CN105203764A - Method and kit for human influenza virus typing detection based on magnetic resolution and colorful quantum dot labelling - Google Patents

Abstract

The invention discloses a method and a kit for human influenza virus typing detection based on magnetic resolution and colorful quantum dot labelling. The kit comprises anti-human influenza virus immune nano-magnetic beads, anti-human influenza virus nanoprobes, quality control samples, a sample treating liquid and a PBST buffer solution, wherein the anti-human influenza virus immune nano-magnetic beads are rich in human influenza virus antigen; the anti-human influenza virus nanoprobes are labelled by colorful quantum dots; the quality control samples are a positive quality control sample and a negative quality control sample; the positive quality control sample is obtained by drying an inactived A-type human influenza virus and an inactived B-type human influenza virus, and combining the dried human influenza viruses on a swab; the negative quality control sample is a pharynx swab of a person whose detection results for the A-type human influenza virus and the B-type human influenza virus are both negative through clinical confirmation. The method and the kit are simple, convenient, fast, and high in sensitivity. The invention further provides a preparation method and a use method of the kit.

Description

Based on magnetic resolution and color quantum point mark human influenza virus's somatotype detect method and kit

Technical field

The present invention relates to technical field of medical detection, be specially a kind of method for quick of detection human influenza virus antigen based on magnetic resolution and color quantum point mark and detection kit, and the method for preparation and use of this detection kit.

Background technology

Influenza virus; be called for short influenza virus; that one causes the mankind and animal to suffer from grippal RNA virus; on taxonomy; influenza virus belongs to Orthomyxoviridae family; it can cause acute upper respiratory infection, and propagates rapidly by air, often has periodically be very popular all over the world.Virus was found by British Wilson's Smith (WilsonSmith) in 1933 the earliest, and he is called H1N1.H represents hemagglutinin; N represents neuraminidase.Digitized representation is dissimilar.

Influenza virus is spherical in shape or thread, diameter 80 ~ 120nm.Virus is formed by three layers, and internal layer is virus nucleocapsid, containing nucleoprotein (NP), P albumen and RNA.NP is soluble antigen (S antigen), has type specificity, and antigenicity is stablized.P albumen (P1, P2, P3) may be rna transcription and copy required polymerase.Middle level is virus envelope, is made up of one deck lipoid and skim albumen (MP), and MP antigenicity is stablized, and also has type specificity.Skin is the radial projection that two kinds of different sugar albumen are formed, i.e. hemagglutinin (hemagglutinin, H) and neuraminidase (neuraminidase, N).H can cause red cell agglutination, be viral absorption in the instrument on sensitive cells surface, N then can be hydrolyzed mucus protein, and the N-acetyl-neuraminate of hydrolyzed cellular surface receptor specificity glycoprotein end is the instrument of disengaging cell surface after virus replication completes.H and N all has variation property, therefore the antigenicity only having strain special, its antibody has protective effect.

According to NP antigenicity, influenza virus is divided into first, second, the third three types.Different by H with N antigen, homologous virus divides again some hypotypes.The overwhelming majority popular is clinically A type and B-mode, and the third type is comparatively rare.The Clinical symptoms of influenza virus is relevant to virus stain, population ages, physiological situation etc., main manifestations heating, chilly, dry cough, have a headache, have a sore throat, the whole body is uncomfortable, weak, have a stuffy nose and runny nose etc.This disease is short for latent period, and infectiousness is strong, propagates rapidly, larger to infant, old person and Low people harm.Threaten maximum with Flu-A in three type influenzas.Because influenza virus pathogenicity is strong, easily morphs, if crowd lacks immunity to variant, then easily cause outbreak of epidemic, existing tens of millions of people is dead, very large to social influence.

Because flu-like symptom and parainfluenza virus pneumonia, respiratory syncytial virus pneumonia and adenovirus pneumonia almost cannot be distinguished clinically, do not do etiological examination, be difficult to make a definite diagnosis influenza infection.Therefore method that is easy, quick, feasible, energy early diagnosis influenza infection is set up very necessary.

The detection method of current influenza virus mainly contains 3 classes: one is isolated culture, its be confirm infect " goldstandard ", but due to cultivation cycle longer, operation steps is more, causes this method can not carry out quick diagnosis clinically; Two is serological methods, namely adopts euzymelinked immunosorbent assay (ELISA), colloidal gold immunization, micro-Immunofluorescence assay and indirect hemagglutination test etc., detects Antibody of Influenza level in examinee's serum, indirectly can point out the existence of influenza infection.But serological test can only provide a kind of retrospective diagnosis, and sometimes needs paired sera.In addition, the opportunity that antibody occurs not easily is grasped, and there is again the difference of influenza virus specific antibody, therefore the Detection job of existing serological method is subject to a definite limitation between children and adolescents and adult; Three is the existence utilizing Protocols in Molecular Biology to detect influenza virus DNA, wherein that the most frequently used is PCR (PCR), the method is quick, sensitive, special, it is the important means studying human influenza virus's infection at present, but due to PCR to experimental facilities and operation requirements higher, and easily there is false positive, can't as conventional methods for clinical diagnosis in China.Therefore, human influenza virus's specific antigen diagnostic method is set up very necessary.At present, the method of the detection human influenza virus antigen of open report is mainly double crush syndrome method, colloidal gold immunity chromatography, indirect immunofluorescence etc., but these methods or have that operation steps is various, detection time is long, or there is the problems such as detection sensitivity is low, testing cost is high.

Summary of the invention

For these technical matterss existed in background technology, the invention provides detection method and kit that a kind of easy, quick, highly sensitive somatotype of energy marked based on magnetic resolution and color quantum point detects human influenza virus's antigen, and the method for preparation and use of this kit.

The present invention is achieved through the following technical solutions:

Based on a method for human influenza virus's antigens genotyping detection that magnetic resolution and color quantum point mark, it is characterized in that: said method comprising the steps of:

1) respectively anti-human for rabbit first, influenza B virus NP protein polyclone antibody and nanometer magnetic bead are passed through covalent coupling, prepare anti-human influenza A virus immune nanometer magnetic bead and anti-human influenza B virus immune nanometer magnetic bead respectively; By anti-human influenza A virus immune nanometer magnetic bead and anti-human influenza B virus immune nanometer magnetic bead mixed in equal amounts i.e. obtained anti-human influenza virus immunization nanometer magnetic bead;

2) by mouse-anti influenza virus A hominis NP protein monoclonal antibody and emission wavelength be the nano-quantum point of 520nm by covalent coupling, prepare quantum dot-labeled anti-human influenza A virus nano-probe; By mouse-anti people influenza B virus NP protein monoclonal antibody and emission wavelength be the nano-quantum point of 600nm by covalent coupling, prepare quantum dot-labeled anti-human influenza B virus nano-probe; By the anti-human influenza virus nano-probe that anti-human influenza A virus nano-probe and anti-human influenza B virus nano-probe mixed in equal amounts i.e. obtained color quantum point mark;

3) human respiratory secretion sample (including but not limited to throat swab) is got, after dissolving by sample treatment liquid, add step 1) the anti-human influenza virus immunization nanometer magnetic bead for preparing, abundant mixing, Magneto separate is carried out after reaction 10-45min, after PBST buffer solution 2 times, step 2 is added in the sediment that Magneto separate obtains) the anti-human influenza virus nano-probe of the color quantum point for preparing mark, Magneto separate is carried out after reaction 10-45min, after PBST buffer solution 2 times, fluorescence microplate reader is used to be all 405nm at Ex, Em reads fluorescent value under being respectively the parameter of 520nm and 600nm respectively, in described sample treatment liquid, each component concentration is respectively: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, 0.3g/LNaN ₃, 5ml/LNonidetP-40,5ml/LTritonX-100, described sample treatment liquid pH=7.4, in described PBST damping fluid, each component concentration is respectively: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, 0.3g/LNaN ₃, 0.5ml/LTween-20, described PBST pH of buffer=7.4,

