CN105785026A - Kit for detecting enterovirus 71 IgM antibodies and detection method - Google Patents
Kit for detecting enterovirus 71 IgM antibodies and detection method Download PDFInfo
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Abstract
The invention discloses a kit for detecting enterovirus 71 IgM antibodies and a detection method. The kit comprises one or more solid carriers and a polypeptide aggregation 1 individually connected to the one or more solid carriers. According to the detection method, when it is detected that any one or more polypeptides in the above polypeptide aggregation 1 response to enterovirus 71 antibody IgM in a blood sample from a subject, it is determined that the subject is an enterovirus 71 infection patient. The kit of the invention has good accuracy and high specificity, is used for detecting enterovirus 71 IgM antibody infection, and is further used in auxiliary diagnosis of hand-foot-and-mouth disease.
Description
Technical field
The invention mainly relates to Measurement for Biotechnique and medical science.Specifically, the present invention relates to the test kit of detection enterovirns type 71 IgM antibody and detection method.
Background technology
The infection of enterovirus EV 71 type is the general name of the disease caused by human enterovirus 71 (EV71), main manifestations is hand-foot-mouth disease clinically, the many eruption and prevalences in infant of hand-foot-mouth disease, part EV71 infects infant and shows as herpangina, and viral encephalitis, viral cerebrospinal meningitis, pulmonary edema, pneumorrhagia etc. occurs in patient with severe symptoms.Cause the cause of disease of hand-foot-mouth disease, be most commonly that coxsackie virus A 16 and enterovirns type 71；Infected the large percentage of the disease generation severe infection caused by enterovirns type 71, case fatality rate is also higher, and severe cases case fatality rate is up to 10%-25%, and therefore etiological diagnosis is significant.
Enterovirns type 71 (EV71) genome is the single-stranded positive RNA containing about 7.5Kb, including 5' end noncoding region, 3' end noncoding region and by Pl, P2, the polyprotein that P3 forms, altogether coding 11 kinds of albumen, wherein 4 structural protein VPl, VP2, VP3, VP4,7 NS2 Protein A, 2B, 2C, 3A, 3B, 3C, 3D.Wherein VP1, VP2, VP3 are exposed to the surface of virus coat, for antigenic variation position main for EV71；VP4 is embedded in the inner side of virus coat and is combined with internal RNA, and the anchor of the similar virus coat of its function, in order to the structure of fixed virus albumen.NS2 Protein A is division initial stage pioneer's protein, also relevant with terminating host cell itself desirable proteins of synthesis；3C then participates in the reaction of most protein cleavage；3D is RNA polymerase, dominates when viral RNA replicates and transcribes and replication reaction；Four kinds of albumen such as 2B, 2C, 3A, 3B are then relevant with the duplication of viral RNA.
Three aspects of the clinical diagnosis Main Basis of enterovirus EV 71: one is clinical manifestation, two is serological evidence, and three is Etiological Evidence.Due to enterovirus EV 71 type, to infect clinical manifestation many, and therefore the Serologic detection of enterovirus EV 71 and pathogeny detection are current topmost foundations.
The detection of enterovirns type 71 IgM antibody has had pertinent literature to report, substantially adopts colloidal gold methods or euzymelinked immunosorbent assay (ELISA) more, and at cost, the aspect such as specificity still has many weak points.Protein chip has become as the trend of technical development due to the advance of technology, but for having no the report of the enterovirns type 71 IgM antibody detection kit based on protein chip method on market.
Summary of the invention
It is an object of the invention to provide a kind of accuracy good, test kit and the detection method for detecting enterovirns type 71 IgM antibody that specificity is high.
Current inventor provides 4 polypeptide.Surprisingly, the inventors discovered that, although described 4 polypeptide individually detect hand-foot-mouth disease infect in IgM antibody time sensitivity all between 35.2%～80.6%, far away not up to the requirement as detection instrument, but, when the biological specimen that the present invention originates using experimenter whether at least one in described 4 polypeptide or one or more have response as judgement experimenter whether by the index of Infected With Plasmodium, enterovirns type 71 IgM antibody can be detected with the sensitivity (false positive rate is low to moderate 2.6%) up to 95.2% to infect, and then the auxiliary for hand-foot-mouth disease diagnoses.
