CN109406659A - A kind of method and its detection kit for identifying bronchiectasis biomarker - Google Patents

Abstract

The present invention provides for identifying biomarker and its application of bronchiectasis.The biomarker is the free glucose and mannose that patients serum obtains by high performance liquid chromatography (HPLC) derived from 1- phenyl -5- methylpyrazole quinoline ketone (PMP) before column.Technical solution of the present invention has pre-treatment simple, analysis time is short, instrument price is reasonable, meet conventional use, operating procedure is easy to learn, and testing result accuracy is high, and need to only take a blood sample carries out the analysis of HPLC instrument and can distinguish normal person and patient, and the advantages that required serum amount is few, and blood sampling volume is less than 1mL.Acquired results show that this analysis method can quickly analyze the mannose and glucose to dissociate in serum, and mannose, glucose sugar concentration and the two peak area can be calculated, there is very important meaning for the marker of the relationship between research free serum mannose and glucose and disease, the novel bronchiectasis Disease Clinical detection of searching.

Description

A kind of method and its detection kit for identifying bronchiectasis biomarker

Technical field

The invention belongs to pharmaceutical technology fields, and in particular to for identifying biomarker and its inspection of bronchiectasis Survey application.

Background technique

Bronchiectasis (branch expands, Bronchiectasis) refers to a kind of disease of bronchiole irreversibility abnormal dilatation Disease.Due to bronchus and its surrounding lung tissue chronic suppurative inflammation and fibrosis, make the muscle and elastic fibrous tissue of bronchial wall It destroys, bronchus is caused to deform and persistently expand.Bronchiectasis may be caused by multi-infection and posteriority reason, including Pneumonia, pulmonary tuberculosis, immune system problem and genetic disease cystic fibrosis.Typical symptom has chronic cough, cough a large amount of It phlegm and spits blood repeatedly.In nearly all case, cystic fibrosis eventually leads to serious bronchiectasis.There is no capsule fine There is the reason of 10-50% unclear in the patient of dimensionization.Due to excessive inflammatory reaction, the air flue (bronchus) being related to becomes It is bigger, so that secretion can not be removed.These secretion will increase the quantity of Pulmonary bacterial, cause air flue blocking and air flue into The rupture of one step.The bronchiectasis course of disease is in chronic process more, can betide any age.This disease is more common in women, Increase with advancing age.It is that the terminal structure that numerous diseases are in progress changes that branch, which expands, and disease incidence is 53~5,66/,100,000, And as the raising disease incidence of the increase at age and diagnostic techniques increases increasingly.Branch expands course of disease length and complicated, diverse clinical manifestations, Light and heavy degree is different, influences patient's various aspects, and underlying diseases are further aggravated and cause decline in pulmonary function, poor prognosis seriously affects life Bioplasm amount, case fatality rate increase.CT images technology is inspection method more commonly used in clinic.The disease that branch is expanded in current daily diagnosis and treatment Because diagnosis and Severity are inadequate, it should further pay attention to layering diagnosis, identify high-risk patient early, propose rational therapy side Case.Therefore, establishing reliable bronchiectasis early diagnosis screening method has preferable clinical meaning.How branch is accurately identified The crowd of tracheaectasy so as to early intervention, or can screen effect that suitable biomarker is treated with predictive disease or The worth research such as the progress of predictive disease.

