mark cloth.The reference value of the new handbook of World Health Organization use may result in clinical practice reclassify it is numerous not
Pregnant Mr. and Mrs.In particular, being classified as the Mr. and Mrs with male factor infertility before those will have on Sperm Parameters
New term of reference, but will be diagnosed now lower than old value as Unexplained infertiiity or female acyesis.New edition handbook, which has been formulated, works as
95% reference interval of preceding fertility semen parameter, meanwhile, the 5th percentage bit line of clinical criteria is also defined, essence is proposed
The lower limit of liquid feature.At present it is not clear in this case, whether this is reclassified has more preferably the assessment of infertile couples
Effect, and the effect of specific diagnosis and male factor evaluation to infertile couples.
Biological marker related with unexplained male infertility is found to assist or substitute seminal parameters analysis and compel very much
It cuts.The technological progress of male's science can effectively make up the deficiency of Sperm routine analysis, mention for the diagnosis of unexplained male infertility
It has supplied new method and new visual angle can be provided for the mechanism of unexplained male infertility.Diagnosis for UMI, works as business
The anxious method for being to provide a kind of pair of fecundity and carrying out objective evaluation.Omics technology contains genomics, transcription group, egg
Bai Zuxue and metabolism group.Metabolism group is a kind of emerging technology, can be used for the discovery of the biological marker of medical diagnosis on disease simultaneously
And it can reveal that the pathogenetic mechanism of disease.Metabolism group discloses the molecule thing occurred under gene, transcription and albumen level
Part, it is considered to be recently close to the group of disease phenotype.Currently, the refining metabolism group of unexplained male infertility changes still not
Know, it is a kind of sample of hurtless measure that refining, which has adjusted the chemical component of sperm, is particularly suitable for being used to find unknown cause male not
Educate the biological marker of diagnosis and Mechanism Study.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of building for OPLS-DA diagnostic model based on refining metabolism group
Cube method and its application, and provide following technical solution:
A kind of method for building up of the OPLS-DA diagnostic model based on refining metabolism group, includes the following steps: in the present invention
1) screen modeling object and sample acquisition: respectively choose unknown sterility Infertility male case group (UMI group) and
Reproductive function normal male control group carries out semen sample acquisition according to the method for WHO standard, and refining is separated and is stored in-
Freezing is sample in 80 DEG C of refrigerators；
2) Fabrication quality control sample: taking 5 μ L to mix in each sample for participating in analysis, Quality control samples be made, point
25 μ L Quality control samples are not taken to be placed in different 1.5mL centrifuge tubes, -80 DEG C of preservations；
3) after sample to be placed to 30min thawing at room temperature, 25 μ L samples sample extraction: are drawn in the centrifuge tube of 1.5mL
In, above-mentioned Quality control samples are melted, sample and Quality control samples be added internal standard 0.3mg/mL chlorophenylalanine and
The 10 μ L of Heptadecanoic acide blended liquid of 1mg/mL is mixed, and adds 300 μ L of organic solvent, vortex oscillation 30S obtains mixed liquor, will mix
Liquid is placed in -20 DEG C of precipitating 10min, is then centrifuged 10min in the case where acceleration is 10,000g, draws 300 μ L of supernatant respectively in sample introduction
In bottle, it is dried in vacuo at room temperature；
4) sample derivatization: 80 μ L of 15mg/mL methoxamine pyridine is added after draining in above-mentioned sample injection bottle, vibrates 30 seconds, in
It is reacted 90 minutes at 30 DEG C, then adds 70 DEG C of 80 μ LBSTFA (1%TMCS) and react 60 minutes；
5) chromatography-mass spectral analysis (GC-TOF analysis): according to every 10 samples being one by sample and Quality control samples
Batch is inserted into 1 Quality control samples and is analyzed by gas chromatograph-mass spectrometer (GC-MS)；
6) use of Quality control samples: calculating interior target RSD in all-mass control sample, which requires to be less than
0.3, if it does not meet the requirements, then the sample for changing excessive batch is reanalysed；If met the requirements, according to internal standard peak
Area establishes Quality Control figure, is limited by way of caution with mean ± 2SD, limits using mean ± 3SD as control, twice in succession beyond warning
Even if limit, beyond control limit it is primary, continuous 5 times increase or reduce it is out of control, it is out of control after sample batch needs reanalyse；Due to