Therefore, the present invention includes:
1., for detecting a test kit for enterovirns type 71 IgM antibody, it includes one or more solid carrier, and is independently connected to the following polypeptide set 1 on the one or more solid carrier:
Polypeptide shown in SEQIDNO:1；
Polypeptide shown in SEQIDNO:2；
Polypeptide shown in SEQIDNO:3；With
Polypeptide shown in SEQIDNO:4.
2. test kit according to claim 1, it is used for detecting enterovirns type 71 antibody IgM.
3. test kit according to claim 1, wherein, whole polypeptide are independently connected on same solid carrier.
4. the following polypeptide set 1 purposes in preparing the test kit for detecting enterovirns type 71 antibody IgM:
Polypeptide shown in SEQIDNO:1；
Polypeptide shown in SEQIDNO:2；
Polypeptide shown in SEQIDNO:3；With
Polypeptide shown in SEQIDNO:4.
5. detecting the method whether experimenter is enterovirns type 71 infected patient, the method includes:
Use whether the enterovirns type 71 antibody IgM that the test kit according to any one of claim 1～3 detects in the blood sample that experimenter is originated by the polypeptide in described polypeptide set 1 has response；
When the enterovirns type 71 antibody IgM in the blood sample that experimenter is originated by any one or the more than one polypeptide that detect in described polypeptide set 1 has response, it is determined that this experimenter is enterovirns type 71 infected patient.
6. method according to claim 5, wherein, described blood sample is whole blood, blood plasma or serum.
7 methods according to claim 5, wherein, described experimenter is the experimenter with hand-foot-mouth disease clinical symptoms.
The detailed description of the invention of invention
Polypeptide set
In this specification, polypeptide set 1 refers to the whole polypeptide being positioned at the first detection zone, and described polypeptide set 1 comprises independent following polypeptide:
Peptide sequence shown in SEQIDNO:1 is SLDFALSLLRRNIRQVQTDQ；
Peptide sequence shown in SEQIDNO:2 is APTGQNTQVSSHRLDTGKVP；
Sequence shown in SEQIDNO:3 is polypeptide A REKVEFLNNLKQLPLLENQ；With
Sequence shown in SEQIDNO:4 is polypeptide GHFTMLGVRDRLAVLPRHSQ；
Described polypeptide can be suitable for adopting the known method such as (1) chemical synthesis process or (2) enzyme reaction synthetic method to obtain, and wherein chemosynthesis is more easy.In the polypeptide situation of the chemosynthesis present invention, by using peptide synthesizer synthesis or this polypeptide semi-synthetic to carry out.As chemical synthesis process, it is possible to list such as peptide solid-phase synthesis etc..So the peptide of synthesis can adopt conventional means such as ion exchange chromatography, reverse phase high performance liquid chromatograph, affinity chromatography etc. to be purified.Such peptide solid phase synthesis process with and subsequent peptide purification be all well-known in the art.
Additionally, when being produced the polypeptide of the present invention by enzyme reaction, it is possible to adopt such as No. WO2004/011653 described method of International Publication pamphlet.Namely; can so produce: the aminoacid esterified for the carboxyl terminal of the aminoacid of a side or dipeptides or amidatioon obtained or dipeptides and aminoacid are in the aminoacid (aminoacid of such as carboxy protective) of free state and react under the existence of peptide synthetase, the dipeptides of generation or tripeptides.As peptide synthetase, it is possible to list: have the bacterial disposing thing or this microbe-derived peptide synthetase that generate the culture of microorganism of ability of peptide, this culture microbial cells separated or this microorganism.
Particularly the chemosynthesis of polypeptide has been realized in commercialization, it is possible to by entrusting the Peptide systhesis company of specialty to synthesize described polypeptide.
