CN109187794A - Refining deoxycytidine and cytidine detection are used as idiopathic male infertility diagnosis marker and its application - Google Patents

Abstract

The invention belongs to analytical chemistry and clinical medicine domains, disclose refining deoxycytidine and cytidine detection as idiopathic male infertility diagnosis marker and its application.The marker is deoxycytidine and/or cytidine, has higher sensitivity and specificity in diagnosis idiopathic male infertility, can be used for preparing idiopathic male infertility diagnosis or monitoring reagent.

Description

Refining deoxycytidine and cytidine detection as idiopathic male infertility diagnosis marker and It is applied

Invention field

The invention belongs to analytical chemistry and clinical medicine domain, it is related to based in UPLC-Q exactive MS detection refining Deoxycytidine relevant to idiopathic male infertility and cytidine and its application.

Background technique

1988, WHO was put forward for the first time the concept of healthy reproduction, i.e., the mankind and its individual in entire life process with life Grow the health status of physiology in the structure, function and action process of relevant body, psychology and the satisfactory harmony of society.Mesh Before, there are about the couple at child-bearing age of 10-15% to suffer from growing barrier in the whole world.China due to large population base, in newly-married couple it is infertile not The patient numbers educated are far more than million, and wherein infertility caused by male factor is higher than 50%.Current research suggests that inherent cause Such as Y chromosome AZF gene region sequence changes, daz gene copy number changes, sex hormone receptor gene order changes.Environment Factor such as adverse factor exposure, nutrition and living habit etc. is related with male sterility.It is worth noting that, male sterility has phase When a part shows as no method interpretation cause of disease, this makes diagnosis idiopathic male infertility become extremely difficult, has also affected treatment adversely Opportunity.The diagnostic criteria of the WHO of male sterility is that man and wife lives together 1 year or more after marriage, contraceptives is not taken, due to the bridegroom's or husband's side Cause the infertile person in the wife's side.However, live together up to 1 year observing time, delayed significantly to male sterility carry out early treatment and The time of intervention；Many men and wives do not ensure that it is stringent live together 1 year so that last male sterility judgement becomes to be stranded very much It is difficult；In order to exclude wife's side reason, the wife's side also needs to carry out detailed inspection, brings heavy economy and medical burden.It is existing Male sterility coherence check rely on be traditional Sperm recovery, it only focuses on sperm quantity, vigor, semen volume, pH With the conventional parameters such as liquefying time.Because being influenced by factors such as abstinence times, Sperm parameters conventional analysis result table It is now biggish fluctuation.Thus, clinical diagnosis is generally required with reference to multiple semen routine analysis.More importantly tradition Seminal parameters inspection cannot comprehensively reflect whole situations of sperm.Therefore, male sterility often shows as conventional sperm ginseng Number Non Apparent Abnormality, but also traditional seminal parameters inspection can not efficient diagnosis male sterility.Thus, it clinically needs to use In the new diagnostic method of idiopathic male infertility.

Metabolism group (metabonomics) is to be detected with high throughput based on group index analysis and data processing is Means, using information modeling and system combination as a branch of the systems biology of target, be after genomics, transcription group, New another important research field of systems biology after proteomics, it is research biosystem produced by by outside stimulus All metabolites variation science, it is contemplated that the change of small molecule metabolites of the metabolic cycles middle-molecular-weihydroxyethyl less than 1000 Change, reflection is responding control of the body under morbid state or environmental stimulus.Metabolism group table in the diagnosis of complex disease Reveal high application potential and value, since the sample of its analysis can be these noninvasive and low wounds of the various body fluid of blood urine Sample, be greatly improved the compliance of patient, and have the characteristics that high sensitivity is stable.The basic reason of male sterility is essence The problem of liquid.Refining as the liquid component of sperm can be well reflected sperm quality change, and have it is noninvasive, be easy The advantages of acquisition.However, using metabonomic analysis refining metabolism small molecule in the diagnostic monitoring of idiopathic male infertility Using not paid close attention to accordingly also.