4) according to step 1)-step 3) method detect four parts respectively and determine for people's first, influenza B virus is the respiratory secretions sample of negative crowd through clinical, read respectively Em be respectively 520nm and 600nm under fluorescent value; The respiratory secretions sample that described people's first, influenza B virus is negative crowd is called for short human influenza virus's negative control sample; Described four parts of human influenza virus's negative control sample are that the mean value of the fluorescent value of 520nm and 3 times of standard deviation sums are designated as CUT-OFF1 value at Em; Described four parts of human influenza virus's negative control sample are that the mean value of the fluorescent value of 600nm and 3 times of standard deviation sums are designated as CUT-OFF2 value at Em; If step 3) in human respiratory secretion sample be greater than CUT-OFF1 value at the detection fluorescent value that Em is 520nm, to be then judged as in human respiratory secretion sample that influenza virus A hominis's antigen is for the positive; If step 3) in human respiratory secretion sample be less than CUT-OFF1 value at the detection fluorescent value that Em is 520nm, to be then judged as in human respiratory secretion sample that influenza virus A hominis's antigen is for feminine gender; If step 3) in human respiratory secretion sample be greater than CUT-OFF2 value at the detection fluorescent value that Em is 600nm, to be then judged as in human respiratory secretion sample that people's influenza B virus antigen is for the positive; If step 3) in human respiratory secretion sample be less than CUT-OFF2 value at the detection fluorescent value that Em is 600nm, to be then judged as in human respiratory secretion sample that people's influenza B virus antigen is for feminine gender.

Based on human influenza virus's antigens genotyping detection kit of magnetic resolution and color quantum point mark, it is characterized in that: described kit is made up of the anti-human influenza virus nano-probe, quality-control product, sample treatment liquid and the PBST damping fluid that have the anti-human influenza virus immunization nanometer magnetic bead of enrichment human influenza virus antigen function, color quantum point marks; Described quality-control product comprises positive quality control product and negative quality-control product; Described positive quality control product is attached on swab by the common drying of people's first, second influenza virus of deactivation and forms; Described negative quality-control product is through the clinical throat swab determining the crowd being feminine gender for people's first, second influenza virus.

For the preparation of a method for the human influenza virus's antigens genotyping detection kit based on magnetic resolution and color quantum point mark, it is characterized in that: described preparation method comprises the following steps:

1) preparation of anti-human influenza virus immunization nanometer magnetic bead:

1.1) get 5mg magnetic bead, with 1mlMES buffer solution three times, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant; Described magnetic bead is with superparamagnetism Fe ₃o ₄for the carboxyl magnetic bead that kernel, particle diameter are 1150nm; 2-(N-morpholino) ethyl sulfonic acid of described MES damping fluid to be mass concentration be 2g/L; The pH=6.0 of described MES damping fluid; The magnetic intensity of described nano magnetic separation vessel is 0.4T;

1.2) add successively by step 1.1) in the concentration of MES buffer be 8-12mg/ml EDC solution and by step 1.1) in the concentration of MES buffer be each 0.5ml of sulfo-NHS solution of 6-10mg/ml, in rotary mixer, 1hr is activated with 10-40rpm/min, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, by 1ml step 1.1) in MES damping fluid resuspended, obtain activate after magnetic bead;

1.3) 5 centrifuge tubes are got, 200 μ L steps 1.2 are added in each centrifuge tube) magnetic bead after the activation that obtains, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, adding by the concentration of PBS damping fluid dilution in each centrifuge tube is each 1ml of rabbit anti-human influenza A virus NP protein polyclone antibody solution of 50-200 μ g/ml, in rotary mixer, 2-6h is reacted with 15rpm/min under room temperature, be placed in after removing supernatant after nano magnetic separation vessel carries out Magneto separate, respectively add the above-mentioned PBS damping fluid of 1ml containing 1mg/ml monoethanolamine, under room temperature with 15rpm/min react in rotary mixer 2h with on closed magnetic bead not with the carboxyl of antibody response, in described PBS damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, the pH=7.4 of described PBS damping fluid,

1.4) after capping completes, these 5 centrifuge tubes are placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, respectively wash three times with 1ml lavation buffer solution; In described lavation buffer solution, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, 0.5ml/LTween-20, the pH=7.4 of described lavation buffer solution;

1.5) in each centrifuge tube, add 1ml respectively preserve the resuspended magnetic bead of damping fluid, be placed in 4 DEG C and save backup, so far obtained anti-human influenza A virus immune nanometer magnetic bead; In described preservation damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, 0.3g/LNaN ₃, 5g/L bovine serum albumin(BSA) (BSA), the pH=7.4 of described preservation damping fluid;

1.6) by and step 1.1)-1.5) identical method utilizes rabbit anti-human influenza B virus NP protein polyclone antibody to obtain anti-human influenza B virus immune nanometer magnetic bead; By the 1:1 mixing by volume of above-mentioned two kinds of immune nanometer magnetic bead suspensions, i.e. obtained anti-human influenza virus immunization nanometer magnetic bead;

2) preparation of the anti-human influenza virus nano-probe of color quantum point mark:

Its concrete preparation method comprises:

2.1) in microcentrifugal tube, add 4nmol carboxyl water-soluble quantum dot, 600nmolN-N-Hydroxysuccinimide sulfo-NHS and 600nmol carbodiimide EDC successively, with phosphate buffer constant volume for 2ml, mixed solution, after 37 DEG C of reaction 30min, excessive sulfo-NHS and EDC as activator is removed in dialysis, obtains the quantum dot after activating; The emission wavelength of described carboxyl water-soluble quantum dot is 520nm; In described phosphate buffer, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride; The pH=7.4 of described phosphate buffer;

2.2) in step 2.1) in the quantum dot of activation that obtains, add the mouse-anti influenza virus A hominis NP protein monoclonal antibody of 4-16nmol, lucifuge reaction 2h, adds single-ended amination polyglycol PEG2000-NH ₂be 1% to final concentration, close unreacted activated carboxyl site, continue lucifuge reaction 1h;

2.3) by 0.2 μm of PES frit removing step 2.2) in antibody aggregation thing, then filtrate is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, there is not the antibody of coupling reaction and the accessory substance in reacting in removing;

2.4) step 2.3 is collected) middle ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleansing solution, again this solution is transferred in a new 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, collect ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1ml phosphate conserving liquid, be placed in 4 DEG C and save backup, so far obtained quantum dot-labeled anti-human influenza A virus nano-probe; In described phosphate cleansing solution, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, 5ml/L Tween-20,0.3g/L Sodium azide, the pH=7.4 of described phosphate cleansing solution; In described phosphate conserving liquid, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/L bovine serum albumin(BSA), 0.3g/L Sodium azide; The pH=7.4 of described phosphate conserving liquid;

2.5) by and step 2.1)-2.4) identical method utilizes the carboxyl water-soluble quantum dot that mouse-anti people influenza B virus NP protein monoclonal antibody and emission wavelength are 600nm to obtain quantum dot-labeled anti-human influenza B virus nano-probe; Above-mentioned two kinds are contained the solution 1:1 mixing by volume of the quantum dot-labeled nano-probe of different emission, i.e. the anti-human influenza virus nano-probe of obtained color quantum point mark;

3) preparation of PBST damping fluid:

Its concrete compound method comprises:

Get 8gNaCL, 0.2gKCl, 0.24KH ₂pO ₄, 1.44gNa ₂hPO ₄, 0.3gNaN ₃, 0.5mlTween-20 is dissolved in 800ml distilled water, adjusts pH to 7.4, then be settled to 1000ml with 5MNaOH;

4) preparation of sample treatment liquid:

Get 8gNaCL, 0.2gKCl, 0.24KH ₂pO ₄, 1.44gNa ₂hPO ₄, 0.3gNaN ₃, 5mlNonidetP-40,5mlTritonX-100, adjust pH to 7.4 with 5MNaOH, then be settled to 1000ml;

5) preparation of quality-control product:

5.1 positive quality control product: positive quality control product is attached on swab by the common drying of people's first, influenza B virus of deactivation and forms;

5.2 negative quality-control products: negative quality-control product is namely through the clinical throat swab determining the crowd being feminine gender for people's first, second influenza virus.