The content highest of glucose in human serum free monosaccharide, the range of normal Check-up crowd is 3.90~ 6.16mmol/L.Currently, hospital laboratory directly can detect concentration of glucose by surveying biochemical indicator, it temporarily cannot be direct Detect mannose concentration.The concentration range of free mannose is 20~80 μm of ol/L in health adult's blood plasma.The content of mannose About the 1% of glucose content.Its major part is considered as from the glucose isomerization in cell.And there is research table recently Bright, the free mannose flowed out in cell is the main source of free mannose in mammalian.This two parts source Free mannose maintain the stabilization of mannose in serum.D-MANNOSE is glycoprotein, cell surface glycoconjugates and glycosyl Change required monosaccharide in the structures such as phosphatidylinositols anchorin.Wherein, mannose is N- sugar chain in various polysaccharide compounds Important component, glucose or mannose of the mannose residue in blood in N- glycan.Free mannose is normal Plasma composition.Currently, the measuring method of serum mannose mainly has enzyme process, gas-liquid chromatography, high resolution liquid chromatography, gas-liquid color Spectrum-mass spectrography and capillary electrophoresis etc..But there is certain shortcoming in these methods.For example, using enzyme process, gas-liquid color When spectrometry and high resolution liquid chromatography, to avoid the influence needs of glucose of high concentration from removing it, cause pretreatment process It is comparatively laborious；Gas-liquid chromatography-mass spectrography instrument price is expensive, is not suitable for routine purpose；Required serum sample amount is big.Currently without High performance liquid chromatography derived from 1- phenyl -5- methylpyrazole quinoline ketone (PMP) detects bronchiectasis simultaneously before document report column The mannose and glucose to dissociate in disease patients serum.

Therefore, establish in the serum of efficiently and accurately dissociate mannose detection method, for research free serum monosaccharide with The marker of relationship, searching Disease Clinical detection between disease has very important meaning.

The PMP derivatization method of monosaccharide is current more common monosaccharide detection method.This method including the following steps: (1) alkaline condition is created for PMP, (2) are added PMP, are derived at 70 DEG C, and (3) adjust the pH value of solution after deriving, will be remaining PMP is extracted.103969371 B of patent of invention CN discloses that " a kind of blood degrades to obtain and detect the method for monosaccharide in cancer Application in disease detection ".Polysaccharide therein and glycoprotein are degraded to by the patent of invention first by blood serum sample by acid degradation Monosaccharide component；Then the 8 kinds of monosaccharide obtained after PMP is derivative and efficient liquid phase is to degradation detect.Before degradation Serum, sample component and property have occurred and that great variety.Although the PMP of monosaccharide spreads out, method is wider in every field application It is general, but there is no inspection of the report by this method applied to dissociate in the serum of patients with bronchiectasis mannose and glucose at present It surveys.

Summary of the invention

The object of the present invention is to provide for identifying biomarker and its application of bronchiectasis, the present invention is adopted The high performance liquid chromatography derived from PMP before column (HPLC) detects the monosaccharide in patients with bronchiectasis serum, more particularly to The detection of free mannose and glucose in serum.Technical solution of the present invention can be used for detecting patients with bronchiectasis or Normal Check-up crowd free serum glucose and mannose concentration and to the two calculated by peak area ratio, can be used for identifying branch gas Enlargement of pipe disease patient.

To achieve the purpose of the present invention, the present invention is achieved by the following technical programs:

For identifying that the biomarker of bronchiectasis, the biomarker are patients serum by PMP before column The free mannose and glucose that derivative high performance liquid chromatography obtains.

For identifying the quantitative analysis method of the biomarker of bronchiectasis, it is characterised in that: this method is column High performance liquid chromatography derived from preceding PMP, comprising the following specific steps

(1) adjusting pH is 7-14: taking blood serum sample to be detected to add ultrapure water in EP pipe, sodium hydroxide is added, be vortexed It mixes；

(2) PMP is derivative: PMP is added in each sample, is vortexed and mixes, and centrifugation is placed on baking oven reaction；

(3) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and hydrochloric acid is added in each sample, is vortexed mixed It is even；

(4) extract: organic solvent is added in every pipe, is vortexed, and centrifugation removes lower layer's organic solvent, retains supernatant；

(5) sample is centrifuged, takes supernatant in sample bottle, covering tightly lid in the brown equipped with interpolation pipe, is centrifuged, is ready to use in High-efficient liquid phase analysis；

(6) standard curve of mannose and glucose is prepared；

(7) standard curve in the chromatogram for obtaining step (5) and step (6) compares simultaneously analytical calculation, determines sweet dew The ratio of sugar, the concentration of glucose and glucose and mannose concentration.

