CN103954748B - The method of H7N9 biomarker and application thereof in the outer blood plasma of screen body - Google Patents
The method of H7N9 biomarker and application thereof in the outer blood plasma of screen body Download PDFInfo
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Abstract
The invention discloses method and the application thereof of H7N9 biomarker in the outer blood plasma of a kind of screen body.1) plasma sample of many cases H7N9 patient is prepared, 2) cytokine and the chemotactic factor of plasma sample are detected, obtain cytokine and the data of chemotactic factor, 3) cytokine in H1N1 patient, normal healthy controls blood plasma and chemotactic factor are compared, analyzing and processing data, obtain H7N9 biomarker, 4) from the H7N9 biomarker described in step 3), determine the biomarker of mortality risk.The present invention carries out detection from immune molecule angle analyze cytokine and the chemotactic factor of H7N9 patient, thus sets up effective H7N9 immune molecule collection of illustrative plates, and uses it for evaluating in vitro H7N9 death risk.The sensitivity of the method and specificity are superior to traditional detection method.
Description
Technical field
The present invention relates to technical field of biological, particularly relate to new immunological marker thing-H7N9 patients blood plasma's cytokine and chemotactic factor, the risk of H7N9 death can be detected in vitro.
Background technology
Human avian flu virus is different with neuraminidase (N) protein antigenicity according to influenza virus A avian adventitia hemagglutinin (H), has now been found that and be defined as 17 H hypotypes (H1 ~ H17) and 10 N hypotypes (N1～N10).Above-mentioned H hypotype and N hypotype are mutually combined the influenza virus A avian defining hundreds of different HxNx hypotypes, and birds particularly aquatic bird is usually the natural reservoir (of bird flu viruses) of these influenza virus, and main existence and spread and epidemic in birds.
Before 2013, H7N9 subtype influenza virus only finds in birds, on Holland, Japan and the U.S. and other places, epidemic outbreaks between fowl once occurred, but does not finds remarkable infection conditions.In by the end of March, 2013, and novel H7N9 subtype avian influenza virus takes the lead in Shanghai and two places, Anhui finding.From in April, 2013 on March 7th, 2014, people from whole nation infects H7N9 bird flu confirmed cases and amounts to 384 examples, the most dead 100 examples.In the 111 example patients before reported, the patient of 76.6% hospitalizes ICU treatment, and the patient evolution wherein having 70% is adult respiratory distress syndrome.Patient's mean age is 61 years old, and over-65s Proportion of patients reaches 42.3%, and 31.5% patient is women.Although having carried out comprehensive treatment, mortality is still up to 27%.
There is cytokinemia in H7N9 the infected's peripheral blood.It is reported and the blood of H5N1 infected patient there is also cytokinemia.But, there is no the biomarker report of the predictive disease order of severity and prognosis.Serious symptom H7N9 the infected's mortality rate is high, although in the urgent need to progression of disease and the biomarker of prognosis of predicting potential fatal avian influenza infection, but there is no disclosed report at present.
Liquid-phase chip, also referred to as microsphere suspending chip, it it is biochip technology platform based on xMAP technology, it is to carry out Ag-Ab, enzyme-substrate, the association reaction of ligand-receptor and nucleic acid hybridization reaction on the microsphere that difference is fluorescence-encoded, detect microsphere respectively by two bundle laser red, green to encode and reporter fluorescence reaches qualitative and quantitative purpose, up to 100 kinds different biologicallies can be completed in one reacting hole, be the high flux molecular detection technology platform of new generation after gene chip, protein chip.
Summary of the invention
In order to overcome the deficiencies in the prior art, it is an object of the invention to provide method and the application thereof of H7N9 biomarker in the outer blood plasma of screen body.The present invention establishes a set of effective method, for vitro detection H7N9 cytokine and Chemokines Levels in the blood plasma separated in order to assess the risk of H7N9 death, i.e. by measuring H7N9 patient, H1N1 patient and the cytokine of normal healthy controls group and chemotactic factor, carry out variation analysis, find new immunological marker thing, thus H7N9 patient is carried out more fully early screening and detection.
The technical solution adopted for the present invention to solve the technical problems is as described below.