As preferably, the present invention is in step 1.2) in, add successively by step 1.1) in the concentration of MES buffer be 10mg/ml EDC solution and by step 1.1) in the concentration of MES buffer be each 0.5ml of sulfo-NHS solution of 8mg/ml, in rotary mixer, 1hr is activated with 15rpm/min, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, by 1ml step 1.1) in MES damping fluid resuspended, obtain activate after magnetic bead;

Described step 1.3) in, adding by the concentration of PBS damping fluid dilution in each centrifuge tube is each 1ml of rabbit anti-human influenza A virus NP protein monoclonal antibody solution of 100 μ g/ml, in rotary mixer, 3h is reacted with 15rpm/min under room temperature, be placed in after removing supernatant after nano magnetic separation vessel carries out Magneto separate, respectively add the above-mentioned PBS damping fluid of 1ml containing 1mg/ml monoethanolamine;

Described step 2.2) in, in step 2.1) in the quantum dot of activation that obtains, add 12nmol mouse-anti influenza virus A hominis NP protein monoclonal antibody, lucifuge reaction 2h.

As preferably, quantum dot of the present invention is water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer is modified.

As preferably, magnetic bead of the present invention is with superparamagnetism Fe ₃o ₄for the carboxyl magnetic bead that kernel, shell material are polystyrene, surface functional group is carboxyl, particle diameter is 1150nm.

Based on a using method for human influenza virus's antigens genotyping detection kit that magnetic resolution and color quantum point mark, it is characterized in that: described using method comprises the following steps:

1) after sample 0.5ml sample treatment solution to be checked being dissolved, lysate is proceeded in the common centrifuge tube of 1.5ml; In described sample treatment solution, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, 0.3g/LNaN ₃, 5ml/LNonidetP-40,5ml/LTritonX-100; The pH=7.4 of described sample treatment solution;

2) to step 1) in centrifuge tube in add based on magnetic resolution and color quantum point mark human influenza virus's antigens genotyping detection kit in anti-human influenza virus immunization nanometer magnetic bead 50-200 μ l, take off after reacting 10-45min with 10rpm/min on rotary mixer under room temperature, centrifuge tube is inserted nano magnetic separation vessel Magneto separate 3min, with pipettor sucking-off supernatant;

3) the PBST damping fluid 1ml added in kit washs twice, and sucking-off cleansing solution after employing nano magnetic separation vessel Magneto separate, finally uses the resuspended magnetic bead of 1mlPBS damping fluid, obtained immune nanometer magnetic bead-viral antigen compound; In described PBS damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄; The pH=7.4 of described PBS damping fluid;

4) 100 μ l steps 3 are got) immune nanometer magnetic bead-viral antigen compound of obtaining is in another centrifuge tube, add again 100 μ l based on magnetic resolution and color quantum point mark human influenza virus's antigens genotyping detection kit in color quantum point mark anti-human influenza virus nano-probe, on rotary mixer, 10-45min is reacted with 15rpm/min under room temperature, be combined by the immunity of the viral antigen of the antibody on quantum dot on immune nanometer magnetic bead, quantum dot is tagged to viral antigen surface, form magnetic bead-viral antigen-quantum dot " sandwich " compound,

5) after having reacted, adopt nano magnetic separation vessel Magneto separate 3min, remove the anti-human influenza virus nano-probe of unnecessary color quantum point mark, 2 times are cleaned with the PBST damping fluid liquid in kit, compound is dispersed in 100 μ lPBS damping fluids again, use fluorescence microplate reader to be all 405nm at Ex, Em detects its fluorescent value under being respectively the parameter of 520nm and 600nm respectively respectively; In described PBS damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄; The pH=7.4 of described PBS damping fluid;

6) by four parts that provide in above-mentioned same method detection kit negative quality-control product samples and a positive quality control product sample, read Em respectively and be respectively fluorescent value under 520nm and 600nm; Four parts of negative quality-control product samples are that the mean value of the fluorescent value of 520nm and 3 times of standard deviation sums are designated as CUT-OFF1 value at Em; Four parts of negative quality-control product samples are that the mean value of the fluorescent value of 600nm and 3 times of standard deviation sums are designated as CUT-OFF2 value at Em; If step 5) in measuring samples be greater than CUT-OFF1 value at the detection fluorescent value that Em is 520nm, to be then judged as in sample to be checked that influenza virus A hominis's antigen is for the positive; If step 5) in sample to be checked be less than CUT-OFF1 value at the detection fluorescent value that Em is 520nm, then the influenza virus A hominis's antigen in sample to be checked that judges to behave is feminine gender; If step 5) in sample to be checked be greater than CUT-OFF2 value at the detection fluorescent value that Em is 600nm, to be then judged as in sample to be checked that people's influenza B virus antigen is for positive; If step 5) in sample to be checked be less than CUT-OFF2 value at the detection fluorescent value that Em is 600nm, to be then judged as in sample to be checked that people's influenza B virus antigen is for negative; If the fluorescent value of positive quality control product sample is less than CUT-OFF1 or CUT-OFF2 value, then show that kit lost efficacy.

As preferably, step 2 proposed by the invention) in, to step 1) in centrifuge tube in add based on magnetic resolution and color quantum point mark human influenza virus's antigens genotyping detection kit in anti-human influenza virus immunization nanometer magnetic bead 140 μ l, take off after reacting 15min with 10rpm/min on rotary mixer under room temperature, centrifuge tube is inserted magnetic frame and be separated 3min, with pipettor sucking-off supernatant;

Described step 4) in, get 100 μ l steps 3) immune nanometer magnetic bead-viral antigen compound of obtaining is in another centrifuge tube, add again 100 μ l based on magnetic resolution and color quantum point mark human influenza virus's antigens genotyping detection kit in color quantum point mark anti-human influenza virus nano-probe, on rotary mixer, 15min is reacted with 15rpm/min under room temperature, be combined by the immunity of the viral antigen of the antibody on quantum dot on immune nanometer magnetic bead, quantum dot is tagged to viral antigen surface, form magnetic bead-viral antigen-quantum dot " sandwich " compound.

As preferably, sample to be checked provided by the present invention includes but not limited to throat swab.

Emission wavelength required for the present invention is the carboxyl water-soluble nano quantum dot of 520nm, 600nm, the carboxyl magnetic bead of 1150nm diameter, mouse-anti influenza virus A hominis NP protein monoclonal antibody, mouse-anti people influenza B virus NP protein monoclonal antibody, rabbit anti-human influenza A virus NP protein polyclone antibody, rabbit anti-human influenza B virus NP protein polyclone antibody can arrive research unit, company's purchase of relevant speciality or customize; Required instrument, equipment, medicine all have commercially available.

The present invention compared to existing technology tool has the following advantages:

1, present invention utilizes immune nanometer magnetic bead to sample can enriching, the speed be separated is fast, efficiency advantages of higher, incorporating quantum point photochemical stability is high simultaneously, the characteristics such as fluorescence intensity is high, detection system is possessed effect that multiple signal works in coordination with amplification, thus the detection sensitivity with superelevation is (as it is 0.1ng/ml to the detection bottom line of influenza virus A hominis (ATCC numbering VR-1743), also be 0.1ng/ml to the detection bottom line of people's influenza B virus (ATCC numbering VR-790)), thus the quality requirements greatly reduced clinical sampling, improve positive rate, with its to the testing result of clinical sample with at present to no difference of science of statistics compared with detection " the goldstandard "-cultivation of this pathogen.

2, detection method is simple, detect fast, be easy to judge, testing cost is cheap, the synchronous inspection altogether of amphitypy human influenza virus can be carried out, overcome that prior art Positive rate is low, cost is high, complicated operation is loaded down with trivial details, length consuming time, the deficiency of clinical practice of cannot carrying out.

