CN107255713B - It can be used for diagnosing the kit of human enterovirus infection - Google Patents
It can be used for diagnosing the kit of human enterovirus infection Download PDFInfo
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- CN107255713B CN107255713B CN201710207593.2A CN201710207593A CN107255713B CN 107255713 B CN107255713 B CN 107255713B CN 201710207593 A CN201710207593 A CN 201710207593A CN 107255713 B CN107255713 B CN 107255713B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/56983—Viruses
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
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Abstract
The present invention provides the kit that can be used for diagnosing human enterovirus infection.Detection kit of the invention includes polypeptide shown in one or more solid carriers and NO:1~10 SEQ ID being independently connected on the solid carrier.When using whether IgM of the polypeptide shown in NO:1~10 SEQ ID on the whole in the blood sample to subject source have response as index that whether subject has been infected by human enterovirus is determined in the case where, can be infected with 94% or more sensitivity and 7% false positive rate diagnosis human enterovirus below.
Description
Technical field
The invention mainly relates to detection kits.Specifically, the present invention relates to can be used for detecting human enterovirus infection
Kit.
Background technique
Enterovirus infection is the general name of the disease as caused by human enterovirus (EV), is clinically mainly shown as brothers mouthful
Disease, hand-foot-and-mouth disease eruption and prevalence mostly in infant, part EV infection infant show as herpangina, and patient with severe symptoms occurs
Viral encephalitis, viral cerebrospinal meningitis, pulmonary edema, empsyxis etc..Cause the cause of disease of hand-foot-and-mouth disease, most commonly Ke's Sa
Odd virus A 16 and enterovirns type 71；The large percentage of severe infection, case fatality rate occur for the disease as caused by enterovirus infection
Also higher, severe cases case fatality rate is up to 10%-25%, therefore etiological diagnosis is significant.
The clinical diagnosis of enterovirus is mainly according to three aspects: first is that clinical manifestation, second is that serological evidence, third is that sick
Original learns evidence.Since enterovirus EV infection clinical manifestation is more, the Serologic detection and pathogeny detection of enterovirus are
Current most important foundation.EV infection generates immunoprotection, and IgM and IgG antibody can be detected after infection.
Summary of the invention
The purpose of the present invention is to provide the new kits and diagnostic method that can be used for diagnosing human enterovirus infection.
The present inventor is it has surprisingly been found that when with whether polypeptide is on the whole to tested shown in NO:1~10 SEQ ID
The case where index whether IgM in the blood sample in person source has response to be infected by human enterovirus as judgement subject
Under, it can be with 94% or more sensitivity and 7% false positive rate diagnosis human enterovirus infection below.
Therefore, the present invention includes:
1. a kind of detection kit comprising one or more solid carriers, and be independently connected to the solid and carry
Polypeptide shown in NO:1~10 SEQ ID on body.
2. according to detection kit described in item 1, wherein polypeptide shown in NO:1~10 the SEQ ID independently connects
It is connected on same solid carrier.
3. according to detection kit described in item 1, wherein the solid carrier is SJ modified silica-gel.
4. being used to diagnose human enterovirus infection according to detection kit described in item 1.
5. it is a kind of diagnosis subject whether be people's enterovirus infection method, this method comprises:
Use polypeptide pair shown in the detection of detection kit described in any one of item 1~4 NO:1~10 the SEQ ID
Whether the IgM in the blood sample in subject source has response；Wherein,
IgM of the polypeptide shown in NO:1~10 the SEQ ID on the whole in the blood sample to subject source has
When response, determining should subject is a human enterovirus infection；Otherwise, it is determined that the subject is inhuman enterovirus infection.
6. according to method described in item 5, wherein the blood sample is whole blood, blood plasma or serum.
7. according to method described in item 5, wherein the subject is the subject with hand-foot-and-mouth disease clinical symptoms.