The high performance liquid chromatography is 1260 efficient liquid phase system of Agilent, Agilent Poroshell EC-C18 color It composes column (4.6mm × 100mm, 2.7 μm), the chromatographic condition in the step (5) are as follows:

Detection wavelength: 254nm, bandwidth 4nm；

Reference wavelength: 350nm, bandwidth 100nm；

Column temperature: 37 DEG C；

Flow velocity: 1mL/min；

Sampling volume: 20 μ L；

Mobile phase: A phase is acetonitrile, and B phase is 0.1mol/L ammonium acetate-acetic acid buffer solution that pH is 5.5.

The change of gradient of mobile phase in the step (5) are as follows:

0min, A phase 15%, B phase 85%；

10min, A phase 22%, B phase 78%；

15min, A phase 24%, B phase 76%；

15.1min, A phase 15%, B phase 85%；

20min, A phase 15%, B phase 85%.

The concentration of hydrochloric acid is 0.3mol/L in the step (3).

Organic solvent is at least one in chloroform, methylene chloride, ethyl acetate and n-hexane in the step (4) Kind.

Biomarker is preparing the purposes in the kit for detecting bronchiectasis.

Biomarker is preparing the purposes in the kit for detecting bronchiectasis, it is characterised in that: described It include concentration in kit is 91.17 μm of ol/L mannoses and concentration is the titer of 5258 μm of ol/L glucose.

A kind of kit for identifying bronchiectasis, which is characterized in that the kit contains biomarker, i.e. serum The free mannose and glucose obtained by high performance liquid chromatography derived from PMP before column.

It include concentration in the kit is 91.17 μm of ol/L mannoses and concentration is 5258 μm of ol/L glucose Titer.

Identify that the kit of bronchiectasis is preparing the purposes in the kit for detecting bronchiectasis.

Compared with prior art, high performance liquid chromatography derived from PMP before column is used for bronchiectasis trouble by the present invention The detection of person's free serum monosaccharide, advantageous effects are:

1, pre-treatment is simple.

2, analysis time is short.

3, required amount of serum is few, simply needs to be less than the blood volume of 1mL every time.

4, instrument price is reasonable, meets conventional use.

5, operating procedure is easy to learn.

6, detection is accurate.

The present invention uses hplc simultaneous determination free serum mannose and glucose derived from PMP before column, Simplify operation, and amount of serum is few, and the measurement for having proven to the method mannose concentration will not be by high concentration glucose Influence.Pass through the ratio (G/M ratio) to free serum glucose, mannose concentration and glucose and mannose peak area Statistical data analysis, establish the relationship with bronchiectasis.Using technical solution of the present invention, by detecting serum In dissociate mannose and glucose, calculate mannose and concentration of glucose and glucose and mannose peak area ratio, Normal population and patients with bronchiectasis can quickly be distinguished.

Detailed description of the invention

Fig. 1 is two types column chromatography figure

Fig. 2 is the chromatogram of different eluent gradients

Fig. 3 is patients with bronchiectasis and normal Check-up crowd free serum concentration of glucose, mannose concentration and Portugal The scatter plot of grape sugar and mannose peak area ratio

Fig. 4 is patients with bronchiectasis and normal Check-up crowd glucose and mannose peak area ratio, as serum The ROC curve figure of sugared biomarker

Fig. 5 is patients with bronchiectasis and normal Check-up crowd free serum concentration of glucose, mannose concentration and Portugal The 3 dimensional drawing of grape sugar and the building of mannose peak area ratio

Specific embodiment

Technical solution of the present invention is further described in detail in the following with reference to the drawings and specific embodiments.

All blood samples involved in embodiment are provided by affiliated hospital, University Of Qingdao, Qingdao City.

Embodiment 1

Technical solution of the present invention the following steps are included:

(1) precision weighs mannose (Man), gucosamine (GlcN), galactosamine (GalN), glucuronic acid (GlcUA), glucose (Glc), galactolipin (Gal), xylose (Xyl), fucose (Fuc) in right amount, add deionized water to prepare two parts Containing the above monosaccharide 0.1mg/mL hybrid standard product solution, matching while using；

(2) it creates alkaline environment: taking 40 μ L mixing monosaccharide standard to manage in 1.5mL EP, 40 μ L 0.3mol/L hydrogen are added Sodium oxide molybdena is vortexed and mixes；

(3) PMP is derivative: 60 μ L PMP (1- phenyl -5- methylpyrazole quinoline ketone) is added in each sample, is vortexed and mixes, centrifugation 70 DEG C of baking ovens react 1h afterwards；

(4) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample Hydrochloric acid is vortexed and mixes；

(5) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight It is 3 times multiple；

(6) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed In bottle, lid is covered tightly, is centrifuged, two parts of samples carry out high-efficient liquid phase analysis, efficient liquid phase using different chromatographic column A and B respectively Chromatography is 1260 efficient liquid phase system of Agilent, and gained chromatogram is shown in Fig. 1.