What 3, detect due to detection kit is human influenza virus's antigen and non-antibody (appearance of antibody needs to infect a few Zhou Yihou), so can carry out early diagnosis and control, clinical diagnosis coincidence rate is high.The method has very high practical value in the clinical diagnosis, aetology discriminating, epidemiology survey etc. of human influenza virus.

4, the clinical sample that detection method is used be respiratory secretions as sputum etc., and non-blood, can exempt the psychological burden of misery that infant patient takes a blood sample and the head of a family, therefore comparatively be easy to promote.

Embodiment

The present invention is according to the double antibodies sandwich principle in immunology, utilize immune nanometer magnetic bead to sample can enriching, separation fast, the efficiency advantages of higher of speed, the characteristics such as incorporating quantum point photochemical stability is high, fluorescence intensity is high, the a set of new method possessing multiple signal and work in coordination with amplification, have the fast typing of hypersensitivity and high degree of specificity detection human influenza virus set up, has wide market application foreground.

The present invention is further described in detail by following examples.

The preparation of various reagent and the explanation of material requested

1.PBS damping fluid: take 1.44g sodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, 8g sodium chloride, 0.2g potassium chloride, is dissolved in the deionized water of 900ml, with deionized water is settled to 1000ml after adjusting pH to 7.4 with 1mol/LNaOH.

2. rabbit anti-human influenza A virus NP protein polyclone antibody: be Abcam Products, article No. ab91648, with the dilution of PBS damping fluid, shakes up, makes polyclonal antibody weight percent concentration in solution be 1mg/ml.

3. rabbit anti-human influenza B virus NP protein polyclone antibody: purchased from Chao Yan bio tech ltd, Shanghai product, article No. ab70783, with the dilution of PBS damping fluid, shakes up, makes polyclonal antibody weight percent concentration in solution be 1mg/ml.

4. mouse-anti influenza virus A hominis NP protein monoclonal antibody: be Abcam Products, article No. ab128193, with the dilution of PBS damping fluid, shakes up, makes polyclonal antibody weight percent concentration in solution be 1mg/ml.

5. mouse-anti people influenza B virus NP protein monoclonal antibody: be Abcam Products, article No. ab20711, with the dilution of PBS damping fluid, shakes up, makes polyclonal antibody weight percent concentration in solution be 1mg/ml.

6. quantum dot: two amounts point used in the present invention is water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer is modified, its emission wavelength is respectively 520nm and 600nm, buy from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., name of product is carboxyl water-soluble quantum dot-520 and carboxyl water-soluble quantum dot-600.

7. magnetic bead: in the present invention, magnetic bead used is with superparamagnetism Fe ₃o ₄for kernel, shell material be polystyrene, surface functional group is carboxyl, particle diameter is be respectively 50nm, 180nm, 350nm, 1150nm, the carboxyl magnetic bead of 3 μm, can buy from Shanxi North America Gene Co., Ltd, Aorun Weina New Material Science and Technology Co., Ltd., Shanghai.

8. influenza virus A hominis: H1N1 hypotype, purchased from American Type Tissue Collection (ATCC), is numbered ATCCVR-1743.

9. people's influenza B virus: purchased from American Type Tissue Collection (ATCC), is numbered ATCCVR-790.

10. the equal purchased from American Type Tissue Collection (ATCC) of the microbiological specimens used by the present invention.

The preparation of the anti-human influenza virus immunization nanometer magnetic bead of embodiment 1

1. the optimization of anti-human influenza virus polyclonal antibody coupled bead reaction conditions:

Using coupling, the magnetic bead of anti-human influenza A virus NP protein polyclone antibody is as solid phase carrier, quantum dot-labeled anti-human influenza A virus NP protein monoclonal antibody is as detection antibody, detect influenza virus A hominis's antigen by double-antibody method principle, observe magnetic bead and many anti-coupling situations.Respectively to the particle diameter of magnetic bead, and the coupling condition such as EDC/NHS activator concentration, coupled antibody concentration, coupling time, sealer kind has carried out a series of optimum choice.

The selection of 1.1 magnetic bead particle diameters

Selection particle diameter is 50nm, 180nm, 350nm, 1150nm, the carboxyl magnetic bead of 3 μm, after the PBS damping fluid all added containing 4mg/mlEDC and 4mg/mlNHS carries out priming reaction, anti-human influenza A virus NP protein polyclone antibody carries out coupling reaction with rabbit respectively.Respectively the immune nanometer magnetic bead prepared is detected the influenza virus A hominis (ATCC numbering VR-1743) of 10ng/mL, observations under fluorescent microscope, selection fluorescence intensity is large, and background fluorescence interference is few, and the very fast person of velocity of separation is the suitableeest magnetic bead particle diameter under magnetic fields.Result shows the magnetic bead requirement the most according to the invention of particle diameter 1150nm, determines that the suitableeest magnetic microsphere particle diameter is 1150nm.

The selection of 1.2EDC/NHS activator concentration

Carry out concentration gradient combination after EDC and NHS concentration in reaction system is set to l ~ 10mg/ml separately, activate the carboxyl magnetic bead of particle diameter 1150nm respectively.The immune nanometer magnetic bead for preparing is detected the influenza virus A hominis (ATCC numbering VR-1743) of 10ng/mL, the most powerhouse of selection fluorescence is the suitableeest activation concentration of EDC and NHS solution.Result shows that coupling effect is best when EDC concentration be 5mg/ml, NHS concentration is 4mg/ml.

The selection of 1.3 coupled antibody concentration

The rabbit anti-human influenza A virus NP protein polyclone antibody of 20 μ g, 40 μ g, 60 μ g, 80 μ g, 100 μ g, 120 μ g, 140 μ g, 160 μ g, 200 μ g is carried out coupling with 1mg by the magnetic bead that the particle diameter that above-mentioned best practice activates is 1150nm respectively.The immune nanometer magnetic bead prepared is detected the influenza virus A hominis (ATCC numbering VR-1743) of 10ng/mL, found that, when the injected volume of antibody is less than 100 μ g/mg, fluorescence intensity increases along with the concentration increase of antibody, and when the mass concentration of antibody is greater than 100 μ g/mg, fluorescence intensity is substantially constant even slightly to be reduced, and therefore the present embodiment selects the coupling amount of rabbit anti-human influenza A virus NP protein polyclone antibody to be 100 μ g/mg.

The selection of 1.4 coupling times

After determining the particle diameter of magnetic bead, EDC/NHS activator concentration and antibody coupling amount, the coupling reaction time of antibody and magnetic bead is set to 0.5h, 1h, 2h, 3h, 4h, 5h respectively, the immune nanometer magnetic bead prepared is detected the influenza virus A hominis (ATCC numbering VR-1743) of 10ng/ml.Found that, when coupled during >3h, fluorescence intensity tends towards stability, and after this extends coupling time again, and fluorescence no longer strengthens.Therefore, the suitableeest coupling reaction time determining rabbit anti-human influenza A virus NP protein polyclone antibody and magnetic bead is 3h.Coupling time is far fewer than the 24h of traditional E LISA method.

The selection of 1.5 sealers

Coupling reaction is carried out after selecting the particle diameter of magnetic bead, EDC/NHS activator concentration, antibody coupling amount and coupling time according to the above-mentioned optimal conditions determined.After coupling terminates, select BSA, monoethanolamine, Tris and D-Glucosamine Hydrochloride, as immune nanometer magnetic bead sealer, obtain finished product immune nanometer magnetic bead.The immune nanometer magnetic bead prepared is detected the influenza virus A hominis (ATCC numbering VR-1743) of 10ng/mL.Found that, adopt monoethanolamine the highest as the detection fluorescent value of the immune nanometer magnetic bead of sealer.Infer because the molecule of monoethanolamine is less, can consume preferably due to the sterically hindered surface carboxyl groups be not combined with antibody, make closed more complete, and effectively reduce space steric effect to the structure influence connecting antibody.

The optimum results of the rabbit anti-human influenza A virus NP protein polyclone antibody that the optimum results of rabbit anti-human influenza B virus NP protein polyclone antibody coupled bead reaction conditions and above-mentioned steps 1.1-1.5 describe and magnetic bead coupling reaction correlated response condition is completely the same.