The specific embodiment of invention
Polypeptide
Polypeptide sequence shown in SEQ ID NO:1 is YHSSVYSLPPDPDHFDGYKQ；
Polypeptide sequence shown in SEQ ID NO:2 is HPYVLDAGIPISQLTVCPHQ；
Polypeptide sequence shown in SEQ ID NO:3 is ISDLLASVDSEEVRQYCRDQ；
Polypeptide sequence shown in SEQ ID NO:4 is GHFTMLGVRDRLAVLPRHSQ；
Polypeptide sequence shown in SEQ ID NO:5 is AREKVEFLNNLKQLPLLENQ；
Polypeptide sequence shown in SEQ ID NO:6 is GKGELCAVFRADPGRNGPWQ；
Polypeptide sequence shown in SEQ ID NO:7 is EDTHPPYKQTQPGADGFELQ；
Polypeptide sequence shown in SEQ ID NO:8 is SLDFALSLLRRNIRQVQTDQ；
Polypeptide sequence shown in SEQ ID NO:9 is NKEPAVLHSRDPRLEVDFEQ；
Polypeptide sequence shown in SEQ ID NO:10 is GHFTMLGVRDPLAVLPRHSQ.
The polypeptide can be suitable for being obtained using the known methods such as (1) chemical synthesis process or (2) enzyme reaction synthetic method
, wherein chemical synthesis is more easy.In chemical synthesis polypeptide of the invention, by using peptide synthesizer synthesis or
The semi-synthetic polypeptide carries out.As chemical synthesis process, can enumerate such as peptide solid-phase synthesis.The peptide synthesized in this way
It can be purified using conventional means such as ion-exchange chromatography, reverse phase high performance liquid chromatography, affinity chromatography etc..Such peptide
Solid phase synthesis process with and subsequent peptide purification be all well-known in the art.
In addition, such as International Publication pamphlet can be used in the case where producing polypeptide of the invention by enzyme reaction
Method described in No. WO2004/011653.I.e., it is possible to produce in this way: by the amino acid of a side or the carboxyl terminal quilt of dipeptides
Amino acid or dipeptides obtained from esterification or amidation, amino acid (such as the carboxy protective for being in amino acid free state
Amino acid) it is reacted in the presence of peptide synthetase, the dipeptides or tripeptides of generation.It as peptide synthetase, can enumerate: tool
There are the culture of the microorganism for the ability for generating peptide, the thallus of the microbial cells or the microorganism that are separated by the culture
Manage object or the microbe-derived peptide synthetase.
The chemical synthesis of especially polypeptide has been realized in commercialization, can by commission profession Peptide systhesis company come
Synthesize the polypeptide.
Kit
The present invention provides a kind of detection kit (kit of the invention) comprising one or more solid carriers, with
And it is independently connected to polypeptide shown in NO:1~10 SEQ ID on the solid carrier.
Preferably, the present invention also provides a kind of detection devices and the detection device in preparing kit of the invention
Purposes, the detection device includes one or more solid carriers, and the SEQ being independently connected on the solid carrier
Polypeptide shown in NO:1~10 ID.
It is surprising that kit of the invention can be with 94% or more sensitivity and 7% false positive rate below
Diagnose human enterovirus infection.In this specification, sensitivity refers to: in the positive sample with the confirmation of " goldstandard " method, by it
His method is measured as the ratio of positive sample.False positive rate refers to: in the negative sample with the confirmation of " goldstandard " method, by other
Method is measured as the ratio of positive sample." goldstandard " method that the art detects human enterovirus infection is detection of nucleic acids
Method, i.e. PCR method.In this specification, " diagnosis ", which can be, is made a definite diagnosis, and is also possible to make a definite diagnosis and providing reference information.
The infection of the so-called enterovirus infection of this specification, including but not limited to enterovirns type 71.
In the present invention, solid carrier is not particularly limited, as long as (e.g. may be used as solid or insoluble material
Material separated from reaction mixture by filtering, precipitating, Magnetic Isolation etc.) carrier.