In the outer blood plasma of a kind of screen body, the method for H7N9 biomarker, comprises the following steps:
1) plasma sample of many cases H7N9 patient is prepared,
2) cytokine and the chemotactic factor of plasma sample are detected, it is thus achieved that cytokine and the data of chemotactic factor,
3) cytokine in H1N1 patient, normal healthy controls blood plasma and chemotactic factor, analyzing and processing data are compared, it is thus achieved that H7N9 biomarker,
4) from the H7N9 biomarker described in step 3), determine the biomarker of mortality risk
Step 2) described in detection use liquid-phase chip technology.
Analyzing and processing data described in step 3) uses SPSS software to process.
H7N9 biomarker described in step 3) is at least to include combination multiple in following biomarker, described biomarker selected from MIF,
SCF, MCP-1, HGF, SCGF-beta, IL-18, IP-10 and IFN-γ.
The appraisal procedure of the order of severity of a kind of H7N9 disease, monitoring of diseases progress or the method evaluating treatment, learn disease severity, or monitoring of diseases progress by the H7N9 biomarker of the quantitative or qualitative patient of comparison Yu normal control, or evaluate treatment.
Described H7N9 biomarker is at least to include combination multiple in following biomarker, and described biomarker is selected from MIF, SCF, MCP-1, HGF, SCGF-beta, IL-18, IP-10 and IFN-γ.
A kind of test kit assessing H7N9 disease severity, essentially consists in detection by quantitative H7N9 biomarker.
Described H7N9 biomarker is at least to include combination multiple in following biomarker, and described biomarker is selected from MIF, SCF, MCP-1, HGF, SCGF-beta, IL-18, IP-10 and IFN-γ.
The invention has the beneficial effects as follows: due to present invention application liquid-phase chip technology, from immune molecule angle, cytokine and the chemotactic factor of H7N9 patient are carried out detection and analyze, thus set up effective H7N9 immune molecule collection of illustrative plates, and use it for evaluating in vitro H7N9 death risk.The sensitivity of the method and specificity are superior to traditional detection method.
Accompanying drawing explanation
The present invention will be further described with embodiment below in conjunction with the accompanying drawings.
Fig. 1 (A) is to compare 6 example normal healthy controls groups, 21 example H1N1 cytokines/Chemokines Levels；
Fig. 1 (B) is to compare 6 example normal healthy controls groups, 21 example H1N1 patients and 46 example H7N9 cytokines/Chemokines Levels；
Wherein, in Fig. 1 (A) and Fig. 1 (B),
The 1st week group of a: matched group and H1N1 morbidity compares,
The 2nd week group of b: matched group and H1N1 morbidity compares,
C: matched group and H1N1 morbidity after 15 days group compare,
D:H1N1 morbidity the 2nd week group of the 1st week group and morbidity compares,
E:H1N1 morbidity the 1st week group with fall ill 15 days after group compare,
F:H1N1 morbidity the 2nd week group with fall ill 15 days after group compare,
The 1st week group of g: matched group and H7N9 morbidity compares,
The 2nd week group of h: matched group and H7N9 morbidity compares,
I: matched group and H7N9 morbidity after 15 days group compare,
J:H7N9 morbidity the 2nd week group of the 1st week group and morbidity compares,
K:H7N9 morbidity the 1st week group with fall ill 15 days after group compare,
L:H7N9 morbidity the 2nd week group with fall ill 15 days after group compare,
The 1st week group of m:H1N1 morbidity compares with the 1st week group of H7N9 morbidity,
The 2nd week group of n:H1N1 morbidity compares with the 2nd week group of H7N9 morbidity,
0:H1N1 morbidity organizes after 15 days and compares with group after H7N9 morbidity 15 days；
Fig. 2 is and the cytokine/chemotactic factor of H7N9 the infected's CT value height correlation；
Fig. 3 is and H7N9 the infected APACHE
Cytokine/the chemotactic factor of II scoring height correlation；
Fig. 4 is the cytokine/chemotactic factor relevant to H7N9 patient's prognosis；
Fig. 5 is the ROC curve of H7N9 morbidity second week blood plasma MIF；
Fig. 6 is the ROC curve of H7N9 morbidity second week blood plasma SCF；
Fig. 7 is the ROC curve table of H7N9 morbidity second week blood plasma MCP-1；
Fig. 8 is the ROC curve table of H7N9 morbidity second week blood plasma HGF；
Fig. 9 is the ROC curve table of H7N9 morbidity second week blood plasma SCGF-beta；
Figure 10 is H7N9 morbidity's second week blood plasma
The ROC curve table of IP-10；
Figure 11 is H7N9 morbidity's second week plasma IL-18
ROC curve table；
Figure 12 is H7N9 morbidity second week blood plasma IFN-gamma
ROC curve table；
Figure 13 is the ROC curve table of H7N9 morbidity second week CRP；
Figure 14 is H7N9 morbidity's second week
The ROC curve of PaO2/FiO2.