2. coupling process:

Get 5mg magnetic bead (with superparamagnetism Fe ₃o ₄carboxyl magnetic bead for kernel, particle diameter are 1150nm) in the common centrifuge tube of 1.5ml, with 1mlMES damping fluid (2g/LMES, pH6.0) wash three times, be placed in after nano magnetic separation vessel carries out Magneto separate (0.4T) and remove supernatant, to add by the concentration of above-mentioned MES buffer be successively the EDC solution of 10mg/ml and be each 0.5ml of sulfo-NHS solution of 8mg/ml by the concentration of above-mentioned MES buffer, in rotary mixer, 1hr is activated with 15rpm/min, remove supernatant after Magneto separate, the MES damping fluid above-mentioned with 1ml is resuspended; Get 5 centrifuge tubes, in each centrifuge tube, add the magnetic bead of the above-mentioned activation of 200 μ L, sucking-off supernatant after Magneto separate, add in each pipe with PBS damping fluid (8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄the concentration of pH7.4) diluting is each 1ml of rabbit anti-human influenza A virus NP protein polyclone antibody solution of 100 μ g/ml, in rotary mixer, 3h is reacted with 15rpm/min under room temperature, after Magneto separate removes supernatant, respectively add 1ml containing the above-mentioned PBS damping fluid of 1mg/ml monoethanolamine, under room temperature with 15rpm/min react in rotary mixer 2h with on closed magnetic bead not with the carboxyl of antibody response.Each pipe supernatant is removed after Magneto separate, each with 1ml lavation buffer solution (8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, 0.5ml/LTween-20, pH7.4) and wash three times, finally each 1ml preserves damping fluid (8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, 0.3g/LNaN ₃, 5g/LBSA, pH7.4) and resuspended magnetic bead, be placed in 4 DEG C and save backup, so far obtained anti-human influenza A virus immune nanometer magnetic bead.

Rabbit anti-human influenza B virus NP protein polyclone antibody is utilized to obtain anti-human influenza B virus immune nanometer magnetic bead by the identical method of above-mentioned steps.By the 1:1 mixing by volume of above-mentioned two kinds of immune nanometer magnetic bead suspensions, i.e. obtained anti-human influenza virus immunization nanometer magnetic bead.

The preparation of the anti-human influenza virus nano-probe of embodiment 2 color quantum point mark

1. the optimization of nanometer carboxylic quantum dot-labeled mouse-anti influenza virus A hominis NP protein monoclonal antibody reaction conditions:

1.1, the determination of the quantum dot-labeled antibody probe optimum mark pH of carboxyl

Phosphate buffer pH in labeled reactant is set to 5,6,7,8,9 respectively, utilizes full spectrometer to carry out fluorescent strength determining to marked product, observe the impact of different pH value on coupling reaction, the Optimal pH determining the reaction of quantum dot-labeled monoclonal antibody is 7.0-8.0.This experimental selection pH7.4.

1.2, the determination of carboxyl quantum dot-labeled antibody probe optimum mark amount

Quantum dot volumetric molar concentration and the ratio of monoclonal antibody concentration are set to 1:1 respectively, 1:2,1:3 and 1:4, after carrying out labeled reactant, full spectrometer is utilized to carry out fluorescent strength determining to marked product, the impact of both observations variable concentrations comparison coupling reaction, determines that optimum molar concentration ratio that quantum dot-labeled mouse-anti influenza virus A hominis NP protein monoclonal antibody reacts be quantum dot and antibody molar ratio is 1:3.This optimal concentration ratio of this experimental selection determines labelled amount.

1.3, the determination of the best sealer kind of the quantum dot-labeled antibody probe of carboxyl

With monoethanolamine, Tris, PEG2000-NH ₂or BSA is as sealer, after carrying out labeled reactant, utilize full spectrometer to carry out fluorescent strength determining to marked product, observe the impact of different sealers for labeled reactant, found that, PEG2000-NH ₂for best sealer, it can significantly improve colloidal stability and the immunocompetence of labeled complex.

The optimum results that the nanometer carboxylic quantum dot-labeled mouse-anti influenza virus A hominis NP protein monoclonal antibody that the condition optimizing result of nanometer carboxylic quantum dot-labeled mouse-anti people influenza B virus NP protein monoclonal antibody reaction and above-mentioned steps 1.1-1.3 describe reacts correlated condition is all completely the same.

2. labeling process:

4nmol carboxyl water-soluble quantum dot (emission wavelength 520nm), 600nmolN-N-Hydroxysuccinimide (sulfo-NHS) and 600nmol carbodiimide (EDC) is added successively in microcentrifugal tube, with phosphate buffer (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, pH7.4) constant volume is 2ml, ceaselessly mixed solution, after 37 DEG C of reaction 30min, excessive sulfo-NHS and the EDC as activator is removed in dialysis.In the quantum dot of activation, add the mouse-anti influenza virus A hominis NP protein monoclonal antibody of 12nmol, lucifuge reaction 2h, adds single-ended amination polyglycol (PEG2000-NH ₂) to final concentration be 1%, close unreacted activated carboxyl site, continue lucifuge reaction 1h.With 0.2 μm of PES frit removing antibody aggregation thing, then filtrate transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, there is not the antibody of coupling reaction and the accessory substance in reacting in removing.Collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleansing solution (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, 5ml/L Tween-20, 0.3g/L Sodium azide, pH7.4) in, again this solution is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1ml phosphate conserving liquid (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/LBSA, 0.3g/L Sodium azide, pH7.4) in, be placed in 4 DEG C to save backup, so far obtained quantum dot-labeled anti-human influenza A virus nano-probe.

The carboxyl water-soluble quantum dot that mouse-anti people influenza B virus NP protein monoclonal antibody and emission wavelength are 600nm is utilized to obtain quantum dot-labeled anti-human influenza B virus NP protein nano probe by above-mentioned same procedure.By nano-probe solution quantum dot-labeled for above-mentioned two kinds of different emission 1:1 mixing by volume, i.e. the anti-human influenza virus nano-probe of obtained color quantum point mark.

Embodiment 3 immune nanometer magnetic bead carries out the optimization of immunocapture condition to human influenza virus's antigen

Using coupling, the immune nanometer magnetic bead of rabbit anti-human influenza A virus NP protein polyclone antibody is as solid phase carrier, quantum dot-labeled mouse-anti influenza virus A hominis NP protein monoclonal antibody is as detection antibody, by double-antibody method principle, set up the detection system of human influenza virus's antigen.Respectively to the consumption of immune nanometer magnetic bead in detection system, the conditions such as capture time have carried out a series of optimum choice.

1. the selection of immune nanometer magnetic bead addition

By 20 μ l, 40 μ l, 60 μ l, 80 μ l, 100 μ l, 120 μ l, 140 μ l, 160 μ l, 180 μ l, 200 μ l join the sample treatment liquid (8g/LNaCL of influenza virus A hominis (ATCC numbering VR-1743) of 0.5ml containing 10ng/mL respectively by the made immune nanometer magnetic bead got ready of embodiment 1,0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, 0.3g/LNaN ₃, 5ml/LNonidetP-40,5ml/LTritonX-100, pH7.4) in, carry out immunocapture, the more quantum dot-labeled probe described by embodiment 2 detects, record fluorescent value.Found that, along with the increase of immune nanometer magnetic bead addition, fluorescent value increases gradually, and when immune nanometer magnetic bead addition reaches 140 μ l, fluorescent value reaches maximum.Continue the amount increasing immune nanometer magnetic bead again, fluorescent value reduces on the contrary.Therefore this experimental selection 140 μ l is as the optimal addn of immune nanometer magnetic bead.

2. the selection of immunocapture time

After determining the addition of magnetic bead, get four parts of made immune nanometer magnetic beads got ready of embodiment 1, at room temperature with 10r/min, the human influenza virus (ATCC numbering VR-1743) of 10ng/mL is carried out to the immunocapture of 10min, 15min, 20min, 30min, 45min and 60min, quantum dot-labeled probe again described by embodiment 2 detects, record fluorescent value.Found that, fluorescent value reaches maximal value when immunocapture 15min, therefore this experimental selection 15min is as the Best Times of immunocapture.