The material for constituting solid carrier includes but is not limited to: silica gel (dimethyl silicone polymer, PDMS), cellulose, Teflon
It is grandTM, NC Nitroncellulose, agarose, glucan, chitosan, polystyrene, polyacrylamide, polyester, polycarbonate, polyamide,
Polypropylene, nylon, polyvinylidene fluoride, latex, silica, glass, glass fibre, gold, platinum, silver, copper, iron, stainless steel, iron
Oxysome, silicon wafer, polyethylene, polyethyleneimine, polylactic acid, resin, polysaccharide, albumen (albumin etc.), carbon or their group
Close etc..
The shape of solid carrier includes but not be limited to: pearl, magnetic bead, film, microcapillary, filter membrane, plate, micro plate, carbon
Nanotube, sensor chip etc..Just as known in the art, it can be set on the flat solid carrier such as film or plate
Set pit, groove, filter membrane bottom etc..
Magnetic bead can have about 25nm~about 1mm range sphere diameter.In a preferred embodiment, magnetic bead has about
The diameter of the μ m of 50nm~about 10.The size of magnetic bead can be selected according to specific purposes.It is contour by Sepharose
Pearl made of crosslinked spherical agarose has the diameter of about 24 μm~about 165 μ ms.Preferably, high crosslinked spherical agarose
Pearl has the diameter of about 24 μm~about 44 μ ms.The size of high crosslinked spherical sepharose 4B can according to specific purposes come into
Row selection.
The example of solid carrier with hydrophobic surface include can from Polysciences, Warrington, PA or
The polystyrene latex beads such as the product of Spherotech, Liberville, IL purchase.
Silica (SiO2)-processing or silica (SiO2) base solid carrier example include can be from
The extraordinary magnetic silica pearl etc. of Polysciences, Warrington, PA purchase, can be used for capturing nucleic acid (such as
DNA).It can be from Dynal Biotech M-280 bought etc. alternatively, can also use.
Magnetic bead with hydrophilic surface can be used for capturing the bacterial cell, nucleic acid and other ingredients of proliferation period.As
The example of the magnetic bead can enumerate Polysciences, the pearl (title: Biomag (registration of Warrington, PA sale
Trade mark) carboxyl) or Bangs Laboratory, Inc., Fishers, IN entitled MC02N/2928 pearl.Alternatively,
The M-270 etc. of Dynal Biotech sale can be used.
In a preferred embodiment, the solid carrier is SJ modified silica-gel, is that Yvonne poly- biomaterial in Suzhou has
A kind of microarray solid support material (the iPDMS film, referring to Chinese patent of silicone rubber material of limit company exploitation
CN101265329A).This material be based on the common PDMS of biological study, be added wherein specific initiator at
Divide and (make the material that can realize surface-functionalized modification by surface initiated polymerization (SIP)), using polyethylene glycol methyl
What acrylate (poly (oligo (ethylene glycol) methacrylate), pOEGMA) surface modification obtained.SJ changes
Property silica gel have outstanding anti-protein non-specific adsorption (Nonspecific protein adsorption, NPA) ability,
Non-specific protein absorption control in complicated protein immunization being detected is to close to " absolute 0 " is horizontal (to be near or below
The detectable limit of instrument), the trouble that can not only exempt closing and be cleaned multiple times can also be by using stronger amplification of signal
Means improve the sensitivity of protein microarray.And the essence of its silicon rubber impart the stronger mechanical performance of the material and
Good operability.SJ modified silica-gel is successfully applied to the more of 11 tumor markers compositions by the poly- company of Suzhou Yvonne
Index joint inspection microarray ELISA kit realizes high-throughput and highly 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, can pass through control
The modification reaction time adjusts its surface topography in a certain range.