Test kit
The present invention provides a kind of detection kit (test kit of the present invention), and it includes one or more solid carrier, and is independently connected to the following polypeptide set 1 on the one or more solid carrier:
Polypeptide shown in SEQIDNO:1；
Polypeptide shown in SEQIDNO:2；
Polypeptide shown in SEQIDNO:3；With
Polypeptide shown in SEQIDNO:4.
In this specification, sensitivity refers to: with in the positive sample of " goldstandard " method confirmation, be determined as the ratio of positive sample by additive method.False positive rate refers to: with in the negative sample of " goldstandard " method confirmation, be determined as the ratio of positive sample by additive method." goldstandard " method that the art detection human enterovirus 71 infects is nucleic acid detection method, i.e. PCR method.In this specification, " detection " can be make a definite diagnosis, it is also possible to is for making a definite diagnosis offer reference information.
In the present invention, solid carrier is not particularly limited, as long as the carrier of solid or insoluble material (such as can be by the material that filtration, precipitation, Magnetic Isolation etc. separate from reactant mixture).
The material constituting solid carrier includes but not limited to: silica gel (polydimethylsiloxane, PDMS), cellulose, polytetrafluoroethyleneTM, NC Nitroncellulose, agarose, glucosan, chitosan, polystyrene, polyacrylamide, polyester, Merlon, polyamide, polypropylene, nylon, polyvinylidene fluoride, latex, silicon dioxide, glass, glass fibre, gold, platinum, silver, copper, ferrum, rustless steel, ferrite, silicon wafer, polyethylene, polymine, polylactic acid, resin, polysaccharide, albumen (albumin etc.), carbon or their combination etc..
The shape of solid carrier includes but is not limited to: pearl, magnetic bead, thin film, micro cautery, filter membrane, plate, micro plate, CNT, sensor chip etc..Just as known in the art, the solid carrier that thin film or plate etc. are smooth can be arranged bottom pit, groove, filter membrane etc..
Magnetic bead can have the sphere diameter of about 25nm～about 1mm scope.In a preferred embodiment, magnetic bead has the diameter of about 50nm～about 10 μ m.The size of magnetic bead can select according to specific purposes.The pearl being made up of the contour crosslinked spherical agarose of Sepharose has the diameter of about 24 μm～about 165 μ m.Preferably, high crosslinked spherical sepharose 4B has the diameter of about 24 μm～about 44 μ m.The size of high crosslinked spherical sepharose 4B can select according to specific purposes.
The polystyrene latex beads such as having that the example of the solid carrier of hydrophobic surface includes can from Polysciences, Warrington, PA or Spherotech, Liberville, the goods that IL buys.
Silicon dioxide (SiO2)-process or silicon dioxide (SiO2) example of solid carrier of base includes the extraordinary magnetic silica pearl etc. that can buy from Polysciences, Warrington, PA, it may be used for catching nucleic acid (such as DNA).Or, it is also possible to using can from the DynalBiotech M-280 etc. bought.
The magnetic bead with hydrophilic surface can be used for catching the bacterial cell of proliferation period, nucleic acid and other composition.Example as magnetic bead, Polysciences can be listed, Warrington, the pearl (title: Biomag (registered trade mark) carboxyl) of PA sale or BangsLaboratory, Inc., the name of Fishers, IN is called the pearl of MC02N/2928, or can use the DynalBiotech M-270 etc. sold.