The preparation of embodiment 4PBST damping fluid

Get 8gNaCL, 0.2gKCl, 0.24KH ₂pO ₄, 1.44gNa ₂hPO ₄, 0.3gNaN ₃, 0.5mlTween-20 is dissolved in 800ml distilled water, adjusts pH to 7.4, then be settled to 1000ml with 5MNaOH.

The preparation of embodiment 5 sample treatment liquid

Get 8gNaCL, 0.2gKCl, 0.24KH ₂pO ₄, 1.44gNa ₂hPO ₄, 0.3gNaN ₃, 5mlNonidetP-40,5mlTritonX-100, adjust pH to 7.4 with 5MNaOH, then be settled to 1000ml.

The preparation of embodiment 6 quality-control product

1. positive quality control product: the influenza virus A hominis (0.25 μ g) with 1% formalin-inactivated and the common drying of people's influenza B virus (0.25 μ g) are attached on swab, are positive quality control product.

2. negative quality-control product: negative quality-control product is namely through the clinical Pharyngeal swab samples determining the crowd being feminine gender for people's first, influenza B virus.

The preparation of embodiment 7 kit

The anti-human influenza virus nano-probe of the anti-human influenza virus immunization nanometer magnetic bead described by embodiment 1, color quantum point mark described by embodiment 2, the PBST damping fluid described by embodiment 4, the sample treatment liquid described by embodiment 5, quality-control product described by embodiment 6 form the human influenza virus's antigens genotyping detection kit based on magnetic resolution and color quantum point mark jointly.

The using method of embodiment 8 kit

Clinical means obtains people's throat swab routinely, after dissolving the clinical sample on throat swab by the sample treatment liquid in 0.5ml kit, lysate is proceeded in the common centrifuge tube of 1.5ml, the anti-human influenza virus immunization nanometer magnetic bead 140 μ l in kit is added in this centrifuge tube, take off after reacting 15min with 10rpm/min on rotary mixer under room temperature, centrifuge tube is inserted magnetic frame and be separated 3min, with pipettor sucking-off supernatant.The PBST damping fluid 1ml added in kit washs twice, and sucking-off cleansing solution after Magneto separate, finally uses the resuspended magnetic bead of 1mLPBS damping fluid.Get 100 μ l above-mentioned immune nanometer magnetic bead-viral antigen compound in another centrifuge tube, add the quantum dot-labeled anti-human influenza virus nano-probe in 100 μ l kits again, on rotary mixer, 15min is reacted with 15rpm/min under room temperature, be combined by the immunity of the viral antigen of the antibody on quantum dot on immune nanometer magnetic bead, quantum dot is tagged to viral antigen surface, forms magnetic bead-viral antigen-quantum dot " sandwich " compound.After having reacted, Magneto separate 3min, remove unnecessary quantum dot-labeled probe, and by PBST buffer solution for cleaning 2 times, compound is dispersed in 100 μ lPBS damping fluids again, use fluorescence microplate reader to be all 405nm at Ex, Em detects its fluorescent value under being respectively the parameter of 520nm and 600nm respectively respectively; Again by four parts that provide in above-mentioned same method detection kit negative quality-control product samples and a positive quality control product sample, read Em respectively and be respectively fluorescent value under 520nm and 600nm; Four parts of negative quality-control product samples are that the mean value of the fluorescent value of 520nm and 3 times of standard deviation sums are designated as CUT-OFF1 value at Em; Four parts of negative quality-control product samples are that the mean value of the fluorescent value of 600nm and 3 times of standard deviation sums are designated as CUT-OFF2 value at Em; If above-mentioned clinical people's throat swab sample is greater than CUT-OFF1 value at the detection fluorescent value that Em is 520nm, be then judged as that in above-mentioned clinical people's throat swab sample, influenza virus A hominis's antigen is for positive; If above-mentioned clinical people's throat swab sample is less than CUT-OFF1 value at the detection fluorescent value that Em is 520nm, be then judged as that in above-mentioned clinical people's throat swab sample, influenza virus A hominis's antigen is for negative.If above-mentioned clinical people's throat swab sample is greater than CUT-OFF2 value at the detection fluorescent value that Em is 600nm, be then judged as that in above-mentioned clinical people's throat swab sample, people's influenza B virus antigen is for positive; If above-mentioned clinical people's throat swab sample is less than CUT-OFF2 value at the detection fluorescent value that Em is 600nm, be then judged as that in above-mentioned clinical people's throat swab sample, people's influenza B virus antigen is for negative.

If the fluorescent value of positive quality control product sample is less than CUT-OFF1 or CUT-OFF2 value, then show that kit lost efficacy.

The detection sensitivity of embodiment 9 kit and specific test

After influenza virus A hominis H1N1 hypotype strain (ATCC numbering VR-1743) sample sample treatment liquid is carried out serial dilution, detect with this kit, it is 0.1ng/ml that result shows that it detects the lowest limit.After people's influenza B virus (ATCC numbering VR-790) sample sample treatment liquid is carried out serial dilution, detect with this kit, it is also 0.1ng/ml that result shows that it detects bottom line.Simultaneously, with Ureaplasma urealyticum (ATCC numbering 27618), mycoplasma hominis (ATCC numbering 23114), Chlamydia pneumoniae (AR-39 strain, ATCC numbering 53592), (the GB strain of streptococcus pneumonia (ATCC numbering 49619) adenovirus hominis 3 type, ATCC numbering VR-3), (the Gomen strain of adenovirus hominis 7 type, ATCC numbering VR-7), haemophilus influenzae (ATCC numbering 53781), moraxelle catarrhalis (ATCC numbering 25238), Respiratory Syncytial Virus(RSV) (ATCC numbering VR26), people I type parainfluenza virus (ATCC numbering VR-94), people II type parainfluenza virus (ATCC numbering VR-92), human III type parainfluenza virus (ATCC numbering VR-93) etc. detects, the sample treatment liquid liquid that kit detects containing these microorganisms is all negative.

The contrast test of embodiment 10 susceptibility

After the chick embryo culture supernatant sample treatment liquid of human influenza virus H1N1 hypotype strain (ATCC numbering VR-1743) is done serial dilution, do contrast by kit described in the embodiment of the present invention 7 and Quidel company of U.S. detection kit respectively to detect, found that, the detection bottom line of Quidel company of U.S. kit is 800 times of dilutions, and the detection bottom line of kit provided by the invention is 32000 times of dilutions.

Embodiment 11 clinical trial example

" goldstandard " isolated culture is detected as reference using human influenza virus, in epidemic season, get the detection that 200 routine clinical oropharyngeal swab specimens carry out influenza virus A hominis's antigen, cultivation positive rate is 10.5% (21/200), this kit is 11.0% (22/200), and the coincidence rate of 2 kinds of methods is 98.5% (197/200).Concrete outcome is as shown in table 1.

The testing result of table 1 clinical samples

It is pointed out that and the foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments, equivalent replacement etc. made within the present invention's spirit and principle all should be included within protection scope of the present invention.