The connection of polypeptide and solid carrier can be using well known to a person skilled in the art the connections of polypeptide and solid carrier
Method carries out.For example, 1- ethyl -3- (3- can be passed through for the connection of protein/polypeptide and modified silica-gel surface
Dimethyl aminopropyl)-carbodiimides [1-ethyl-3- (3-dimethyl ami-nopropyl) carbodiimide, EDC]
Reaction with n-hydroxysuccinimide (N-hydroxysuccinimide, NHS) will be on the macromolecular chain of modified silica-gel surface
Carboxyl (- COOH) group is changed to activated group, the activated group can be reacted with the amino (- NH2) of institute's band on protein/polypeptide to
It realizes and protein/polypeptide is fixed on solid carrier surface.
The concentration of polypeptide in the sampling liquid that uses when point sample is not particularly limited, those skilled in the art can be according to normal
Rule selection, preferably 1 μ of μ g~1000 g/mL, the more preferable 10 μ μ of g~500 g/mL.In addition, dividing on a solid carrier for polypeptide
The density of cloth is not particularly limited, and those skilled in the art can be according to conventional selection, and preferably 1~100 point/10mm2, more preferably
5~50 points/10mm2。
In the present specification, " independent " refers to: the polypeptide forms a point on the solid carrier, does not wrap in the point
It preferably only include the polypeptide containing other polypeptides on the influential amount of detection.
Solid carrier in kit of the invention can be one, be also possible to multiple, for example, more than two but excellent
It is selected as 1.
The kit can also include:
1. the serum dilution or serum dilution component solution that prepare: serum dilution, such as there is Beijing to match biology of speeding
The Sample dilution (product number 070021-S2) of Science and Technology Ltd., Zhengzhou Bo Weijia Biotechnology Co., Ltd sample-adding
Change colour Sample dilution (product number bwj010103) etc..The serum dilution is used to dilute serum, the serum of kit detection
Dilute suitable multiple, such as 2~200 times, preferably 10~100 times.
The kit can also include:
2. washing lotion is concentrated: after solid carrier surface is incubated for serum and ELIAS secondary antibody, solid carrier table need to be washed with washing lotion
Face unbonded antibody and ELIAS secondary antibody.Concentration washing lotion is, for example, 1% polysorbas20 aqueous solution, when use need to dilute 2~40 times,
It is preferred that 5~20 times.
The kit can also include:
3. ELIAS secondary antibody solution: IgM and solid carrier (such as SJ modified silica-gel) in human enterovirus infected person anteserum
On polypeptide combine, ELIAS secondary antibody can be in conjunction with IgM, and the marker on ELIAS secondary antibody can be reacted with luminous substrate liquid, thus
Issue detectable light.ELIAS secondary antibody can be the goat-anti people IgM of such as horseradish peroxidase-labeled.To ELIAS secondary antibody in enzyme
Concentration in mark two corresponding anti-solution is not particularly limited, and can be such as 1ng~1000ng/mL.
The kit can also include:
4. luminous substrate liquid component solution: luminous substrate liquid can be anti-with the horseradish peroxidase that marks on ELIAS secondary antibody
It answers, so that reaction issues the detectable chemical light of instrument.Luminous substrate liquid is mixed by two kinds of solution, is A liquid-respectively
Hydrogenperoxide steam generator and the luminous ammonia solution of B liquid-.Luminol (luminol) is only crossed with oxidizer treatment and can just be shone.Usually
Use hydrogen peroxide and a kind of mixed aqueous solution of hydroxide bases as exciting agent.Under horseradish peroxidase enzyme catalytic, dioxygen
Water decomposition is oxygen and water:
2H2O2→O2+2H2O
A pairs of anion is generated when luminol is reacted with hydroxide, it can be by oxygen oxygen that hydrogen peroxide decomposites
Change, product is an organic peroxide.The peroxide is very unstable, decomposites nitrogen immediately, generates the 3- ammonia of excitation state
Base phthalic acid.During excitation state to ground state converts, the energy of release exists in the form of photon, and wavelength is located at the indigo plant of visible light
Light part.The example of luminescent solution component solution such as Thermo Seientific companyELISA Femto
Maximum Sensitivity Substrate, article No. 37074.
The kit can also include:
5. one or more reaction cavity.