Chromatographic column A:Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm)；

Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm) chromatographic condition:

Detection wavelength: 254nm, bandwidth 4nm；

Reference wavelength: 350nm, bandwidth 100nm；

Column temperature: 37 DEG C；

Flow velocity: 1mL/min；

Sampling volume: 20 μ L；

Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；

Change of gradient:

Time	Acetonitrile	0.1mol/L ammonium acetate pH 5.5
			0min	15%	85%
10min	22%	78%
			15min	24%	76%
15.1min	15%	85%
			20min	15%	85%

Chromatographic column B:Agilent ZORBAX XDB-C18 chromatographic column (4.6mm × 150mm, 5 μm)；

Agilent ZORBAX XDB-C18 chromatographic column (4.6mm × 150mm, 5 μm) chromatographic condition:

Detection wavelength: 254nm, bandwidth 4nm；

Reference wavelength: 350nm, bandwidth 100nm；

Column temperature: 37 DEG C；

Flow velocity: 1mL/min；

Sampling volume: 20 μ L；

Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；

Change of gradient:

Time	Acetonitrile	0.1mol/L ammonium acetate pH 5.5
			0min	15%	85%
40min	22%	78%
			40.1min	15%	85%
55min	15%	85%

It will be seen from figure 1 that the Agilent ZORBAX XDB-C18 chromatogram column analysis time is long, chromatographic peak separating degree is poor, The Agilent Poroshell EC-C18 chromatogram column analysis time is the 1/3 of ZORBAX XDB-C18 chromatographic column, and chromatographic peak is good. Abscissa is time (min), and ordinate is DAD signal strength.1,2,3,4,5,6,7,8 successively Man, GlcN, GalN, GlcUA, The chromatographic peak of Glc, Gal, Xyl, Fuc.

Embodiment 2

(1) precision weighs mannose (Man), rhamnose (Rha) and glucose (Glc) in right amount, and deionized water is added to prepare phase Same 5 parts contain the above monosaccharide 0.1mg/mL hybrid standard product solution, matching while using；

(2) it creates alkaline environment: taking 40 μ L mixing monosaccharide standard to manage in 1.5mL EP, 40 μ L 0.3mol/L hydrogen are added Sodium oxide molybdena is vortexed and mixes；

(3) PMP is derivative: 60 μ L 0.5mol/L PMP are added in each sample, are vortexed and mix, 70 DEG C of baking oven reactions after centrifugation 1h；

(4) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample Hydrochloric acid is vortexed and mixes；

(5) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight It is 3 times multiple；

(6) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed In bottle, lid is covered tightly, is centrifuged, 5 parts of samples are used for high-efficient liquid phase analysis, gained chromatography under different mobile phase variable gradients respectively Figure, is shown in Fig. 2.

High performance liquid chromatography is 1260 efficient liquid phase system of Agilent, Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm)；

Chromatographic condition:

Detection wavelength: 254nm, bandwidth 4nm；

Reference wavelength: 350nm, bandwidth 100nm；

Column temperature: 37 DEG C；

Flow velocity: 1mL/min；

Sampling volume: 20 μ L；

Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；

Mobile phase variable gradient A:

Time	Acetonitrile	0.1mol/L ammonium acetate pH5.5
			0min	15%	85%
20min	15%	85%

Mobile phase variable gradient B:

Time	Acetonitrile	0.1mol/L ammonium acetate pH5.5
			0min	20%	80%
20min	20%	80%

Mobile phase variable gradient C:

Time	Acetonitrile	0.1mol/L ammonium acetate pH5.5
			0min	22%	78%
20min	22%	78%

Mobile phase variable gradient D:

Time	Acetonitrile	0.1mol/L ammonium acetate pH5.5
			0min	15%	85%
10min	22%	78%
			15min	15%	85%
20min	15%	85%

Mobile phase variable gradient E:

Time	Acetonitrile	0.1mol/L ammonium acetate pH5.5
			0min	15%	85%
10min	22%	78%
			15min	24%	76%
15.1min	15%	85%
			20min	15%	85%

Figure it is seen that only Man is eluted out under the conditions of gradient A；Under the conditions of gradient B, although three Can be separated and, but the peak of solvent PMP and Man from it is relatively close；And under the conditions of gradient C, solvent peak and the peak Man weight It closes；Under the conditions of gradient D, the overlong time that the peak Glc is eluted out leads to cannot to be formed point and high peak.Only in gradient E item Under part, appearance is symmetrical, and separating degree is good.Abscissa is time (min), and ordinate is DAD signal strength.1,2 and 3 is successively The chromatographic peak of Man, Rha and Glc.

Embodiment 3

(1) precision weighs mannose and proper amount of glucose, and deionized water is added to be configured to each 0.5mg/mL containing the above monosaccharide, 0.25mg/mL, 0.1mg/mL, 0.05mg/mL, 0.01mg/mL, 0.005mg/mL, 0.0025mg/mL, 0.001mg/mL, The hybrid standard product solution of 0.0005mg/mL, matching while using；

(2) it creates alkaline environment: taking 40 μ L mixing monosaccharide standard to manage in 1.5mL EP, 40 μ L 0.3mol/L hydrogen are added Sodium oxide molybdena is vortexed and mixes；

(3) PMP is derivative: 60 μ L 0.5mol/L PMP are added in each sample, are vortexed and mix, 70 DEG C of baking oven reactions after centrifugation 1h；

(4) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample Hydrochloric acid is vortexed and mixes；

(5) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight It is 3 times multiple；

(6) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed In bottle, lid is covered tightly, is centrifuged, is ready to use in high-efficient liquid phase analysis.

High performance liquid chromatography is 1260 efficient liquid phase system of Agilent, Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm)；

Chromatographic condition:

Detection wavelength: 254nm, bandwidth 4nm；

Reference wavelength: 350nm, bandwidth 100nm；

Column temperature: 37 DEG C；

Flow velocity: 1mL/min；

Sampling volume: 20 μ L；

Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；

Mobile phase variable gradient:

(7) standard curve of mannose and glucose is drawn.

Embodiment 4

(1) it creates alkaline environment: taking 95 patients with bronchiectasis made a definite diagnosis and 95 age-sex's matchings respectively 10 μ L of normal Check-up crowd blood serum sample add 30 μ L ultrapure waters in 1.5mL EP pipe, be added 40 μ L 0.3mol/L hydroxides Sodium is vortexed and mixes；

(2) remaining step is the same as embodiment 3, analytical calculation glucose, the concentration of mannose and glucose and mannose peak The ratio of area.Statistic mixed-state is as a result, be shown in Fig. 3.Fig. 3 result is summarized to Tables 1 and 2.

1. patients with bronchiectasis free serum monosaccharide of table and G/M peak area ratio with normal Check-up crowd it is opposite become Gesture

	Glucose	Mannose	G/M ratio
				Bronchiectasis	-NS	↑**	↓***

Annotation: there was no significant difference compared with normal Check-up crowd for " NS " representative, " * * * p < 0.001 ", " * * p < 0.01 " " * p < 0.05 ", which is respectively represented, has significant difference compared with normal Check-up crowd, and " ↑ " and " ↓ " respectively represents and normal physical examination Compared to rising or falling, "-" is indicated with normal Check-up crowd without significant change crowd.

2 kinds of free monosaccharide concentration (μm ol/L) and its peak in 2. patients with bronchiectasis of table and normal Check-up crowd serum Area ratio result

	Glucose	Mannose	G/M ratio
				Normal Check-up crowd	5272±1466	65.6±20.48	81.8±15.95
Bronchiectasis	5258±3746	91.17±71.01	49.71±15.69

In addition, to thering is the mannose of significant difference and glucose peaks area ratio to carry out ROC song in table 1 with normal person Line analysis finds the higher index of area under the curve (AUC), sensitivity and specificity with expectation, to determine best screening sun Property critical value (cutoff value), AUC illustrate that diagnosis effect is better closer to 1.Testing result is shown in Fig. 4, and Fig. 4 result is summarized To table 3.