Include internal standard in each sample to be tested, invalid data processing is assert beyond the data of control limit to internal standard in sample；
7) deconvolution processing the identification of compound: is carried out to obtain substance to spectral peak using Chromatof (LECO) software
Mass spectrogram and spectrum library searching is carried out according to the mass spectrogram, according to retention time and spectrum storehouse matching degree carry out it is qualitative, judge through peak
After obtain retention time, substance title (containing substance is not identified) and peak area, after carrying out data processing to each sample, obtain every
The peak area for each substance that a sample is included carries out sample and the substance detected whole according to the appraisal of substance
It closes, forms the response intensity of material composition in behavior sample, be classified as the matrix table of the response intensity of substance in the sample, it will be above-mentioned
Treated matrix table imported into 11.5 software package of SIMCA-P (Umetrics,Sweden it) is used for multidimensional statistics,
Data carry out centralization in SIMCA-P software and pareto homogenization processing (extracts square root the peak area of each spectral peak
Processing, to reduce the big spectral peak bring deviation of peak area)；
8) OPLS-DA is modeled: the variance factor point that multidimensional data in step (7) is first found as required before compression
Group is preset before modeling with finding with the maximally related variable of factor for being used for grouping and the other influence factors of shielding
The Y value of above-mentioned control sample is set as by one Y value for extracting in metabolin matrix with the maximally related data information of Y value
The Y value of O, case group sample are set as 1, establish OPLS-DA model, find associated model and contribute biggish metabolin variable, and
Diagnostic model is established by being used in combination for these metabolin variables.
Further, unknown sterility infertility case group (UMI group) and reproductive function normal male in the step (1)
Control group chooses 80 respectively.
Further, when carrying out chromatography-mass spectral analysis in the step (5), instrument parameter: injector temperature 270
DEG C, sample volume is 1 μ L, and input mode is Splitless injecting samples, carrier gas: 99.999% ultrapure helium, flow 1mL/min；Time
Program: initial temperature is 80 DEG C of initial temperature, retains 2min, rises to 180 DEG C with 10 DEG C/min, then rise to 240 DEG C with 5 DEG C/min, so
290 DEG C are risen to 25 DEG C/min afterwards, retains 9min, transmission line temperature is 260 DEG C, chromatographic column: DB-5MS capillary column；Mass spectrum item
Part: ion source temperature is 200 DEG C, scanning range 30-600, and solvent delay is 5min, scanning speed 20Hz.
Further, it needs to judge internal standard peak used in detection substance each in sample in step (7)；Matter
The peak of substance in sample is controlled respectively with initial data, initial data and chlorophenylalanine ratio, initial data and Heptadecanoic acide ratio
Three kinds of processing modes of value calculate RSD, select correcting mode of the RSD reckling as the substance in three kinds of processing modes.
A kind of method for building up of heretofore described OPLS-DA diagnostic model based on refining metabolism group male not
The infertile research application of bright reason.
The beneficial effects of the present invention are: the present invention carries out chromatography-matter of seminal plasma sample to a large amount of case groups and control group
Spectrum analysis, since there are many compound that metabolin is analyzed simultaneously, to reduce chromatography peak stretching and compression analysis time, therefore generation
It thanks to a group credit analysis and generally has more total effluent, place of deconvoluting can be carried out to total eluting peak by Chromatof software
Reason, since GC-TOF analysis has high acquisition rate (per second to can get 20 maps), software can go to roll up by result spectrogram
Product carries out processing appropriate to data, by deconvoluting, after qualitative, integral, carries out to obtained compound combination library searching
Identification, after RSD < 0.3 is screened in Quality control samples, met the requirements to obtain compound.Using obtained
Metabolism group compound data establishes the OPLS-DA diagnostic model based on refining metabolism group, differentiation effect of the model to UMI
It is built better than using parameters such as semen volume, sperm concentration, sperm motility rate, sperm motility and sperm motility modes in seminal parameters
Vertical OPLS-DA model, the former sensitivity and specificity is respectively 98% and 95%, and the sensitivity and specificity of the latter is only
67% and 73%.