The common technology of metabolism group research at present includes liquid chromatograph mass spectrography (LC-MS), chromatography of gases-mass spectrum connection With (GC-MS) and nuclear magnetic resonance technique (NMR).Nuclear magnetic resonance technique feature is to component to be measured without destruction, Sample pretreatment letter It is single, but sensitivity is lower；Gas chromatography-mass spectrography has preferable sensitivity and reproducibility, but generally to use derivatization Method carries out pre-treatment to sample, so that experimental procedure becomes complicated.And LC-MS has sample process simple, high sensitivity is faced The practical feature of bed.UPLC-Q exactive MS is the combination of high resolution mass spectrum of new generation Yu ultra high efficiency liquid phase, is had Compared to the stronger sensitivity of traditional LC-MS, specificity and stability.So carrying out refining using UPLC-Q exactive MSS It is metabolized the metabonomic analysis of small molecule, if stable special refining metabolism relevant to idiopathic male infertility morbidity can be found Small molecule researches and develops the UPLC-Q exactive MS detection method of corresponding metabolism small molecule mark as biomarker, Tremendous contribution will be made for male reproductive health.

Summary of the invention

The purpose of the present invention is provide relevant deoxycytidine and cytidine for idiopathic male infertility.

Another object of the present invention is for refining deoxycytidine and cytidine are used for idiopathic male infertility diagnosis and provide base In the application of UPLC-Q exactive MS.

A further object of the present invention is to provide chromatographic mass spectrometry detection and diagnosis for above-mentioned refining deoxycytidine and cytidine Kit.

The purpose of the present invention is what is realized by following technical measures:

The relevant refining marker of idiopathic male infertility, the marker are deoxycytidine and/or cytidine.

The refining marker is preparing the application in idiopathic male infertility diagnosis or monitoring reagent.

A kind of kit diagnosed or monitor idiopathic male infertility, the kit contain deoxycytidine in detection refining And/or the reagent of cytidine.

The kit, the kit contain using deoxycytidine in UPLC-Q exactive MS method detection refining And/or the reagent of cytidine.

The kit, the kit contain following reagent:

Deoxycytidine and/or cytidine standard items；

Internal standard A: creatinine, valine, niacin, thymidine, glutaric acid, L- phenylpropylamine acid, N- acetaminophen, horse The Isotopic Internal Standard of one or more substances in uric acid；

Internal standard B: the Isotopic Internal Standard of pentadecanoic acid；

Internal standard C: the Isotopic Internal Standard of lignoceric acid.

The kit, the kit also contain:

Hypersil GOLD C18 chromatographic column；

Reagent A: protein precipitation is used, and contains 100% methanol；

Reagent B: mobile phase is used, the water containing 0.1% formic acid；

Reagent C: mobile phase is used, the acetonitrile containing 0.1% formic acid；

Reagent D: redissolving and use, ultrapure water.

A method of above-mentioned refining biomarker relevant to idiopathic male infertility being detected, this method uses UPLC-Q exactive MS method detects deoxycytidine and/or cytidine content in refining.

The method, in this method:

One, liquid-phase condition:

Liquid-phase chromatographic column is Hypersil GOLD C18 chromatographic column, and column temperature is 40 DEG C；

Mobile phase A is the water containing 0.1% formic acid, and Mobile phase B is the acetonitrile containing 0.1% formic acid, and flow velocity is 400 μ L/min；

Instrument gradient are as follows: 0-3min 1%B, 3-10min 1% arrives 99%B, 10-13min 99%B, 13-13.1min 99% arrives 1%B, 13.1-17min 1%B；

Input mode: 5 μ l of volume；

Two, Mass Spectrometry Conditions

It is analyzed using heating electrospray ionisation mode (HESI), positive ion mode spray voltage: 3.5kV；Anion Mode spray voltage: 2.5kV；Capillary temperature under both of which: 250 DEG C, heter temperature: 425 DEG C, sheath gas air-flow: 50AU, Assist gas air-flow: 13AU, blowback air air-flow: 0AU；Lens voltage: 60V；Using mode is swept entirely, scanning range: 70 arrive 1050m/ z；Resolution ratio: 70000.