	AUC	Cutoff value	Sensitivity	Specificity
					G/M ratio	0.9230	62.35	51.61%	91.94%

From Fig. 3, table 1, table 2 can be seen that the matched normal Check-up crowd of age-sex and compare, bronchiectasis patient's blood There was no significant difference for concentration of glucose free clearly, and mannose concentration significantly rises, and glucose and mannose peak area ratio are significant Decline.In addition, table 3 is shown, the specificity and sensitivity of mannose and glucose peaks area ratio are higher, illustrate free serum Glucose and mannose peak area ratio system related with bronchiectasis disease, G/M ratio can distinguish normal person and branch gas Enlargement of pipe disease patient.Therefore, branch gas can be detected as biomarker using glucose free clearly and mannose peak area ratio Enlargement of pipe disease patient.

Embodiment 5

It is used to detect the application of bronchiectasis using biomarker of the present invention, specifically includes following step It is rapid:

(1) it creates alkaline environment: 10 μ L of blood serum sample to be detected being taken to add 30 μ L ultrapure waters in 1.5mL EP pipe, be added 40 μ L 0.3mol/L sodium hydroxides are vortexed and mix；

(2) PMP is derivative: 60 μ L PMP are added in each sample, are vortexed and mix, and 70 DEG C of baking ovens react 1h after centrifugation；

(3) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample Hydrochloric acid is vortexed and mixes；

(4) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight It is 3 times multiple；

(5) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed In bottle, lid is covered tightly, is centrifuged, is ready to use in high-efficient liquid phase analysis.High performance liquid chromatography is 1260 efficient liquid phase system of Agilent, Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm)；

Chromatographic condition are as follows:

Detection wavelength: 254nm, bandwidth 4nm；

Reference wavelength: 350nm, bandwidth 100nm；

Column temperature: 37 DEG C；

Flow velocity: 1mL/min；

Sampling volume: 20 μ L；

Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；

Change of gradient:

Time	A (acetonitrile)	B (0.1mol/L ammonium acetate pH 5.5)
			0min	15%	85%
10min	22%	78%
			15min	24%	76%
15.1min	15%	85%
			20min	15%	85%

(6) draw the standard curve of glucose and mannose: precision weighs glucose and appropriate mannose, adds deionized water It is configured to each 0.5mg/mL, 0.25mg/mL, 0.1mg/mL, 0.05mg/mL, 0.01mg/mL, 0.005mg/mL containing the above monosaccharide, The hybrid standard product solution of 0.0025mg/mL, 0.001mg/mL, 0.0005mg/mL, matching while using；Take 40 μ L mixing monosaccharide marks Quasi- product are managed in 1.5mL EP, and 40 μ L 0.3mol/L sodium hydroxides are added, and are vortexed and are mixed；Remaining step with (2), (3), (4), (5)。

(7) standard curve in the chromatogram for obtaining step (5) and step (6) compares simultaneously analytical calculation, determines grape The ratio of sugar, the concentration of mannose and glucose and mannose peak area.And it is made between three by 9 software of Origin 3 dimensional drawing, normal person and pneumothorax patient can be distinguished more intuitively to observe triple combination, see Fig. 5.

ROC curve is made by Prism software, to determine a possibility that G/M ratio is as marker, as a result, it has been found that quick Perception is 51.61%, and specificity is 91.94%, sensibility and specificity with higher.

If the free glucose and mannose peak area ratio in test serum sample determine to be measured less than 49.71 Blood serum sample is patients with bronchiectasis.

Result above shows: this method pre-treatment is simple；Analysis time is short；Required amount of serum is few, every time only The blood volume of 1mL need to be less than；Instrument price is reasonable, meets conventional use；Operating procedure is easy to learn；Detection is accurate.Institute of the present invention The method stated has very the marker of relationship, searching Disease Clinical detection between research free serum monosaccharide and pneumothorax Important meaning will be suitble to clinically be used for the diagnosis of patients with bronchiectasis.