In a preferred embodiment, described solid carrier is SJ modified silica-gel, and it is the microarray solid support material (iPDMS thin film, referring to Chinese patent CN101265329A) of a kind of silicone rubber material that Suzhou consor thing Materials Co., Ltd develops.This material is based on the PDMS that biological study is commonly used, add specific initiator composition (make this material can pass through surface initiated polymerization (SIP) and realize surface-functionalized modification) wherein, obtain then through polyethylene glycol methacrylate-styrene polymer (poly (oligo (ethyleneglycol) methacrylate), pOEGMA) finishing.SJ modified silica-gel has outstanding anti-protein non-specific adsorption (Nonspecificproteinadsorption, NPA) ability, non-specific protein absorption in complicated protein immunization detection can be controlled to close to " absolute 0 " level (being near or below the detectable limit of instrument), it is possible not only to exempt the trouble closed and repeatedly clean, it is also possible to by using higher amplification of signal means to improve the susceptiveness of protein microarray.And the essence of its silicone rubber imparts the stronger mechanical performance of this material and good operability.SJ modified silica-gel has successfully been applied to the combination assay microarray ELISA kit of 11 tumor markers compositions by the poly-company in Suzhou, achieve high flux and high-sensitive detection, it was demonstrated that this material is a kind of outstanding protein microarray solid support material.Meanwhile, this material also has the adjustable characteristic of surface nature, it is possible to adjust its surface topography within the specific limits by the controlled modification response time.
The connection of polypeptide and solid carrier can adopt the method for attachment that well known to a person skilled in the art polypeptide and solid carrier to carry out.Such as, for the connection on protein/polypeptide and modified silica-gel surface, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides [1-ethyl-3-(3-dimethylami-nopropyl) carbodiimide can be passed through, EDC] and N-hydroxy-succinamide (N-hydroxysuccinimide, NHS) reaction changes the carboxyl (-COOH) group on the macromolecular chain of modified silica-gel surface into activated group, this activated group can react thus realizing protein/polypeptide is fixed on solid carrier surface with the amino (-NH2) be with on protein/polypeptide.
In the sampling liquid used during for point sample, the concentration of polypeptide is not particularly limited, and those skilled in the art can conventionally select, it is preferred to 1 μ g～1000 μ g/mL, more preferably 10 μ g～500 μ g/mL.Additionally, the density being distributed on a solid support for polypeptide is not particularly limited, those skilled in the art can conventionally select, it is preferred to 1～100 points/10mm2, more preferably 5～50 points/10mm2。
In this manual, " independent " refers to: this polypeptide forms a point on described solid carrier, does not comprise other polypeptide detecting influential amount, it is preferable that only comprise this polypeptide in this point.
Solid carrier in the test kit of the present invention can be one, it is also possible to is multiple, it is preferred to two or more.
This test kit can also include:
1. the serum dilution prepared or serum dilution component solution: serum dilution, for instance have the application of sample variable color Sample dilution (production code member bwj010103) etc. of the Sample dilution (production code member 070021-S2) of Sai Chi bio tech ltd, Beijing, Bo Weijia bio tech ltd, Zhengzhou.This serum dilution is used for dilute serum, and the serum of test kit detection to dilute suitable multiple, for instance 2～200 times, it is preferable that 10～100 times.
This test kit can also include:
2. concentration washing liquid: after solid carrier surface hatches serum and ELIAS secondary antibody, the unconjugated antibody of solid carrier surface and ELIAS secondary antibody need to be washed by washing liquid.Concentration washing liquid is such as the polysorbas20 aqueous solution of 1%, need to dilute 2～40 times, preferably 5～20 times during use.
This test kit can also include:
3. ELIAS secondary antibody solution: the polypeptide in the polypeptide set I on solid carrier (such as SJ modified silica-gel) of the IgM in human enterovirus 71 infected person anteserum is combined, ELIAS secondary antibody can be combined with IgM, and the label in ELIAS secondary antibody can react with luminous substrate, thus sending detectable light.ELIAS secondary antibody can be the goat-anti people IgM of such as horseradish peroxidase-labeled.ELIAS secondary antibody concentration in ELIAS secondary antibody solution is not particularly limited, it is possible to be example 1ng～1000ng/mL.