Detailed description of the invention
Fig. 1 is target quality control chart in the chlorophenylalanine of Quality control samples in the specific embodiment of the invention；
Fig. 2 is target quality control chart in the chlorophenylalanine of sample in the specific embodiment of the invention；
Fig. 3 is target quality control chart in the Heptadecanoic acide of Quality control samples in the specific embodiment of the invention；
Fig. 4 is target quality control chart in the Heptadecanoic acide of sample in the specific embodiment of the invention；
Fig. 5 is the OPLS-DA model established in the specific embodiment of the invention using refining metabolism group data；
Fig. 6 is the OPLS-DA model established using seminal parameters data.
Specific embodiment
To be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, below with reference to
Specific embodiment, the present invention is further explained.
The embodiment of the present invention meets statutory regulation and has passed through the approval of Nanjing Medical University's ethics meeting.Case male comes
From 12 months or more the Mr. and Mrs that can not be pregnant of affiliated hospital of Nanjing Medical University outpatient service, women spouse has carried out a series of clinics
It checks and reproductive status is excluded extremely.Case male has carried out complete medical history and physical examination, has carried out hormone determination
And semen routine analysis；Spouse has carried out complete medical history and physical examination, including conventional gynecological inspection, hormone serum level
Measurement, the test of fallopian tubal ovarian function, β hysteroscope, ultrasound detection, immune detection, microorganism detection, karyotype point
Analysis.Control group male recruits and samples with case group in the same period in this example, their reproductive function is normal, and 6-8
Child's birth of their unsoundness of the moon.Semen sample acquires the method according to WHO standard, meets abstinence time requirement.
In the embodiment of the present invention, instrument is preferred are as follows: gas chromatograph-mass spectrometer (GC-MS) GC-TOFMS (Agilent
6890N gas chromatography coupled with Pegasus HT time-of-flight mass
Spectrometer), Leco company (U.S.)；DB-5MS Capillary Column for Gas Chromatography (internal diameter: 0.25mm；Length: 30m；Film
It is thick: 0.25 μm), Anjelen Sci. & Tech. Inc；Centrifuge (LD5-2A type) Beijing Medical Centrifugal Machine Factory；Centrifuge (TGL-
16B) Town in Shanghai pavilion scientific instrument manufactory；Its woods Bell's instrument manufacturing of miniature eddy mixer (QL-901 type) Jiangsu Haimen has
Limit company；Startorius company of electronic analytical balance (BS124S) Germany；Numerical control Ultrasound Instrument (KQ-250DB type) city of Kunshan is super
Sound Instrument Ltd.；Superpure water machine (II type of Mill-Q) Milipore, (Bedford, MA, USA).
In the embodiment of the present invention, agents useful for same is preferred are as follows: chloroform, dehydrated alcohol, and pyridine, sodium hydroxide, anhydrous sodium sulfate,
Above it is all that analysis is pure, is purchased from the general chemical reagent Co., Ltd of Town in Shanghai；L-2- chlorophenylalanine, Heptadecanoic acide, N-O- bis- (three
First silicon substrate) (BSTFA is purchased from SIGMA company (U.S.) containing 1%TMCS), pyridine, methoxamine to trifluoroacetamide.