Such as can be biological incubation reactor more than one or two, it can use according to demand for example two-sided
Biological incubation reactor, Chinese patent ZL 201120177686.3 or ZL201110142518.5；Or the biology of single side is incubated
Educate reactor ZL201220430886.x.
The kit can also include:
6. other detection molecules (such as polypeptide, protein, nucleic acid etc.) for being used to detect human enterovirus infection.
The kit can also include:
7. operation instructions.
The kit can also include:
8. the positive quality control point being connected on one or more of solid carriers or other independent solid carriers (can
Using e.g. people IgM as positive quality control point) and/or negative Quality Control point (such as phosphate buffer, abbreviation PB).
Preferably, it is with the tested of hand-foot-and-mouth disease clinical symptoms that kit of the invention, which can be adapted for the subject,
Person.
Diagnostic method
In another aspect, the present invention provide it is a kind of diagnosis subject whether be people's enterovirus infection method (this
The diagnostic method of invention), this method comprises:
Polypeptide shown in detection kit detection NO:1~10 the SEQ ID using aforementioned present invention carrys out subject
Whether the IgM in the blood sample in source has response；Wherein,
IgM of the polypeptide shown in NO:1~10 the SEQ ID on the whole in the blood sample to subject source has
When response (criterion 1), determining should subject is a human enterovirus infection；Otherwise, it is determined that the subject is non-human enterovirus sense
Dye.
In the present specification, " there is response on the whole " to refer to: the letter of 10 polypeptides shown in NO:1~10 the SEQ ID
Make an uproar than the sum of (SNR) be greater than 5 (preferably greater than 6, more preferably greater than 7, more preferably greater than 8, more preferably greater than 9, more preferably greater than
10,15 are more preferably greater than, is more preferably greater than 20), wherein signal-to-noise ratio=(polypeptide point signal value-negative control point signal value)/yin
Property control point signal value.
In this specification, the positive reaction of kit of the invention is referred to: on the blood sample in subject source meets
State criterion 1；It otherwise is negative reaction.
The blood sample can be whole blood, blood plasma or serum.
In one preferred embodiment, the subject can be the subject with hand-foot-and-mouth disease clinical symptoms.
In the present invention, hand-foot-and-mouth disease clinical symptoms, which are embodied in the positions such as fever and hand, foot, oral cavity, fash or bleb.
Embodiment
1. the preparation and confirmation of polypeptide
Polypeptide used in embodiment is synthesized by gill biochemistry (Shanghai) Co., Ltd., and has been carried out to it really by mass spectrum
Recognize.Wherein,
Polypeptide shown in SEQ ID NO:1: YHSSVYSLPPDPDHFDGYKQ；
Polypeptide shown in SEQ ID NO:2: HPYVLDAGIPISQLTVCPHQ；
Polypeptide shown in SEQ ID NO:3: ISDLLASVDSEEVRQYCRDQ；
Polypeptide shown in SEQ ID NO:4: GHFTMLGVRDRLAVLPRHSQ；
Polypeptide shown in SEQ ID NO:5: AREKVEFLNNLKQLPLLENQ；
Polypeptide shown in SEQ ID NO:6: GKGELCAVFRADPGRNGPWQ；
Polypeptide shown in SEQ ID NO:7: EDTHPPYKQTQPGADGFELQ；
Polypeptide shown in SEQ ID NO:8: SLDFALSLLRRNIRQVQTDQ；
Polypeptide shown in SEQ ID NO:9: NKEPAVLHSRDPRLEVDFEQ；
Polypeptide shown in SEQ ID NO:10: GHFTMLGVRDPLAVLPRHSQ.
2. the preparation of kit
Detection chip is with SJ modified silica-gel (iPDMS film) for solid support material, more by point sample fixation on it
Peptide solution is prepared.SJ modified silica-gel is that with olefin-terminal, surface is added in traditional polydimethyl siloxane material
The initiator of initiated polymerization, and the three-dimensional structure by heat cross-linking (si-h bond bonding) fixed to dimethyl silicone polymer
In, obtain a kind of new material i.e. SJ modified silica-gel.Its manufacturing process is referring to International Patent Publication WO2014/044184.