Detailed description of the invention
The concrete scheme of the present invention is given by following example:
The mensuration of H7N9 patients blood plasma's immune factor
(1) experiment sample
1, H7N9 patient 46 example, patient 35 example in 2 weeks of falling ill, plasma sample 80 parts.61 years old mean age, wherein male 22 people.
2, H1N1 patient 21 examples, 53.9 years old mean age, wherein male 14 people.
3, check sample 6 people, is taken from healthy volunteer.
(2) experiment material
1, reagent Bio-Plex Pro Human Cytokine Array 27-Plex Group I, 21-Plex Group II, filter paper, the 1ml volley of rifle fire, the 200ul volley of rifle fire, 1ml aseptic rifle head, 200ul aseptic rifle head, 10ul aseptic rifle head 2, instrument
Luminex200, level concussion instrument, magnetic frame, 1.5mlEP manages, and 2mlEP manages.
(3) experimental procedure
1, the collection of case
H7N9 patient makes a definite diagnosis according to epidemiology contact history, clinical manifestation and laboratory examination results, can make people and infect the diagnosis of H7N9 bird flu.In the case of epidemiological history is not quite clear, according to clinical manifestation, auxiliary examination and laboratory detection result, particularly from patients with respiratory tract secretions specimen, isolate H7N9 bird flu virus, or H7N9 bird flu virus detection of nucleic acids is positive, or dynamically detection paired sera H7N9 avian influenza virus specific antibody horizontal is 4 times or raises above, can make people and infect the diagnosis of H7N9 bird flu
H1N1 patient makes a definite diagnosis: influenza sample clinical manifestation occur, has one or more laboratory detection result following: (1) influenza A H1N1 influenza virus detection of nucleic acids is positive (can use real-time RT-PCR and RT-PCR method) simultaneously；(2) it is separated to influenza A H1N1 influenza virus；(3) specific antibody level of paired sera influenza A H1N1 influenza virus 4 times or more than 4 times rising.
2, the preparation of blood plasma:
Patient and matched group take peripheral blood 3ml on an empty stomach early morning and are placed in the disposable glass blood taking tube of EDTA anticoagulant,
4 ° of C 3000 leave heart 10min ,-80 DEG C of Refrigerator stores of the blood plasma of isolated.
3, liquid-phase chip processes
It is worthy of note, cytokine and chemotactic factor are detected as a example by liquid-phase chip by the present invention, and other high-throughout method is also sufficient to competent detection analysis task to those skilled in the art.
According to description configuration standard product, coupled
Beads, detection antibodies and Streptavidin-PE.detection
Antibodies configuration in first 15 minutes, Streptavidin-PE configured with first 10 minutes.
1) every hole adds 100ul wash buffer damping, abandons supernatant, blots with filter paper at the bottom of plate.
2) every hole adds 50ul coupled beads, washes plate 2 times.
3) standard substance, sample are added.After sealing lucifuge, orifice plate is placed under shaker at room temperature and hatches 30 minutes.
4) plate is washed 3 times with wash buffer.Every hole adds 25ul detection antibody, is placed under shaker at room temperature by orifice plate and hatches 30 minutes after sealing lucifuge.