A kind of method for building up of the OPLS-DA diagnostic model based on refining metabolism group, includes the following steps: in the present invention
1) modeling object and sample acquisition are screened: choosing 80 above-mentioned unknown sterility Infertility male case groups respectively
(UMI group) and the normal human male control group of 80 reproductive functions carries out semen sample acquisition according to the method for WHO standard, and will
It is sample that refining separation, which is stored in freezing in -80 DEG C of refrigerators,；
2) Fabrication quality control sample: taking 5 μ L to mix in each sample for participating in analysis, Quality control samples be made, point
25 μ L Quality control samples are not taken to be placed in different 1.5mL centrifuge tubes, -80 DEG C of preservations；
3) after sample to be placed to 30min thawing at room temperature, 25 μ L samples sample extraction: are drawn in the centrifuge tube of 1.5mL
In, above-mentioned Quality control samples are melted, sample and Quality control samples be added internal standard 0.3mg/mL chlorophenylalanine and
The 10 μ L of Heptadecanoic acide blended liquid of 1mg/mL is mixed, and adds 300 μ L of organic solvent, vortex oscillation 30s obtains mixed liquor, will mix
Liquid is placed in -20 DEG C of precipitating 10min, is then centrifuged 10min in the case where acceleration is 10,000g, draws 300 μ L of supernatant respectively in sample introduction
In bottle, it is dried in vacuo at room temperature；
4) sample derivatization: 80 μ L of 15mg/mL methoxamine pyridine is added after draining in above-mentioned sample injection bottle, vibrates 30 seconds, in
It is reacted 90 minutes at 30 DEG C, then adds 70 DEG C of 80 μ LBSTFA (1%TMCS) and react 60 minutes；
5) chromatography-mass spectral analysis (GC-TOF analysis): according to every 10 samples being one by sample and Quality control samples
Batch is inserted into 1 Quality control samples and is analyzed by above-mentioned gas chromatograph-mass spectrometer (GC-MS), instrument parameter: injection port temperature
Degree is 270 DEG C, and sample volume is 1 μ L, and input mode is Splitless injecting samples, carrier gas: 99.999% ultrapure helium, flow 1mL/
min；Time-program(me): initial temperature is 80 DEG C of initial temperature, retains 2min, rises to 180 DEG C with 10 DEG C/min, then rise to 5 DEG C/min
240 DEG C, 290 DEG C then are risen to 25 DEG C/min, retains 9min, transmission line temperature is 260 DEG C, chromatographic column: DB-5MS capillary
Column；Mass Spectrometry Conditions: ion source temperature is 200 DEG C, scanning range 30-600, and solvent delay is 5min, scanning speed 20Hz；
6) use of Quality control samples: calculating interior target RSD in all-mass control sample, which requires to be less than
0.3, if it does not meet the requirements, then the sample for changing excessive batch is reanalysed；If met the requirements, according to internal standard peak
Area establishes Quality Control figure, is limited by way of caution with mean ± 2SD, limits using mean ± 3SD as control, twice in succession beyond warning
Even if limit, beyond control limit it is primary, continuous 5 times increase or reduce it is out of control, it is out of control after sample batch needs reanalyse；Due to
Include internal standard in each sample to be tested, invalid data processing is assert beyond the data of control limit to internal standard in sample；
7) it the identification of compound: carries out carrying out deconvolution processing to spectral peak to obtain using Chromatof (LECO) software
The mass spectrogram of substance simultaneously carries out spectrum library searching according to the mass spectrogram, qualitative according to retention time and spectrum storehouse matching degree progress, through peak
Retention time, substance title (containing substance is not identified) and peak area are obtained after judgement, after carrying out data processing to each sample, are obtained
Each sample each substance for being included peak area, according to the appraisal of substance, to sample and the substance detected into
Row integration, forms the response intensity of material composition in behavior sample, is classified as the matrix table of the response intensity of substance in the sample, will
Above-mentioned treated matrix table imported into SIMCA-P11.5 software package (Umetrics,Sweden it) unites for multidimensional
Meter, data carry out centralization in SIMCA-P software and pareto homogenization processing (carries out out the peak area of each spectral peak flat
Side's processing, to reduce the big spectral peak bring deviation of peak area)；
8) OPLS-DA is modeled: the variance factor point that multidimensional data in step (7) is first found as required before compression
Group is preset before modeling with finding with the maximally related variable of factor for being used for grouping and the other influence factors of shielding
The Y value of above-mentioned control sample is set as by one Y value for extracting in metabolin matrix with the maximally related data information of Y value
The Y value of O, case group sample are set as 1, establish OPLS-DA model, find associated model and contribute biggish metabolin variable, and
Diagnostic model is established by being used in combination for these metabolin variables.
Two internal standards are used in this example, it is therefore desirable to judge internal standard peak used in each detection substance.