The above examples are only used to illustrate the technical scheme of the present invention, rather than is limited；Although referring to aforementioned reality Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these are modified or replace It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.

Claims (13)

1. for identifying the biomarker of bronchiectasis, it is characterised in that: the biomarker is that serum passes through column The free glucose and mannose that high performance liquid chromatography (HPLC) derived from preceding 1- phenyl -5- methylpyrazole quinoline ketone (PMP) obtains.

2. according to claim 1 for identifying the biomarker of bronchiectasis, it is characterised in that: this method is High performance liquid chromatography derived from PMP before column, comprising the following specific steps

(1) adjusting pH is 7-14: taking blood serum sample to be detected to add ultrapure water in EP pipe, sodium hydroxide is added, be vortexed and mix；

(2) PMP is derivative: PMP is added in each sample, is vortexed and mixes, and centrifugation is placed on baking oven reaction；

(3) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and hydrochloric acid is added in each sample, is vortexed and is mixed；

(4) extract: organic solvent is added in every pipe, is vortexed, and centrifugation removes lower layer's organic solvent, retains supernatant；

(5) sample is centrifuged, takes supernatant in sample bottle, covering tightly lid in the brown equipped with interpolation pipe, is centrifuged, is ready to use in efficiently Liquid phase analysis；

(6) standard curve of glucose and mannose is prepared；

(7) standard curve in the chromatogram for obtaining step (5) and step (6) compares and analytical calculation, determines glucose, sweet Reveal the concentration of sugar and the ratio of glucose and mannose peak area.

3. according to claim 1 for identifying the biomarker of bronchiectasis, it is characterised in that: described efficient Liquid chromatogram be 1260 efficient liquid phase system of Agilent, Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm), the chromatographic condition in the step (5) are as follows:

Detection wavelength: 254nm, bandwidth 4nm；

Reference wavelength: 350nm, bandwidth 100nm；

Column temperature: 37 DEG C；

Flow velocity: 1mL/min；

Sampling volume: 20 μ L；

Mobile phase: A phase is acetonitrile, and B phase is 0.1mol/L ammonium acetate-acetic acid buffer solution that pH is 5.5.

4. according to claim 3 for identifying the biomarker of bronchiectasis, it is characterised in that: the step (5) change of gradient of mobile phase in are as follows:

0min, A phase 15%, B phase 85%；

10min, A phase 22%, B phase 78%；

15min, A phase 24%, B phase 76%；

15.1min, A phase 15%, B phase 85%；

20min, A phase 15%, B phase 85%.

5. according to claim 2 for identifying the biomarker of bronchiectasis, it is characterised in that: the step (3) concentration of hydrochloric acid is 0.3mol/L in.

6. according to claim 2 for identifying the quantitative analysis method of bronchiectasis biomarker, feature Be: organic solvent is at least one of chloroform, methylene chloride, ethyl acetate and n-hexane in the step (4).

7. the quantitative analysis method of identification bronchiectasis biomarker is in bronchiectasis described in claim 2-6 Application in disease, it is characterised in that the blood serum sample is the serum of patients with bronchiectasis.

8. a kind of kit for identifying bronchiectasis, which is characterized in that the kit contains biology described in claim 1 The free mannose and glucose that marker, i.e. serum are obtained by high performance liquid chromatography derived from PMP before column.

9. the kit of identification bronchiectasis according to claim 8, which is characterized in that include in the kit The titer for the glucose that concentration is 91.17 μm of ol/L mannoses and concentration is 5258 μm of ol/L.

10. the described in any item reagent consumptive materials of claim 2-9 are in preparation for mannose in patients with bronchiectasis serum With the purposes in the kit of glucose quantitation.

11. a kind of kit for identifying bronchiectasis, it is characterised in that pass through the mannose and glucose quantitation in serum Identify bronchiectasis.

12. the kit of the described in any item identification bronchiectasis of claim 8-11 detects bronchiectasis in vitro Purposes.

13. purposes according to claim 12, it is characterised in that identified by mannose in serum and glucose quantitation Bronchiectasis.