5) orifice plate is placed on magnetic frame, supernatant discarded, blots with filter paper at the bottom of plate, use wash
Buffer washes plate 3 times.Every hole adds 50ul streptavidin-PE, is placed under shaker at room temperature by orifice plate and hatches 10 minutes after sealing lucifuge.
6) orifice plate is placed on magnetic frame, supernatant discarded, blots with filter paper at the bottom of plate, use wash
Buffer washes plate 3 times.Every hole adds 125 μ l assay buffer, seals lucifuge, and 1,100 rpm agitator shakes 30 seconds.The upper machine testing of Luminex and data analysis thereof.
4, chip detection and the collection of data
XPONENT 3.1 software is utilized to carry out initial data analysis.SPSS16.0 is utilized to carry out statistical analysis.
5, data analysis
1) foundation of H7N9 patient's related immune factor model
46 parts of H7N9 patients blood plasma's samples, 21 parts of H1N1 patients blood plasma's samples are divided into first week sample (10 parts of H1N1 sample of morbidity by the difference according to distance disease time, 24 parts of H7N9 sample), morbidity second week sample (7 parts of H1N1 sample, 11 parts of H7N9 sample), fall ill 15 days later plasma samples (4 parts of H1N1 sample, 11 parts of H7N9 sample).Normal healthy controls blood plasma 6 example.Set up H7N9 patients blood plasma's differential pattern of immune factor.Utilize the significance of difference between the cytokine of Mann-Whitney U inspection difference group and chemotactic factor.Spearman's
Rank correlation coefficient analysis is for immune factor and virus load and the correlation analysis of disease severity.Benjamini & Hochberg method is for controlling the false positive rate of multiple check.ROC curve is used for forecast analysis.Set two-sided test significance level as P < 0.05
2) H7N9 patient's related immune factor predicts the foundation of fatal prognostic model
Collecting CRP and the APACHE II that patient takes a blood sample the same day, CRP and APACHE II is to H7N9 patient disease severity and the sensitivity of Mortality Prediction, specificity, positive predictive value, negative predictive value in calculating, evaluates the effectiveness of this model.
6, experimental result
1) data analysis of H7N9 patient's related immune factor model: use SPSS16.0 that 46 example H7N9 patients, 21 example H1N1 patients and 6 example healthy volunteer's immune factors are compared and statistical analysis, result show detected 48 kinds of cytokines of H7N9 bird flu peripheral blood in patients and chemotactic factor have 34 kinds morbidity within 1 week, organize relatively normal healthy controls group express there were significant differences (P < 0.05), level significantly raises.The 1st week group of H7N9 bird flu morbidity has 28 kinds of cytokines and Chemokines Levels to be significantly higher than the H1N1 patient fallen ill the same period.The cytokine more than raised includes Th1 cytokines (such as IP-10, IL-2, IFN-γ, TNF-α and IL-1 β etc.), Th2 cytokines (IL-4, IL-6, IL-10 and IL-13) and TH17 cytokines (IL-17), and the 2nd week group of H7N9 bird flu morbidity has similar variation tendency.These all show that H7N9 bird flu patient exists significant cytokine storm.Such as Fig. 1 (A), 1(B) shown in.
2) H7N9 patient's related immune factor and the correlation analysis of patient's virus load: use SPSS16.0 to carry out Spearman's rank correlation coefficient analysis, research finds IP-10, MIG, MIF, HGF and IL-18 are in the virus load positive correlation of morbidity first week, second week and patient；SCF, beta-NGF and
SCGF-beta is in the virus load positive correlation of morbidity's second week Yu patient.Result of study shows H7N9 virus induction cytokinemia, as shown in Figure 2.
3) H7N9 patient's related immune factor and the correlation analysis of patient disease severity: use SPSS16.0 to carry out Spearman's rank correlation coefficient analysis, studying the APACHE II scoring positive correlation finding SCF, HGF, MIF, IL-18, IP-10 and MIG morbidity first week, second week and patient, SCGF-beta is in the APACHE II scoring positive correlation of morbidity's second week with patient.Result of study shows the cytokinemia of H7N9 virus induction and the order of severity positive correlation of disease, as shown in Figure 3.