Herein to the peak of substance in quality-control sample respectively with initial data, initial data and chlorophenylalanine ratio, initial data and ten
Seven alkanoic acid ratio calculation RSD select correcting mode of the RSD reckling as the substance in three kinds of processing modes.Such as: parent mass peak face
RSD of the product in each quality-control sample is minimum, then carries out subsequent statistical analysis and pattern-recognition using original peak area；Original number
According to the RSD minimum with chlorophenylalanine ratio in each quality-control sample, then select chlorophenylalanine as the internal standard of the substance.
Based on above-mentioned, this example isolates to obtain component after Chromatof software carries out deconvolution, qualitative, integral
333, by library searching, 153 compounds are identified altogether, wherein 96 chemicals pass through local reference substance library identification.
It after RSD < 0.3 is screened in Quality control samples, obtains 136 compounds and meets the requirements, wherein 90 compounds obtain
Identification, 70 are identified that detailed data is as shown in the table by local reference substance library
The resulting chemical component list of metabonomic analysis
A. the retention time of the compound
The compound name of b.Chromatof software identification, unknown compound, which starts+three bit digitals number with UN, to be indicated
C. storehouse matching degree is composed
D. √ indicates that the compound passes through local reference substance library verifying
E. RSD of the substance in Quality control samples, calculates according to corrected data
F. the substance corrects situation, and 1 is corrects using Heptadecanoic acide, and 2 is correct using chlorophenylalanine, and 3 be not correct
Directly use initial data.
For this example since sample size is big, analytical cycle is long, therefore uses stringent quality control method.Extremely according to Fig. 1
Fig. 4 carries out Quality Control to the internal standard in sample as it can be seen that all quality-control samples meet quality control requirement, in two internal standards,
The difference of any one and mean value is more than that the sample of mean ± 3SD is removed and is not involved in further data processing, using being obtained
The metabolism group data obtained, we establish OPLS-DA model, which includes 1 prediction ingredient and two orthogonal components
(R2Xcum=0.333, R2Ycum=0.833, Q2cum=0.634), as a result such as Fig. 5；In order to compare, while also using sperm matter
The parameters such as semen volume, sperm concentration, sperm motility rate, sperm motility and sperm motility mode in amount parameter establish other one
A OPLS-DA model (R2Xcum=0.360, R2Ycum=0.423, Q2cum=0.331), such as Fig. 6.The results show that using generation
Thanking to the model that group data are established, (95%) sensitivity 98%, specificity, differentiate that effect is better than establishing using seminal parameters data
OPLS-DA model (sensitivity 67%, specificity 73%).
It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, above-described embodiment and explanation
It is merely illustrated the principles of the invention described in book, without departing from the spirit and scope of the present invention, the present invention also has
Various changes and modifications, these changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention
It is defined by the appending claims and its equivalent thereof.
Claims (4)
1. a kind of method for building up of the OPLS-DA diagnostic model based on refining metabolism group, which is characterized in that including walking as follows
It is rapid:
(1) modeling object and sample acquisition are screened: choosing unknown sterility Infertility male case group and reproductive function just respectively
Normal human male control group carries out semen sample acquisition according to the method for WHO standard, and refining separation is stored in -80 DEG C of refrigerators
Freezing is sample；
(2) it Fabrication quality control sample: takes 5 μ L to mix in each sample for participating in analysis, Quality control samples is made, take respectively
25 μ L Quality control samples are placed in different 1.5mL centrifuge tubes, -80 DEG C of preservations；
(3) sample extraction: after sample to be placed to 30min thawing at room temperature, drawing 25 μ L samples in the centrifuge tube of 1.5mL,
Above-mentioned Quality control samples are melted, the chlorophenylalanine and 1mg/ of internal standard 0.3mg/mL is added with Quality control samples for sample
The 10 μ L of Heptadecanoic acide blended liquid of mL is mixed, and adds 300 μ L of organic solvent, vortex oscillation 30s obtains mixed liquor, mixed liquor is set
In -20 DEG C of precipitating 10min, it then is centrifuged 10min in the case where acceleration is 10,000g, draws 300 μ L of supernatant respectively in sample injection bottle