This test kit can also include:
4. luminescent solution component solution: luminescent solution can react with the horseradish peroxidase of labelling in ELIAS secondary antibody so that reaction sends the chemical light that instrument can detect that.Luminescent solution is mixed by two kinds of solution, is A liquid hydrogenperoxide steam generator respectively, and B liquid luminol solution.Luminol (luminol) is only crossed by oxidizer treatment just can luminescence.Generally use the mixed aqueous solution of hydrogen peroxide and a kind of hydroxide bases as exciting agent.Under horseradish peroxidase enzyme catalytic, decomposing hydrogen dioxide solution is oxygen and water:
2H2O2→O2+2H2O
Generating a pairs of anion with hydroxide when luminol reacts, the dioxygen oxidation that it can be gone out by peroxide decomposition, product is an organic peroxide.This peroxide is very unstable, decomposites nitrogen immediately, generates the 3-aminophthalic acid of excited state.During excited state converts to ground state, the energy of release exists with the form of photon, and wavelength is positioned at the blue light components of visible ray.The example of luminescent solution component solution such as ThermoSeientific companyELISAFemtoMaximumSensitivitySubstrate, article No. 37074.
This test kit can also include:
5. one or two above biological incubation reactors, can adopt such as two-sided biological incubation reactor, Chinese patent ZL201120177686.3 or ZL201110142518.5 according to demand；Or the biological incubation reactor ZL201220430886.x of one side.
This test kit can also include:
6. other are for detecting the detection molecules (such as polypeptide, protein, nucleic acid etc.) that human enterovirus 71 infects.
This test kit can also include:
7. operation instructions.
This test kit can also include:
8. it is connected to the positive quality control point on the one or more solid carrier or other independent solid carriers (can adopt is such as that people IgM is as positive quality control point) and/or negative Quality Control point (such as phosphate buffer is called for short PB).
Detection method
In one aspect of the method, whether the present invention provides a kind of experimenter of detection to be the method (detection method of the present invention) that human enterovirus 71 infects, and the method includes:
Use whether the enterovirns type 71 antibody IgM that the test kit of the invention described above detects in the blood sample that experimenter is originated by the polypeptide in described polypeptide set I has response；
When the enterovirns type 71 antibody IgM in the blood sample that experimenter is originated by any one or the more than one polypeptide that detect in described polypeptide set 1 has response, judge that this experimenter is as enterovirns type 71 infected patient, thus the auxiliary for hand-foot-mouth disease diagnoses.
In this manual, " response " refers to: signal to noise ratio (SNR) is more than or equal to 2, wherein, and signal to noise ratio=(polypeptide point signal value-negative control point signal value)/negative control point signal value.
In this specification, the positive reaction of the test kit of the present invention is referred to: the blood sample in experimenter source meets above-mentioned criterion；It it is otherwise negative reaction.
Described blood sample can be whole blood, blood plasma or serum.
In one preferred embodiment, described experimenter can be the experimenter with hand-foot-mouth disease clinical symptoms.In the present invention, hand-foot-mouth disease clinical symptoms is embodied in the positions such as heating and hands, foot, oral cavity erythra or herpes.
Embodiment
1. the preparation of polypeptide and confirmation
The polypeptide used in embodiment is by the synthesis of gill biochemistry (Shanghai) Co., Ltd., and by mass spectrum, it has been confirmed.Wherein,
SEQIDNO:1 (SLDFALSLLRRNIRQVQTDQ), molecular weight is 2373；
SEQIDNO:2 (APTGQNTQVSSHRLDTGKVP), molecular weight is 2093；
SEQIDNO:3 (AREKVEFLNNLKQLPLLENQ), molecular weight is 2396；
SEQIDNO:4 (GHFTMLGVRDRLAVLPRHSQ), molecular weight is 2289.
2. the preparation of test kit
Detection chip is with SJ modified silica-gel (iPDMS thin film) for solid support material, is prepared from by point sample immobilized polypeptide solution thereon.SJ modified silica-gel is to add with olefin-terminal, surface initiated polymerization initiator in traditional polydimethyl siloxane material, and it is fixed in the three dimensional structure of polydimethylsiloxane by heat cross-linking (si-h bond bonding), obtain a kind of new material and SJ modified silica-gel.Its manufacturing process is referring to International Patent Publication WO2014/044184.