The SJ modified silica-gel film made can be reserved in 4 DEG C of refrigerators.
Using16 people's point sample instruments of PersonalArrayerTM prepare polypeptide microarrays (i.e. on modified silica-gel
Detection chip), process are as follows:
1) it pre-processes
By SJ modified silica-gel thin slice (15 × 15mm2) be immersed in activating solution, taking-up elutes 3 with deionized water after 30min
It is secondary, with being dried with nitrogen, it is immediately used to point sample.
2) point sample
Sampling liquid is diluted good and is transferred in the corresponding micropore of 384 orifice plates, 384 orifice plates with sample are placed in point sample instrument
On base station, while pretreated modified silica-gel thin slice being placed on the base station of point sample instrument, carries out point sample at once.Point sample environmental condition
For room temperature (25 DEG C), humidity set 50%.By polypeptide shown in NO:1~10 SEQ ID on SJ modified silica-gel thin slice point at
Microarray, one point of each polypeptide point sample, in addition one people IgM point of point sample is as positive quality control point and a PB point as yin
Property control point.The point sample amount of each point is about 0.6nL on manufactured polypeptide microarrays, and sampling point radius is 200 μm.
3) chemistry is fixed
The polypeptide microarrays just made will be placed in climatic chamber (26 DEG C, 60% humidity) fixed at least 6h.Chemistry is solid
Process is determined referring to International Patent Publication WO2014/044184.
It will include first to capture the buffer point of peptide molecule on modified silica-gel film by point sample instrument, then buffer
Liquid starts to evaporate, and capture peptide molecule and the surface intimate contact of SJ modified silica-gel and interacts, and passes through and is chemically combined, modified silicon
High molecular end-the COOH of ploy (OEGMA) on glue surface and peptide molecule-- NH2It is formed and stablizes covalent bond, and then will had
Chemically active peptide molecule is fixed on SJ modified silica-gel surface.
4) it assembles
The polypeptide microarrays of fixed 6h must assemble in two days.SJ modified silica-gel thin slice is attached to by gum first
On special reaction column, reaction cavity is covered.Biological incubation reactor can use two-sided biological incubation reactor according to demand,
That is, a reactor is made of two reaction columns and a reaction cavity or single side biological incubation reactor, that is, one anti-
Device is answered to be made of a reaction column and a reaction cavity.
5) it saves
The polypeptide microarrays assembled need to vacuumize sealing, are stored in 4 DEG C of refrigerator, spare.
3. being detected with kit
Checking procedure:
1) it balances temperature of reagent: all reagents is balanced to room temperature.
2) preparation of washing lotion: concentration washing lotion purified water or distilled water are diluted by 1:9, such as: 450mL purified water or steaming
50mL is added in distilled water, washing lotion is concentrated, mixes well.The washing lotion being not used is placed on 2~8 DEG C of preservations, can be reserved for 3 months.
3) dilution of sample: by sample to be tested, with serum dilution, by 1:100 dilution, (such as: 2 μ L serum are added to 198 μ L
Serum dilution mixes well).Sample detection needs the 200 μ L of sample after dilution.
4) wetted chip: biochip incubation reaction device needed for taking out detection.1mL washing lotion is added, infiltrates chip surface 3
Minute.Discard the washing lotion for impregnating chip.
5) it is loaded: in the present embodiment, to be measured by what is diluted using liquid-transfering gun with single side biological incubation reactor sample-adding
Sample is added in single side biological incubation reactor by liquid feeding venthole (any one in two), and each single side biological incubation is anti-
It answers device to add 200 μ L, closes liquid feeding venthole (two) with sealing paste.
6) sample incubation: being placed in 150rpm on horizontal shaker for the single side biological incubation reactor for adding sample, incubation at room temperature
30 minutes.