The present invention is described in detail as follows:

The present inventor acquires standard compliant refining sample with S.O.P. (SOP), and system collects complete crowd Basic information and clinical data, and use the metabolism group method based on UPLC-Q exactive MS and analyzed.

The experimental method specifically studied mainly includes following components:

One, research object selection and group basis

First stage screening stage

It is included in 148 people of 148 people of idiopathic male infertility and normal healthy controls to clarify a diagnosis at random, totally 296 people.

A group: healthy control group (148 people):

1. the age is between 19 to 39 years old；

2. constitutional index is between 17 to 31；

3. the male of fecundity health, and have healthy offspring after 5-8 months；

4. without whole body major disease.

B group: idiopathic male infertility disease group (148 people):

1. the age matches with control group；

2. constitutional index is matched with control group

3. pregnancy is attempted not succeed within 12 months, and spouse does not have the male of infertile disease；.

4. without the clear male sterility cause of disease；

5. Smoking And Drinking history is matched with control group；

6. nationality matches with control group；

7. without whole body major disease.

Second stage Qualify Phase

It is included in 15 people of 15 people of idiopathic male infertility and normal healthy controls to clarify a diagnosis, totally 30 people.

A group: healthy control group (15 people):

1. the age is between 24 to 36 years old；

2. constitutional index is between 19 to 24；

3. the male of fecundity health, and have healthy offspring after 5-8 months；

4. without whole body major disease.

B group: idiopathic male infertility disease group (15 people):

1. the age matches with control group；

2. constitutional index is matched with control group；

3. pregnancy is attempted not succeed within 12 months, and spouse does not have the male of infertile disease；

4. without the clear male sterility cause of disease；

5. Smoking And Drinking history is matched with control group；

6. nationality matches with control group；

7. without whole body major disease.

Two, UPLC-Q exactive MS metabonomic analysis and idiopathic male infertility diagnosis deoxycytidine and cytidine Screening and verifying

1. Sample pretreatment

1.1. 10 μ L refinings are taken, 10 μ L internal standard A are added, 10 μ L internal standard B are added, 10 μ L internal standard C are added, methanol (examination is added Agent A) 40 μ L, vortex 30s, protein precipitation.

1.2. 4 DEG C of 16000g are centrifuged 15min in centrifuge, and supernatant is transferred to 1.5mL import EP pipe, and by supernatant It is concentrated to dryness in centrifugal concentrating drying instrument at room temperature.

1.3. it is redissolved with 10 μ L ultrapure waters (reagent D), it is to be analyzed.

2. instrument detects

2.1. analysis instrument: UPLC Ultimate 3000 system (Dionex) high performance liquid chromatograph；Q- Exactive high-resolution mass spectrometer.

2.2. liquid-phase condition:

2.2.1 liquid-phase chromatographic column be Hypersil GOLD C18 chromatographic column (100mm × 2.1mm, 1.9 μm of partial size, Thermo Scientific, Germany), column temperature is 40 DEG C.

2.2.2 acetonitrile (examination of water (reagent B) and (B) of the mobile phase used for (A) containing 0.1% formic acid containing 0.1% formic acid Agent C), flow velocity is 400 μ L/min.

2.2.3 instrument gradient are as follows: 0-3min 1%B, 3-10min 1% arrives 99%B, 10-13min 99%B, 13- 13.1min99% to 1%B, 13.1-17min 1%B.

2.2.4 input mode: 5 μ l of volume.

2.3. Mass Spectrometry Conditions

2.3.1 heating electrospray ionisation mode (HESI) is analyzed.

2.3.2 using heating electrospray ionisation mode (HESI), positive ion mode spray voltage: 3.5kV；Negative ion mode Spray voltage: 2.5kV；Capillary temperature under both of which: 250 DEG C, heter temperature: 425 DEG C, sheath gas air-flow: 50AU, auxiliary Gas air-flow: 13AU, blowback air air-flow: 0AU；Lens voltage: 60V.Using mode is swept entirely, scanning range: 70 arrive 1050m/z；Point Resolution: 70000.