4) correlation analysis of H7N9 patient's related immune factor and the fatal prognosis of patient: according to the death of patient whether, patient is divided into improvement group and dead group, passes through Mann-Whitney
U inspection finds that Died Patients morbidity second week related immune factor HGF, SCF, IL-18, IP-10, MIF and SCGF-beta level relatively improvement group morbidity second week significantly raise, as shown in Figure 4.
5) prognosis that H7N9 patient's related immune factor prediction patient is fatal: use the related immune factor of the SPSS16.0 all notable risings of calculating and the area under curve of the ROC curve of PaO2/FiO2 ratios and CRP.Research finds MIF, SCF, MCP-1,
HGF, SCGF-beta, IP-10, IL-18 and the IFN-γ area under curve compared with other immune factors ROC are higher, and MIF, SCF, MCP-1, HGF and SCGF-beta relatively PaO2/FiO2
The area under curve of ratios and CRP is higher, more can predict the prognosis that patient is fatal, as shown in Fig. 5-Figure 14.
Claims (5)
1. the method for H7N9 biomarker in the outer blood plasma of screen body, it is characterised in that comprise the following steps:
1) plasma sample of many cases H7N9 patient is prepared；
2) cytokine and the chemotactic factor of plasma sample are detected, it is thus achieved that cytokine and the data of chemotactic factor；
3) compare the cytokine in H1N1 patient, normal healthy controls blood plasma and chemotactic factor, analyzing and processing data, it is thus achieved that H7N9 biomarker, from described H7N9 biomarker, determine the biomarker of mortality risk；
Step 3) specifically includes: A) set up H7N9 patient's related immune factor model, the described related immune factor is above-mentioned cytokine and chemotactic factor: utilize the significance of difference, Spearman's rank correlation between the cytokine of Mann-Whitney U inspection difference group and chemotactic factor
Coefficient analysis is for immune factor and virus load and the correlation analysis of disease severity, and Benjamini & Hochberg method is for controlling the false positive rate of multiple check, and ROC curve is used for forecast analysis；H7N9 patient's related immune factor predicts the foundation of fatal prognostic model, collect CRP and the APACHE II that patient takes a blood sample the same day, CRP and APACHE II is to H7N9 patient disease severity and the sensitivity of Mortality Prediction, specificity, positive predictive value, negative predictive value in calculating, evaluates the effectiveness of this model；
B) data analysis of H7N9 patient's related immune factor model, uses SPSS16.0 to compare H7N9 patient, H1N1 patient and normal healthy controls immune factor and statistical analysis；
H7N9 patient's related immune factor and the correlation analysis of patient's virus load: use SPSS16.0 to carry out Spearman's rank correlation
coefficient analysis；
H7N9 patient's related immune factor and the correlation analysis of patient disease severity, use SPSS16.0 to carry out Spearman's rank correlation
coefficient analysis；
According to the death of patient whether the correlation analysis of H7N9 patient's related immune factor and the fatal prognosis of patient, be divided into improvement group and dead group by patient, by the significance of difference between the cytokine of Mann-Whitney U inspection difference group and chemotactic factor；
The prognosis that H7N9 patient's related immune factor prediction patient is fatal, uses SPSS16.0 to calculate the related immune factor of all notable risings and the area under curve of the ROC curve of PaO2/FiO2 ratios and CRP.
Method the most according to claim 1, it is characterised in that step 2) described in detection use liquid-phase chip technology.
Method the most according to claim 1, it is characterized in that, H7N9 biomarker described in step 3) is at least to include combination multiple in following biomarker, and described biomarker is selected from MIF, SCF, MCP-1, HGF, SCGF-beta, IL-18, IP-10 and IFN-γ.
4. the test kit assessing H7N9 disease severity, it is characterised in that essentially consist in detection by quantitative H7N9 biomarker；Described H7N9 biomarker includes MIF, SCF, MCP-1, HGF, SCGF-beta, IL-18, IP-10 and IFN-γ simultaneously.
5. the biomarker assessing H7N9 disease severity, it is characterised in that simultaneously include MIF, SCF, MCP-1, HGF, SCGF-beta, IL-18, IP-10 and IFN-γ.
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