In, it is dried in vacuo at room temperature；
(4) sample derivatization: 80 μ L of 15mg/mL methoxamine pyridine is added after draining in above-mentioned sample injection bottle, vibrates 30 seconds, in 30
It is reacted 90 minutes at DEG C, then adds 80 μ LBSTFA70 DEG C and react 60 minutes；
(5) sample and Quality control samples chromatography-mass spectral analysis: are inserted into 1 quality according to every 10 samples for a batch
Control sample is analyzed by gas chromatograph-mass spectrometer (GC-MS)；
(6) use of Quality control samples: calculating interior target RSD in all-mass control sample, which requires less than 0.3, such as
Fruit is undesirable, then reanalyses the sample for changing excessive batch；If met the requirements, built according to internal standard peak area
Vertical Quality Control figure, is limited by way of caution with mean ± 2SD, is limited using mean ± 3SD as control, is exceeded warning limit twice in succession, is exceeded
Even if primary, continuous 5 raisings of control limit or reduction are out of control, rear sample batch needs out of control are reanalysed；Due to it is each to
All include internal standard in sample, invalid data processing is assert beyond the data of control limit to internal standard in sample；
(7) deconvolution processing the identification of compound: is carried out to obtain the mass spectrogram of substance simultaneously to spectral peak using Chromatof software
Spectrum library searching is carried out according to the mass spectrogram, it is qualitative according to retention time and spectrum storehouse matching degree progress, retained after peak judges
Time obtains each sample and is wrapped containing the substance title and peak area for not identifying substance after carrying out data processing to each sample
The peak area of each substance contained integrates sample and the substance detected according to the appraisal of substance, forms behavior
The response intensity of material composition in sample, is classified as the matrix table of the response intensity of substance in the sample, will be above-mentioned treated
Matrix table imported into SIMCA-P11.5 software package for multidimensional statistics, data is carried out in SIMCA-P software centralization with
Pareto homogenization processing, is that the peak area of each spectral peak is carried out extraction of square root processing, to reduce the big spectral peak band of peak area
The deviation come；
(8) OPLS-DA is modeled: the variance factor that multidimensional data in step (7) is first found as required before compression is grouped, with
It finds and presets a Y before modeling with the maximally related variable of factor for being used for grouping and the other influence factors of shielding
Value, for extracting in metabolin matrix with the maximally related data information of Y value, is set as O for the Y value of above-mentioned control sample, disease
The Y value of example group sample is set as 1, establishes OPLS-DA model, finds associated model and contributes biggish metabolin variable, and passes through
Diagnostic model is established in being used in combination for these metabolin variables.
2. a kind of method for building up of OPLS-DA diagnostic model based on refining metabolism group according to claim 1, special
Sign is that unknown sterility infertility case group and reproductive function normal male control group choose 80 respectively in the step (1)
Example.
3. a kind of method for building up of OPLS-DA diagnostic model based on refining metabolism group according to claim 1, special
Sign is, when carrying out chromatography-mass spectral analysis in the step (5), instrument parameter: injector temperature is 270 DEG C, sample volume 1
μ L, input mode are Splitless injecting samples, carrier gas: 99.999% ultrapure helium, flow 1mL/min；Time-program(me): initial temperature
For 80 DEG C of initial temperature, retains 2min, 180 DEG C are risen to 10 DEG C/min, then rise to 240 DEG C with 5 DEG C/min, then with 25 DEG C/min liter
To 290 DEG C, retain 9min, transmission line temperature is 260 DEG C, chromatographic column: DB-5MS capillary column；Mass Spectrometry Conditions: ion source temperature
It is 200 DEG C, scanning range 30-600, solvent delay is 5min, scanning speed 20Hz.
4. a kind of method for building up of OPLS-DA diagnostic model based on refining metabolism group according to claim 1, special
Sign is, needs to judge internal standard peak used in detection substance each in sample in step (7)；Substance in quality-control sample
Peak respectively with three kinds of initial data, initial data and chlorophenylalanine ratio, initial data and Heptadecanoic acide ratio processing sides
Formula calculates RSD, selects correcting mode of the RSD reckling as the substance in three kinds of processing modes.
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