3. substance is qualitative

Metabolism small molecule is qualitative to compare Chromatographic information (retention time) and mass spectrum using with standard items deoxycytidine and cytidine Information (accurate molecular weight), and compare the Chromatographic information of Isotopic Internal Standard standard items series in sample in real time with corrected retention time Between.

4. data are analyzed:

Biomarker screening confirms key metabolites using Multivariate Logistic Regression.

5. being metabolized the difference and diagnostic significance of small molecule in healthy control group, idiopathic male infertility group refining sample.

The corrected age, constitutional index, smoking and history of drinking history information, logistic regression find refining The content of deoxycytidine and cytidine is related with idiopathic male infertility.It is examined using random crowd using above-mentioned metabolism small molecule combinatorial Disconnected idiopathic male infertility, sensitivity 93.33%, specificity 93.33%, area is 0.9822 under ROC curve, have compared with High diagnostic value.

Three, diagnostic reagent box preparation method

According to above-mentioned a series of experiments as a result, the present inventor be also prepared for it is a kind of can be used for idiopathic male infertility dynamic supervise The diagnostic kit of survey, the diagnostic kit include measurement subject's refining in be stabilized and detectable deoxycytidine and The standard items of cytidine and the internal standard standard items series of assistant analysis.Diagnostic kit further includes that a set of refining metabolism small molecule mentions It takes and used in chromatograph reagent and equipment.

Beneficial effects of the present invention:

The present inventor compares in normal control and idiopathic male infertility refining by using UPLC-Q exactive MS Metabolism small molecule, it was found that exist in refining can be used for assessing whether with idiopathic male infertility, with diagnostic value Deoxycytidine and cytidine combination and the application of the deoxycytidine and the UPLC-Q exactive MS of cytidine detection, are developed It can be convenient for the idiopathic male infertility diagnosis of clinical application, monitoring reagent box.

The present invention is advantageous in that using the marker that refining metabolism small molecule is evaluated as idiopathic male infertility:

(1) refining metabolism small molecule is a kind of new biomarkers, is associated with disease outcome by force, not only stable, nothing It creates, be easy to detect, and is quantitative accurate, will greatly improve the sensibility and specificity of idiopathic male infertility diagnosis, such small point The successful exploitation of sub- biomarker will start completely new situation for the prevention and treatment of idiopathic male infertility, be other diseases biological marker The development of object is offered reference.

(2) deoxycytidine and cytidine provided by the invention can be used as the diagnosis marker of idiopathic male infertility, for clinic The further testing in depth testing of doctor provides foundation, quick and precisely to grasp the morbid state of patient and coincident with severity degree of condition, adopting in time It takes the control prece of more personalized to provide support, delays and prevent progression of disease.

(3) present invention is verified using the refining sample of idiopathic male infertility and the random crowd of normal healthy controls, it was demonstrated that Deoxycytidine and cytidine level have higher sensitivity and specificity in diagnosis idiopathic male infertility in refining, can be used as Marker uses.

(4) present invention uses tight, multistage verifying and appraisement system, and initial stage screens a variety of refining generations by preliminary experiment Thank to small molecule, carry out independent crowd's verifying using UPLC-Q exactive MS, ensure that the refining metabolism biological marker and The reliability of diagnostic method.

(5) UPLC-Q exactive MS technology sample process is simple, and instrument is analyzed rapidly and accurately, clinic with higher Diagnose practical value.

Detailed description of the invention

Fig. 1 screening stage, the corrected age, constitutional index, smoking and history of drinking history information, Multivariate Logistic Regression Analysis finds that the content of refining deoxycytidine and cytidine and idiopathic male infertility are closely related.^aThe list of Confounding Factor is not adjusted Factor Logistic regression result.^bMultivariate Logistic Regression result after adjusting age, constitutional index, smoking and history of drinking history.

Fig. 2 is metabolized small molecule detection level fluctuation (mean ± standard deviation).

Fig. 3 Qualify Phase, the Normal group and idiopathic male infertility made of refining deoxycytidine content information ROC curve between group.

Fig. 4 Qualify Phase, using refining cytidine content information production Normal group and idiopathic male infertility group it Between ROC curve.