The SJ modified silica-gel thin film made can be saved in 4 DEG C of refrigerators.
AdoptPersonalArrayerTM16 people's point sample instrument prepares polypeptide microarrays (i.e. detection chip) on modified silica-gel, and process is:
1) pretreatment
By SJ modified silica-gel thin slice (15 × 15mm2) be immersed in activating solution, take out with deionized water drip washing 3 times after 30min, dry up with nitrogen, be immediately used to point sample.
2) point sample
Sampling liquid diluted good and transfer in the 384 corresponding micropores of orifice plate, 384 orifice plates with sample being placed on point sample instrument base station, the modified silica-gel thin slice of pretreatment is placed on the base station of point sample instrument simultaneously, carrying out point sample at once.Point sample environmental condition is room temperature (25 DEG C), and humidity set is 50%.Following polypeptide is put into microarray, one point of each polypeptide point sample in polypeptide set I on SJ modified silica-gel thin slice, and additionally one people IgM point of point sample is as positive quality control point and a negative Quality Control point of PB point conduct.On the polypeptide microarrays made, the point sample amount of each point is about 0.6nL, and sampling point radius is 200 μm.
Here, on the modified silica-gel thin slice of test kit, the polypeptide set 1 of point sample is specific as follows:
Polypeptide shown in SEQIDNO:1；
Polypeptide shown in SEQIDNO:2；
Polypeptide shown in SEQIDNO:3；With
Polypeptide shown in SEQIDNO:4；
3) chemistry is fixing
The polypeptide microarrays just made to be placed in climatic chamber (26 DEG C, 60% humidity) and fix at least 6h.Chemistry fixation procedure is referring to International Patent Publication WO2014/044184.
First pass through point sample instrument and will include the buffer point catching peptide molecule on modified silica-gel thin film, then buffer starts evaporation, catch peptide molecule and the surface intimate contact of SJ modified silica-gel and interact, by chemical bond, the high molecular end-COOH of ploy (OEGMA) on the modified silica-gel surface and-NH of peptide molecule2Formed and stablize covalent bond, and then chemically active peptide molecule will be had to be fixed on SJ modified silica-gel surface.
4) assembling
The polypeptide microarrays of fixing 6h must assemble in two days.First pass through gum to be attached on special reaction column by SJ modified silica-gel thin slice, cover reaction cavity.Biological incubation reactor can adopt two-sided biological incubation reactor according to demand, i.e. a reactor is made up of two reaction columns and a reaction cavity, or one side biological incubation reactor, i.e. a reactor is made up of a reaction column and a reaction cavity.
5) preserve
The polypeptide microarrays assembled, it is necessary to evacuation seals, and is saved in the refrigerator of 4 DEG C, standby.
3. detect with test kit
Testing sequence
Testing sequence
1) balance temperature of reagent: by all reagent balance to room temperature.
2) preparation of washing liquid: washing liquid purified water will be concentrated or 1:9 dilution pressed by distilled water, for instance: 450mL purified water or distilled water add 50mL and concentrates washing liquid, fully mix.Do not make the washing liquid being finished be placed on 2～8 DEG C of preservations, can preserve 3 months.
3) dilution of sample: by testing sample serum dilution by 1:100 dilute (as: 2 μ L serum join 198 μ L serum dilutions, fully mixing).Sample detection needs the sample 200 μ L after dilution.
4) wetted chip: take out the biochip incubation reaction device that detection is required.Add 1mL washing liquid, infiltration chip surface 3 minutes.Discard the washing liquid soaking chip.
5) application of sample: in the present embodiment, with one side biological incubation reactor application of sample, liquid-transfering gun is used to be passed through the testing sample that dilute in liquid feeding passage (any one in two) addition one side biological incubation reactor, each one side biological incubation reactor adds 200 μ L, closes liquid feeding passage (two) with sealing paste.