Claims (9)

1. the method that the human influenza virus's antigens genotyping marked based on magnetic resolution and color quantum point detects, is characterized in that: said method comprising the steps of:

1) respectively anti-human for rabbit first, influenza B virus NP protein polyclone antibody and nanometer magnetic bead are passed through covalent coupling, prepare anti-human influenza A virus immune nanometer magnetic bead and anti-human influenza B virus immune nanometer magnetic bead respectively; By anti-human influenza A virus immune nanometer magnetic bead and anti-human influenza B virus immune nanometer magnetic bead mixed in equal amounts i.e. obtained anti-human influenza virus immunization nanometer magnetic bead;

2) by mouse-anti influenza virus A hominis NP protein monoclonal antibody and emission wavelength be the nano-quantum point of 520nm by covalent coupling, prepare quantum dot-labeled anti-human influenza A virus nano-probe; By mouse-anti people influenza B virus NP protein monoclonal antibody and emission wavelength be the nano-quantum point of 600nm by covalent coupling, prepare quantum dot-labeled anti-human influenza B virus nano-probe; By the anti-human influenza virus nano-probe that anti-human influenza A virus nano-probe and anti-human influenza B virus nano-probe mixed in equal amounts i.e. obtained color quantum point mark;

3) human respiratory secretion sample is got, after dissolving by sample treatment liquid, add step 1) the anti-human influenza virus immunization nanometer magnetic bead for preparing, abundant mixing, Magneto separate is carried out after reaction 10-45min, after PBST buffer solution 2 times, step 2 is added in the sediment that Magneto separate obtains) the anti-human influenza virus nano-probe of the color quantum point for preparing mark, Magneto separate is carried out after reaction 10-45min, after PBST buffer solution 2 times, fluorescence microplate reader is used to be all 405nm at Ex, Em reads fluorescent value under being respectively the parameter of 520nm and 600nm respectively, in described sample treatment liquid, each component concentration is respectively: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, 0.3g/LNaN ₃, 5ml/LNonidetP-40,5ml/LTritonX-100, described sample treatment liquid pH=7.4, in described PBST damping fluid, each component concentration is respectively: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, 0.3g/LNaN ₃, 0.5ml/LTween-20, described PBST pH of buffer=7.4,

4) according to step 1)-step 3) method detect four parts respectively and determine for people's first, influenza B virus is the respiratory secretions sample of negative crowd through clinical, read respectively Em be respectively 520nm and 600nm under fluorescent value; The respiratory secretions sample that described people's first, influenza B virus is negative crowd is called for short human influenza virus's negative control sample; Described four parts of human influenza virus's negative control sample are that the mean value of the fluorescent value of 520nm and 3 times of standard deviation sums are designated as CUT-OFF1 value at Em; Described four parts of human influenza virus's negative control sample are that the mean value of the fluorescent value of 600nm and 3 times of standard deviation sums are designated as CUT-OFF2 value at Em; If step 3) in human respiratory secretion sample be greater than CUT-OFF1 value at the detection fluorescent value that Em is 520nm, to be then judged as in human respiratory secretion sample that influenza virus A hominis's antigen is for the positive; If step 3) in human respiratory secretion sample be less than CUT-OFF1 value at the detection fluorescent value that Em is 520nm, to be then judged as in human respiratory secretion sample that influenza virus A hominis's antigen is for feminine gender; If step 3) in human respiratory secretion sample be greater than CUT-OFF2 value at the detection fluorescent value that Em is 600nm, to be then judged as in human respiratory secretion sample that people's influenza B virus antigen is for the positive; If step 3) in human respiratory secretion sample be less than CUT-OFF2 value at the detection fluorescent value that Em is 600nm, to be then judged as in human respiratory secretion sample that people's influenza B virus antigen is for feminine gender.

2., based on human influenza virus's antigens genotyping detection kit of magnetic resolution and color quantum point mark, it is characterized in that: described kit is made up of the anti-human influenza virus nano-probe, quality-control product, sample treatment liquid and the PBST damping fluid that have the anti-human influenza virus immunization nanometer magnetic bead of enrichment human influenza virus antigen function, color quantum point marks; Described quality-control product comprises positive quality control product and negative quality-control product; Described positive quality control product is attached on swab by the common drying of people's first, second influenza virus of deactivation and forms; Described negative quality-control product is through the clinical throat swab determining the crowd being feminine gender for people's first, second influenza virus.

3., for the preparation of the method for human influenza virus's antigens genotyping detection kit based on magnetic resolution and color quantum point mark as claimed in claim 2, it is characterized in that: described preparation method comprises the following steps:

1) preparation of anti-human influenza virus immunization nanometer magnetic bead:

1.1) get 5mg magnetic bead, with 1mlMES buffer solution three times, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant; Described magnetic bead is with superparamagnetism Fe ₃o ₄for the carboxyl magnetic bead that kernel, particle diameter are 1150nm; 2-(N-morpholino) ethyl sulfonic acid of described MES damping fluid to be mass concentration be 2g/L; The pH=6.0 of described MES damping fluid; The magnetic intensity of described nano magnetic separation vessel is 0.4T;

1.2) add successively by step 1.1) in the concentration of MES buffer be 8-12mg/ml EDC solution and by step 1.1) in the concentration of MES buffer be each 0.5ml of sulfo-NHS solution of 6-10mg/ml, in rotary mixer, 1hr is activated with 10-40rpm/min, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, by 1ml step 1.1) in MES damping fluid resuspended, obtain activate after magnetic bead;

1.3) 5 centrifuge tubes are got, 200 μ L steps 1.2 are added in each centrifuge tube) magnetic bead after the activation that obtains, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, adding by the concentration of PBS damping fluid dilution in each centrifuge tube is each 1ml of rabbit anti-human influenza A virus NP protein polyclone antibody solution of 50-200 μ g/ml, in rotary mixer, 2-6h is reacted with 15rpm/min under room temperature, be placed in after removing supernatant after nano magnetic separation vessel carries out Magneto separate, respectively add the above-mentioned PBS damping fluid of 1ml containing 1mg/ml monoethanolamine, under room temperature with 15rpm/min react in rotary mixer 2h with on closed magnetic bead not with the carboxyl of antibody response, in described PBS damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, the pH=7.4 of described PBS damping fluid,

1.4) after capping completes, these 5 centrifuge tubes are placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, respectively wash three times with 1ml lavation buffer solution; In described lavation buffer solution, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, 0.5ml/LTween-20, the pH=7.4 of described lavation buffer solution;

1.5) in each centrifuge tube, add 1ml respectively preserve the resuspended magnetic bead of damping fluid, be placed in 4 DEG C and save backup, so far obtained anti-human influenza A virus immune nanometer magnetic bead; In described preservation damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, 0.3g/LNaN ₃, 5g/L bovine serum albumin(BSA), the pH=7.4 of described preservation damping fluid;

1.6) by and step 1.1)-1.5) identical method utilizes rabbit anti-human influenza B virus NP protein polyclone antibody to obtain anti-human influenza B virus immune nanometer magnetic bead; By the 1:1 mixing by volume of above-mentioned two kinds of immune nanometer magnetic bead suspensions, i.e. obtained anti-human influenza virus immunization nanometer magnetic bead;

2) preparation of the anti-human influenza virus nano-probe of color quantum point mark:

Its concrete preparation method comprises:

2.1) in microcentrifugal tube, add 4nmol carboxyl water-soluble quantum dot, 600nmolN-N-Hydroxysuccinimide sulfo-NHS and 600nmol carbodiimide EDC successively, with phosphate buffer constant volume for 2ml, mixed solution, after 37 DEG C of reaction 30min, excessive sulfo-NHS and EDC as activator is removed in dialysis, obtains the quantum dot after activating; The emission wavelength of described carboxyl water-soluble quantum dot is 520nm; In described phosphate buffer, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride; The pH=7.4 of described phosphate buffer;

2.2) in step 2.1) in the quantum dot of activation that obtains, add the mouse-anti influenza virus A hominis NP protein monoclonal antibody of 4-16nmol, lucifuge reaction 2h, adds single-ended amination polyglycol PEG2000-NH ₂be 1% to final concentration, close unreacted activated carboxyl site, continue lucifuge reaction 1h;

2.3) by 0.2 μm of PES frit removing step 2.2) in antibody aggregation thing, then filtrate is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, there is not the antibody of coupling reaction and the accessory substance in reacting in removing;

2.4) step 2.3 is collected) middle ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleansing solution, again this solution is transferred in a new 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, collect ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1ml phosphate conserving liquid, be placed in 4 DEG C and save backup, so far obtained quantum dot-labeled anti-human influenza A virus nano-probe; In described phosphate cleansing solution, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, 5ml/L Tween-20,0.3g/L Sodium azide, the pH=7.4 of described phosphate cleansing solution; In described phosphate conserving liquid, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/L bovine serum albumin(BSA), 0.3g/L Sodium azide; The pH=7.4 of described phosphate conserving liquid;