7) it cleans: reaction column being taken out from the reaction cavity of single side biological incubation reactor, discards liquid in reactor,
3 times inside repeated flushing reaction column top chip surface and reaction cavity (12mL washing lotion is about used every time, rinse 1-2 minutes).
8) enzyme labelled antibody is incubated for: reaction column and reaction cavity being reconfigured, clamping.Sealing paste is thrown off, each single side is raw
The 200 μ L of goat-anti people IgM solution of object incubation reaction device addition horseradish peroxidase-labeled.Again logical with sealing paste closing liquid feeding
Stomata.It is placed in 150rpm on shaking table, is incubated at room temperature 30 minutes.
9) it cleans again: discarding reaction cavity, carefully cleaned with washing lotion chip surface 3 times.
10) luminous substrate liquid is added: each chip surface is separately added into 15 μ L luminous substrate liquid, keeps luminescent solution uniform
It is laid on chip surface
11) chip is imaged: the chip that joined luminous substrate liquid carries out chip with chemiluminescence image analysis system
It exposes 1 minute and is imaged.When as chemiluminescence image analysis system, it is not particularly limited, it can the micro- battle array of 5500 types using such as day
Column imager, Yvonne victory board TN5500 type Microarray image instrument or Yvonne victory board CLEAR4000 type Microarray image instrument etc..
Result judgement: for every a serum, the signal-to-noise ratio of each polypeptide in kit is counted, and according to above-mentioned
Criterion 1 described in " diagnostic method " part is determined.
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 for the polypeptide point that imager software kit is read, and negative control point signal value refers to imaging
The chemiluminescence intensity value for the negative control point that instrument software kit is read.
To 397 parts of the clinical serum being collected into, using the testing result of mentioned reagent box and using traditional goldstandard method-
The testing result of nucleic acid detection method (using the fluorescent quantificationally PCR detecting kit of Takara) is compared, as a result such as 1 institute of table
Show.
The supplier of 397 parts of above-mentioned clinical serum all has the clinical symptoms of hand-foot-and-mouth disease.
The kit of 1 embodiment of table is compared with the fluorescent quantificationally PCR detecting kit of Takara
Accuracy rate=(249+123)/397=93.7%
Sensitivity=249/265=94.0%
Specificity=123/132=93.2%
False positive rate=9/132=6.8%
The above result shows that the present invention has good accuracy rate for detecting enterovirus antibodies in human serum (IgM),
Sensitivity and specificity, suitable for the aetology laboratory auxiliary diagnosis of enterovirus, and easy to operate, be suitble to medical treatment at different levels and
Disease control department uses.
More than, more specific description is carried out to the present invention by embodiment, but be not the limit to the technology of the present invention range
It is fixed.By the record of this specification, those skilled in the art readily can modify/change to the present invention, these include
Within the technical scope of the present invention.
Sequence table
<110>Shenzhen Children's Hospital
<120>it can be used for diagnosing the kit of human enterovirus infection
<160> 10
<170> PatentIn version 3.2
<210> 1
<211> 20
<212>amino acid
<213>artificial sequence
<400> 1
YHSSVYSLPPDPDHFDGYKQ 20
<210> 2
<211> 20
<212>amino acid
<213>artificial sequence
<400> 2
HPYVLDAGIPISQLTVCPHQ 20
<210> 3
<211> 20
<212>amino acid
<213>artificial sequence
<400> 3
ISDLLASVDSEEVRQYCRDQ 20
<210> 4
<211> 20
<212>amino acid
<213>artificial sequence
<400> 4
GHFTMLGVRDRLAVLPRHSQ 20
<210> 5
<211> 20
<212>amino acid
<213>artificial sequence
<400> 5
AREKVEFLNNLKQLPLLENQ 20
<210> 6
<211> 20
<212>amino acid
<213>artificial sequence
<400> 6
GKGELCAVFRADPGRNGPWQ 20
<210> 7
<211> 20
<212>amino acid
<213>artificial sequence
<400> 7
EDTHPPYKQTQPGADGFELQ 20
<210> 8
<211> 20
<212>amino acid
<213>artificial sequence
<400> 8
SLDFALSLLRRNIRQVQTDQ 20
<210> 9
<211> 20
<212>amino acid
<213>artificial sequence
<400> 9
NKEPAVLHSRDPRLEVDFEQ 20
<210> 10
<211> 20
<212>amino acid
<213>artificial sequence
<400> 10
GHFTMLGVRDPLAVLPRHSQ 20
Claims (4)
1. a kind of detection kit comprising one or more solid carriers, and be independently connected on the solid carrier
NO:1~10 SEQ ID shown in polypeptide.