6) sample incubation: be placed on horizontal shaker by the one side biological incubation reactor adding sample 150rpm, incubated at room 30 minutes.
7) clean: reaction column is taken out from the reaction cavity of one side biological incubation reactor, discards liquid in reactor, repeatedly rinse reaction column top chip surface and internal 3 times (every time about the using 12mL washing liquid, rinse 1-2 minute) of reaction cavity.
8) enzyme labelled antibody is hatched: reaction column and reaction cavity are reconfigured, clamping.Throwing off sealing paste, each one side biological incubation reactor adds the goat-anti people IgM solution 200 μ L of horseradish peroxidase-labeled.Again liquid feeding passage is closed with sealing paste.It is placed on shaking table 150rpm, incubated at room 30 minutes.
9) clean again: discard reaction cavity, carefully clean chip surface by washing liquid 3 times.
10) luminescent solution is added: each chip surface is separately added into 30 μ L luminescent solutions, makes luminescent solution can be laid on chip surface uniformly
11) chip imaging: the chip chemiluminescence image adding luminescent solution is analyzed system chip is exposed imaging in 1 minute.When analyzing system as chemiluminescence image, it does not have particular restriction, use such as sky energy 5500 type Microarray image instrument, prompt board TN5500 type Microarray image instrument or prompt board CLEAR4000 type Microarray image instrument etc..
Result judges: for every a serum, whether each polypeptide in statistics test kit has response (that is, signal to noise ratio (SNR) is be more than or equal to 2) respectively, and the criterion described in above-mentioned " detection method " part judges.That is,
When the enterovirns type 71 antibody IgM in any one in the polypeptide detected in described polypeptide set 1 or the more than one blood sample to experimenter source has response, it is determined that this experimenter is enterovirns type 71 infected patient, i.e. hand-foot-mouth disease infected patient.
Wherein, signal to noise ratio=(polypeptide point signal value-negative control point signal value)/negative control point signal value.Polypeptide point signal value refers to the chemiluminescence intensity value of the polypeptide point that imager software kit reads, and negative control point signal value refers to the chemiluminescence intensity value of the negative control point that imager software kit reads.
Embodiment
To collecting clinical serum 240 parts, adopting the test kit testing result of the present invention to compare with the testing result adopting tradition goldstandard method-nucleic acid detection method (adopting the fluorescent quantificationally PCR detecting kit of Takara), result is as shown in table 1.
The test kit of table 1 present invention and the ratio of fluorescent quantificationally PCR detecting kit adopting Takara
Accuracy rate=(118+113)/240 × 100%=96.3%
Sensitivity=118/124 × 100%=95.2%
Specificity=113/116 × 100%=97.4%
False positive=3/116 × 100%=2.6%
Result above shows, the present invention has good accuracy, sensitivity and specificity for detecting the infection of hands enterovirns type 71, it is adaptable to the nosetiology laboratory auxiliary detection that enterovirns type 71 infects, and simple to operate, is suitable for medical treatment at different levels and disease control department's use.
As a comparison, for any bar in above-mentioned 4 polypeptide, it is prepared for detection device according to the description of the title 4 of International Patent Publication WO2014/044184 embodiment part, use above-mentioned 240 parts of serum samples, detect according to the description of International Patent Publication WO2014/044184 embodiment division header 3, when SNR=2, result show above-mentioned 4 polypeptide individually detect hand-foot-mouth disease infect in IgM antibody time sensitivity all between 35.2%～80.6%, (see table 2) can not meet the requirement as detection kit.
24 polypeptide of table sensitivity results unit in 240 parts of serum: %
| Serial number | 1 | 2 | 3 | 4 |
| Enterovirns type 71 recall rate | 80.6 | 35.2 | 67.5 | 75.1 |
Above, by embodiment, the present invention carried out more specific description, but be not the restriction to the technology of the present invention scope.By the record of this specification, those skilled in the art can be easy to the present invention is modified/changes, and these are included in the technical scope of the present invention.