2.5) by and step 2.1)-2.4) identical method utilizes the carboxyl water-soluble quantum dot that mouse-anti people influenza B virus NP protein monoclonal antibody and emission wavelength are 600nm to obtain quantum dot-labeled anti-human influenza B virus nano-probe; Above-mentioned two kinds are contained the solution 1:1 mixing by volume of the quantum dot-labeled nano-probe of different emission, i.e. the anti-human influenza virus nano-probe of obtained color quantum point mark;

3) preparation of PBST damping fluid:

Its concrete compound method comprises:

Get 8gNaCL, 0.2gKCl, 0.24KH ₂pO ₄, 1.44gNa ₂hPO ₄, 0.3gNaN ₃, 0.5mlTween-20 is dissolved in 800ml distilled water, adjusts pH to 7.4, then be settled to 1000ml with 5MNaOH;

4) preparation of sample treatment liquid:

Get 8gNaCL, 0.2gKCl, 0.24KH ₂pO ₄, 1.44gNa ₂hPO ₄, 0.3gNaN ₃, 5mlNonidetP-40,5mlTritonX-100, adjust pH to 7.4 with 5MNaOH, then be settled to 1000ml;

5) preparation of quality-control product:

5.1 positive quality control product: positive quality control product is attached on swab by the common drying of people's first, influenza B virus of deactivation and forms;

5.2 negative quality-control products: negative quality-control product is namely through the clinical throat swab determining the crowd being feminine gender for people's first, second influenza virus.

4. method according to claim 3, it is characterized in that: described step 1.2) in, add successively by step 1.1) in the concentration of MES buffer be 10mg/ml EDC solution and by step 1.1) in the concentration of MES buffer be each 0.5ml of sulfo-NHS solution of 8mg/ml, in rotary mixer, 1hr is activated with 15rpm/min, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, by 1ml step 1.1) in MES damping fluid resuspended, obtain activate after magnetic bead;

Described step 1.3) in, adding by the concentration of PBS damping fluid dilution in each centrifuge tube is each 1ml of rabbit anti-human influenza A virus NP protein monoclonal antibody solution of 100 μ g/ml, in rotary mixer, 3h is reacted with 15rpm/min under room temperature, be placed in after removing supernatant after nano magnetic separation vessel carries out Magneto separate, respectively add the above-mentioned PBS damping fluid of 1ml containing 1mg/ml monoethanolamine;

Described step 2.2) in, in step 2.1) in the quantum dot of activation that obtains, add 12nmol mouse-anti influenza virus A hominis NP protein monoclonal antibody, lucifuge reaction 2h.

5. the method according to claim 3 or 4, is characterized in that: described quantum dot is water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer is modified.

6. method according to claim 5, is characterized in that: described magnetic bead is with superparamagnetism Fe ₃o ₄for the carboxyl magnetic bead that kernel, shell material are polystyrene, surface functional group is carboxyl, particle diameter is 1150nm.

7., based on the using method of human influenza virus's antigens genotyping detection kit based on magnetic resolution and color quantum point mark as claimed in claim 2, it is characterized in that: described using method comprises the following steps:

1) after sample 0.5ml sample treatment solution to be checked being dissolved, lysate is proceeded in the common centrifuge tube of 1.5ml; In described sample treatment solution, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄, 0.3g/LNaN ₃, 5ml/LNonidetP-40,5ml/LTritonX-100; The pH=7.4 of described sample treatment solution;

2) to step 1) in centrifuge tube in add based on magnetic resolution and color quantum point mark human influenza virus's antigens genotyping detection kit in anti-human influenza virus immunization nanometer magnetic bead 50-200 μ l, take off after reacting 10-45min with 10rpm/min on rotary mixer under room temperature, centrifuge tube is inserted nano magnetic separation vessel Magneto separate 3min, with pipettor sucking-off supernatant;

3) the PBST damping fluid 1ml added in kit washs twice, and sucking-off cleansing solution after employing nano magnetic separation vessel Magneto separate, finally uses the resuspended magnetic bead of 1mlPBS damping fluid, obtained immune nanometer magnetic bead-viral antigen compound; In described PBS damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄; The pH=7.4 of described PBS damping fluid;

4) 100 μ l steps 3 are got) immune nanometer magnetic bead-viral antigen compound of obtaining is in another centrifuge tube, add again 100 μ l based on magnetic resolution and color quantum point mark human influenza virus's antigens genotyping detection kit in color quantum point mark anti-human influenza virus nano-probe, on rotary mixer, 10-45min is reacted with 15rpm/min under room temperature, be combined by the immunity of the viral antigen of the antibody on quantum dot on immune nanometer magnetic bead, quantum dot is tagged to viral antigen surface, form magnetic bead-viral antigen-quantum dot " sandwich " compound,

5) after having reacted, adopt nano magnetic separation vessel Magneto separate 3min, remove the anti-human influenza virus nano-probe of unnecessary color quantum point mark, 2 times are cleaned with the PBST damping fluid liquid in kit, compound is dispersed in 100 μ lPBS damping fluids again, use fluorescence microplate reader to be all 405nm at Ex, Em detects its fluorescent value under being respectively the parameter of 520nm and 600nm respectively respectively; In described PBS damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH ₂pO ₄, 1.44g/LNa ₂hPO ₄; The pH=7.4 of described PBS damping fluid;

6) by four parts that provide in above-mentioned same method detection kit negative quality-control product samples and a positive quality control product sample, read Em respectively and be respectively fluorescent value under 520nm and 600nm; Four parts of negative quality-control product samples are that the mean value of the fluorescent value of 520nm and 3 times of standard deviation sums are designated as CUT-OFF1 value at Em; Four parts of negative quality-control product samples are that the mean value of the fluorescent value of 600nm and 3 times of standard deviation sums are designated as CUT-OFF2 value at Em; If step 5) in measuring samples be greater than CUT-OFF1 value at the detection fluorescent value that Em is 520nm, to be then judged as in sample to be checked that influenza virus A hominis's antigen is for the positive; If step 5) in sample to be checked be less than CUT-OFF1 value at the detection fluorescent value that Em is 520nm, then the influenza virus A hominis's antigen in sample to be checked that judges to behave is feminine gender; If step 5) in sample to be checked be greater than CUT-OFF2 value at the detection fluorescent value that Em is 600nm, to be then judged as in sample to be checked that people's influenza B virus antigen is for positive; If step 5) in sample to be checked be less than CUT-OFF2 value at the detection fluorescent value that Em is 600nm, to be then judged as in sample to be checked that people's influenza B virus antigen is for negative; If the fluorescent value of positive quality control product sample is less than CUT-OFF1 or CUT-OFF2 value, then show that kit lost efficacy.

8. method according to claim 7, it is characterized in that: described step 2) in, to step 1) in centrifuge tube in add based on magnetic resolution and color quantum point mark human influenza virus's antigens genotyping detection kit in anti-human influenza virus immunization nanometer magnetic bead 140 μ l, take off after reacting 15min with 10rpm/min on rotary mixer under room temperature, centrifuge tube is inserted magnetic frame and be separated 3min, with pipettor sucking-off supernatant;

Described step 4) in, get 100 μ l steps 3) immune nanometer magnetic bead-viral antigen compound of obtaining is in another centrifuge tube, add again 100 μ l based on magnetic resolution and color quantum point mark human influenza virus's antigens genotyping detection kit in color quantum point mark anti-human influenza virus nano-probe, on rotary mixer, 15min is reacted with 15rpm/min under room temperature, be combined by the immunity of the viral antigen of the antibody on quantum dot on immune nanometer magnetic bead, quantum dot is tagged to viral antigen surface, form magnetic bead-viral antigen-quantum dot " sandwich " compound.

9. the method according to claim 7 or 8, is characterized in that: described sample to be checked includes but not limited to throat swab.