2. detection kit according to claim 1, wherein polypeptide shown in NO:1~10 the SEQ ID is independently
It is connected on same solid carrier.
3. detection kit according to claim 1, wherein the solid carrier is SJ modified silica-gel.
4. detection kit according to claim 1 is used to diagnose human enterovirus infection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710207593.2A CN107255713B (en) | 2017-03-31 | 2017-03-31 | It can be used for diagnosing the kit of human enterovirus infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710207593.2A CN107255713B (en) | 2017-03-31 | 2017-03-31 | It can be used for diagnosing the kit of human enterovirus infection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107255713A CN107255713A (en) | 2017-10-17 |
| CN107255713B true CN107255713B (en) | 2019-01-08 |
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| CN201710207593.2A Active CN107255713B (en) | 2017-03-31 | 2017-03-31 | It can be used for diagnosing the kit of human enterovirus infection |
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| CN (1) | CN107255713B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108663523A (en) * | 2017-03-31 | 2018-10-16 | 复旦大学 | It can be used for diagnosing the kit of human enterovirus 71 infection |
| 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 |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105675878B (en) * | 2014-12-08 | 2017-09-29 | 苏州偲聚生物材料有限公司 | Kit and diagnostic method available for diagnosis human enterovirus 71 infection |
| CN105732769A (en) * | 2014-12-10 | 2016-07-06 | 苏州偲聚生物材料有限公司 | Polypeptides, detection device comprising the polypeptides, and detection kit comprising the detection device |
| CN105732770A (en) * | 2014-12-10 | 2016-07-06 | 苏州偲聚生物材料有限公司 | Polypeptides, a detection device comprising the polypeptides, and a detection kit comprising the detection device |
| CN105732776A (en) * | 2014-12-10 | 2016-07-06 | 苏州艾比拓生物技术有限公司 | Polypeptide, detection device containing polypeptide and detection kit containing device |
| CN105732772A (en) * | 2014-12-10 | 2016-07-06 | 苏州偲聚生物材料有限公司 | Polypeptide, detection device containing polypeptide and detection kit containing device |
| CN105732771A (en) * | 2014-12-10 | 2016-07-06 | 苏州艾比拓生物技术有限公司 | Polypeptide, detection device containing polypeptide and detection kit containing device |
| CN105732773A (en) * | 2014-12-10 | 2016-07-06 | 苏州偲聚生物材料有限公司 | Polypeptide, detection device containing polypeptide and detection kit containing device |
| CN105732775A (en) * | 2014-12-10 | 2016-07-06 | 苏州艾比拓生物技术有限公司 | Polypeptide, detection device containing polypeptide and detection kit containing device |
| CN105785026B (en) * | 2014-12-24 | 2017-07-18 | 中国科学院苏州纳米技术与纳米仿生研究所 | Kit and detection method for detecting enterovirns type 71 IgM antibody |
| CN105784997B (en) * | 2014-12-24 | 2017-09-05 | 深圳国际旅行卫生保健中心 | Kit and detection method for detecting enterovirns type 71 IgM antibody |
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| CN107255713A (en) | 2017-10-17 |
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Inventor after: Liu Xiaorong Inventor after: Ma Dongli Inventor after: Zhong Shan Inventor before: Ma Dongli Inventor before: Zhong Shan |