Fig. 5 Qualify Phase, the Normal group made of refining deoxycytidine and cytidine content information and idiopathic male ROC curve between property infertility group.

Specific embodiment

The present invention will be further explained by examples below.

The selection of 1 research object of embodiment and group basis

This part research object examines idiopathic male infertility case and health from the head of affiliated hospital of Nanjing Medical University Fertility control.Research contents and informed consent form obtain the approval of Ethics Committee of Nanjing Medical University, meet relevant laws and regulations Requirement.Case and to impinge upon understand content after endorsed informed consent form.All research objects have carried out complete physique It checks, and completing a includes personal basic data, living habit, occupation and environmental exposure, genetic risk factors, sexual function With reproductive function, history of disease and the questionnaire of physical exertion.First stage incorporates satisfactory 148 idiopathic males Sterile case and 148 normal healthy controls；The satisfactory 15 idiopathic male infertility cases of second stage and 15 health are right According to the screening experiment object as idiopathic male infertility refining metabolism small molecule biomarker.

Specific sample group standard is as follows:

First stage screening stage

It is included in 148 people of 148 people of idiopathic male infertility and normal healthy controls to clarify a diagnosis at random, totally 296 people.

A group: healthy control group (148 people):

1. the age is between 19 to 39 years old；

2. constitutional index is between 17 to 31；

3. the male of fecundity health, and have healthy offspring after 5-8 months；

4. without whole body major disease.

B group: idiopathic male infertility disease group (148 people):

1. the age matches with control group；

2. constitutional index is matched with control group

3. pregnancy is attempted not succeed within 12 months, and spouse does not have the male of infertile disease；.

4. without the clear male sterility cause of disease；

5. Smoking And Drinking history is matched with control group；

6. nationality matches with control group；

7. without whole body major disease.

Second stage Qualify Phase

It is included in 15 people of 15 people of idiopathic male infertility and normal healthy controls to clarify a diagnosis, totally 30 people.

A group: healthy control group (15 people):

1. the age is between 24 to 36 years old；

2. constitutional index is between 19 to 24；

3. the male of fecundity health, and have healthy offspring after 5-8 months；

4. without whole body major disease.

B group: idiopathic male infertility disease group (15 people):

1. the age matches with control group；

2. constitutional index is matched with control group；

3. pregnancy is attempted not succeed within 12 months, and spouse does not have the male of infertile disease；

4. without the clear male sterility cause of disease；

5. Smoking And Drinking history is matched with control group；

6. nationality matches with control group；

7. without whole body major disease.

2 UPLC-MS metabolism group idiopathic male infertility deoxycytidine of embodiment and cytidine screening

1. Sample pretreatment

1.1. 10 μ L refinings are taken, 10 μ L internal standard A are added, 10 μ L internal standard B are added, 10 μ L internal standard C are added, methanol (examination is added Agent A) 40 μ L, vortex 30s, protein precipitation.

1.2. 4 DEG C of 16000g are centrifuged 15min in centrifuge, and supernatant is transferred to 1.5mL import EP pipe, and by supernatant It is concentrated to dryness in centrifugal concentrating drying instrument at room temperature.

1.3. it is redissolved with 10 μ L ultrapure waters (reagent D), it is to be analyzed.

2. instrument detects

2.1. analysis instrument: UPLC Ultimate 3000system (Dionex) high performance liquid chromatograph；Q- Exactive high-resolution mass spectrometer.

2.2. liquid-phase condition:

2.2.1 liquid-phase chromatographic column be Hypersil GOLD C18 chromatographic column (100mm × 2.1mm, 1.9 μm of partial size, Thermo Scientific, Germany), column temperature is 40 DEG C.

2.2.2 acetonitrile (examination of water (reagent B) and (B) of the mobile phase used for (A) containing 0.1% formic acid containing 0.1% formic acid Agent C), flow velocity is 400 μ L/min.

2.2.3 instrument gradient are as follows: 0-3min 1%B, 3-10min 1% arrives 99%B, 10-13min 99%B, 13- 13.1min99% to 1%B, 13.1-17min 1%B.