Sequence table
<110>Suzhou Institute of Nano-tech. and Nano-bionics, Chinese Academy of Sciences
<120>for detecting test kit and the detection method of enterovirns type 71 IgM antibody
<130>SJ-EV-1224-1
<160>4
<170>PatentInversion3.5
<210>1
<211>20
<212>PRT
<213>artificial sequence
<220>
<221>misc_feature
<223>Enterovirus 71 antigen polypeptide
<400>1
SerLeuAspPheAlaLeuSerLeuLeuArgArgAsnIleArgGlnVal
151015
GlnThrAspGln
20
<210>2
<211>20
<212>PRT
<213>artificial sequence
<220>
<221>misc_feature
<223>Enterovirus 71 antigen polypeptide
<400>2
AlaProThrGlyGlnAsnThrGlnValSerSerHisArgLeuAspThr
151015
GlyLysValPro
20
<210>3
<211>20
<212>PRT
<213>artificial sequence
<220>
<221>misc_feature
<223>Enterovirus 71 antigen polypeptide
<400>3
AlaArgGluLysValGluPheLeuAsnAsnLeuLysGlnLeuProLeu
151015
LeuGluAsnGln
20
<210>4
<211>20
<212>PRT
<213>artificial sequence
<220>
<221>misc_feature
<223>Enterovirus 71 antigen polypeptide
<400>4
GlyHisPheThrMetLeuGlyValArgAspArgLeuAlaValLeuPro
151015
ArgHisSerGln
20
Claims (7)
1., for detecting a test kit for enterovirns type 71 IgM antibody, it includes one or more solid carrier, and is independently connected to the following polypeptide set 1 on the one or more solid carrier:
Polypeptide shown in SEQIDNO:1；
Polypeptide shown in SEQIDNO:2；
Polypeptide shown in SEQIDNO:3；With
Polypeptide shown in SEQIDNO:4.
2. test kit according to claim 1, it is used for detecting enterovirns type 71 antibody IgM.
3. test kit according to claim 1, wherein, whole polypeptide are independently connected on same solid carrier.
4. the following polypeptide set 1 purposes in preparing the test kit for detecting enterovirns type 71 antibody IgM:
Polypeptide shown in SEQIDNO:1；
Polypeptide shown in SEQIDNO:2；
Polypeptide shown in SEQIDNO:3；With
Polypeptide shown in SEQIDNO:4.
5. detecting the method whether experimenter is enterovirns type 71 infected patient, the method includes:
Use whether the enterovirns type 71 antibody IgM that the test kit according to any one of claim 1 ~ 3 detects in the blood sample that experimenter is originated by the polypeptide in described polypeptide set 1 has response；
When the enterovirns type 71 antibody IgM in the blood sample that experimenter is originated by any one or the more than one polypeptide that detect in described polypeptide set 1 has response, it is determined that this experimenter is enterovirns type 71 infected patient.
6. method according to claim 5, wherein, described blood sample is whole blood, blood plasma or serum.
7. method according to claim 5, wherein, described experimenter is the experimenter with hand-foot-mouth disease clinical symptoms.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410816354.3A CN105785026B (en) | 2014-12-24 | 2014-12-24 | Kit and detection method for detecting enterovirns type 71 IgM antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410816354.3A CN105785026B (en) | 2014-12-24 | 2014-12-24 | Kit and detection method for detecting enterovirns type 71 IgM antibody |
Publications (2)
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| CN107255713A (en) * | 2017-03-31 | 2017-10-17 | 深圳市儿童医院 | Kit available for diagnosis human enterovirus infection |
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| CN113125720A (en) * | 2021-05-07 | 2021-07-16 | 深圳国际旅行卫生保健中心(深圳海关口岸门诊部) | EV71 virus detection chip and preparation method and detection method thereof |
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| CN113125720A (en) * | 2021-05-07 | 2021-07-16 | 深圳国际旅行卫生保健中心(深圳海关口岸门诊部) | EV71 virus detection chip and preparation method and detection method thereof |
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