2.2.4 input mode: 5 μ l of volume.

2.3. Mass Spectrometry Conditions

2.3.1 heating electrospray ionisation mode (HESI) is analyzed.

2.3.2 using heating electrospray ionisation mode (HESI), positive ion mode spray voltage: 3.5kV；Negative ion mode Spray voltage: 2.5kV；Capillary temperature under both of which: 250 DEG C, heter temperature: 425 DEG C, sheath gas air-flow: 50AU, auxiliary Gas air-flow: 13AU, blowback air air-flow: 0AU；Lens voltage: 60V.Using mode is swept entirely, scanning range: 70 arrive 1050m/z；Point Resolution: 70000.

3. substance is qualitative

Metabolism small molecule is qualitative to compare Chromatographic information (retention time) and mass spectrum using with standard items deoxycytidine and cytidine Information (accurate molecular weight), and compare the Chromatographic information of Isotopic Internal Standard standard items series in sample in real time with corrected retention time Between.

4. data are analyzed:

Biomarker screening confirms key metabolites using Multivariate Logistic Regression.

5. being metabolized the difference and diagnostic significance of small molecule in healthy control group, idiopathic male infertility group refining sample.

The corrected age, constitutional index, smoking and history of drinking history information, logistic regression find refining The content of deoxycytidine and cytidine and idiopathic male infertility are closely related (Fig. 1).

The stability analysis of embodiment 3 refining deoxycytidine and cytidine

The method of embodiment 2 is used to be evaluated (interval time 2 to the stability of refining deoxycytidine and cytidine level Week).The results show that deoxycytidine and cytidine measurement horizontal stable (Fig. 2) in refining, have as diagnosis/monitoring marker Characteristic.

4 deoxycytidine of embodiment and cytidine combine the diagnosis to idiopathic male infertility

According to above-mentioned UPLC-Q exactive MS metabolism group method, the present inventor by random 15 case of crowd and The seminal plasma sample detection deoxycytidine and cytidine of 15 controls, the sensitivity and specificity of ROC curve and assessment prediction are drawn with this, And then evaluation capacity of this 2 metabolism small molecule levels to idiopathic male infertility in assessment detection refining.

The sensitivity of deoxycytidine is 93.33%, specificity 73.33%, and area is 0.8756 under ROC curve；Cytidine Sensitivity is 80.00%, specificity 86.67%, and area is 0.9244 under ROC curve.

The sensitivity for combining deoxycytidine and cytidine is 93.33%, specificity 93.33%, and area is under ROC curve 0.9822。

So combining deoxycytidine and cytidine has the ability for preferably diagnosing idiopathic male infertility.

Embodiment 5 is used for the production of idiopathic male infertility refining deoxycytidine and cytidine detection and diagnosis kit

Determine in normal control and idiopathic male infertility refining have by the method for UPLC-Q exactive MS first There is the metabolism small molecule compared with high abundance.Then, it is wherein being sieved by the metabonomic technology based on UPLC-Q exactive MS Select relevant to idiopathic male infertility metabolism small molecule, as whether be idiopathic male infertility diagnosis index.Finally will The quantity of the correspondence refining metabolism small molecule filtered out is controlled at 2, this is the optimization made on the basis of preliminary experiment It simplifies.This kit includes a collection of refining metabolism small molecule detection reagent and consumptive material, wherein metabolism small molecule is qualitative and fixed Amount uses the standard items of deoxycytidine and cytidine, and assistant analysis uses internal standard A: creatinine, valine, niacin, thymidine, penta 2 The deuterium mark Isotopic Internal Standard of acid, eight kinds of L- phenylpropylamine acid, N- acetaminophen, hippuric acid substances.Internal standard B: pentadecanoic acid Deuterium mark Isotopic Internal Standard.Internal standard C: the deuterium mark Isotopic Internal Standard of lignoceric acid.It is other that there are also for UPLC chromatographic isolation Mating reverse chromatograms column (Hypersil GOLD C18 chromatographic column, 100mm × 2.1mm, 1.9 μm of partial size), for precipitating refining egg White reagent (100% methanol), for mobile phase reagent (water containing 0.1% formic acid and containing 0.1% formic acid acetonitrile), For extracting the reagent (100% ultrapure water) of metabolism small molecule.The value of this kit is only to need 10 μ l refinings, Ji Kejian The content of deoxycytidine and cytidine is surveyed, then idiopathic male infertility is diagnosed by content, and be easy to carry out dynamic monitoring and observation Therapeutic effect.

Specific kit forms are as follows:

Deoxycytidine standard items

Cytidine standard items

Internal standard A (creatinine, valine, niacin, thymidine, glutaric acid, L- phenylpropylamine acid, N- acetaminophen, horse The deuterium mark Isotopic Internal Standard aqueous solution of eight kinds of substances of uric acid)

Internal standard B (the deuterium mark Isotopic Internal Standard methanol solution of pentadecanoic acid)

Internal standard C (the deuterium mark Isotopic Internal Standard methanol solution of lignoceric acid)

Further, can also contain:

Chromatographic column (Thermo 100mm × 2.1mm, 1.9 μm of partial size, Hypersil GOLD C18 chromatographic column)

Reagent A (100% methanol)

Reagent B (water containing 0.1% formic acid)

Reagent C (acetonitrile containing 0.1% formic acid)

Reagent D (100% ultrapure water).

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Claims (8)

1. the relevant refining marker of idiopathic male infertility, it is characterised in that the marker is deoxycytidine and/or cytidine.

2. refining marker described in claim 1 is preparing the application in idiopathic male infertility diagnosis or monitoring reagent.

3. the kit of a kind of diagnosis or monitoring idiopathic male infertility, it is characterised in that the kit contains to be taken off in detection refining The reagent of oxygen cytidine and/or cytidine.

4. kit according to claim 3, it is characterised in that the kit contains using UPLC-Q exactive MS Method detects the reagent of deoxycytidine and/or cytidine in refining.

5. kit according to claim 4, it is characterised in that the kit contains following reagent:

Deoxycytidine and/or cytidine standard items；

Internal standard A: creatinine, valine, niacin, thymidine, glutaric acid, L- phenylpropylamine acid, N- acetaminophen, hippuric acid In one or more substances Isotopic Internal Standard；

Internal standard B: the Isotopic Internal Standard of pentadecanoic acid；

Internal standard C: the Isotopic Internal Standard of lignoceric acid.

6. kit according to claim 5, it is characterised in that the kit also contains:

Hypersil GOLD C18 chromatographic column；

Reagent A: protein precipitation is used, and contains 100% methanol；

Reagent B: mobile phase is used, the water containing 0.1% formic acid；

Reagent C: mobile phase is used, the acetonitrile containing 0.1% formic acid；

Reagent D: redissolving and use, ultrapure water.

7. a kind of method for detecting refining biomarker relevant to idiopathic male infertility as described in claim 1, It is characterized in that this method using UPLC-Q exactive MS method, detects deoxycytidine and/or cytidine content in refining.

8. according to the method described in claim 7, it is characterized in that in this method:

One, liquid-phase condition:

Liquid-phase chromatographic column is Hypersil GOLD C18 chromatographic column, and column temperature is 40 DEG C；

Mobile phase A is the water containing 0.1% formic acid, and Mobile phase B is the acetonitrile containing 0.1% formic acid, and flow velocity is 400 μ L/min；

Instrument gradient are as follows: 0-3min 1%B, 3-10min 1% arrives 99%B, 10-13min 99%B, 13-13.1min 99% To 1%B, 13.1-17min 1%B；

Input mode: 5 μ l of volume；

Two, Mass Spectrometry Conditions

It is analyzed using heating electrospray ionisation mode (HESI), positive ion mode spray voltage: 3.5kV；Negative ion mode Spray voltage: 2.5kV；Capillary temperature under both of which: 250 DEG C, heter temperature: 425 DEG C, sheath gas air-flow: 50AU, auxiliary Gas air-flow: 13AU, blowback air air-flow: 0AU；Lens voltage: 60V；Using mode is swept entirely, scanning range: 70 arrive 1050m/z；Point Resolution: 70000.