CN110456041A - A kind of norovirus GI type GII type combined detection reagent and preparation method thereof - Google Patents
A kind of norovirus GI type GII type combined detection reagent and preparation method thereof Download PDFInfo
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- CN110456041A CN110456041A CN201910720354.6A CN201910720354A CN110456041A CN 110456041 A CN110456041 A CN 110456041A CN 201910720354 A CN201910720354 A CN 201910720354A CN 110456041 A CN110456041 A CN 110456041A
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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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- 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
- G01N33/531—Production of immunochemical test materials
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention discloses a kind of norovirus GI type GII type combined detection reagents and preparation method thereof, including PVC bottom plate, left side bonding at the top of the PVC bottom plate is connected with sample pad, left side at the top of the PVC bottom plate and right side bonding for being located at sample pad is connected with colloid gold label pad, the right side in left side and sample pad bottom at the top of the colloid gold label pad, which bonds, to be connected, and bonding is connected with NC film at the center at the top of the PVC bottom plate.Present invention simultaneously provides a kind of simple process, reproducible, finished product structures to stablize, the good norovirus GI type/GII type combined detection reagent preparation method of service performance, due to that can check the norovirus of two kinds of genotype (GI type/GII type) simultaneously, recall rate can be greatly improved, and parting can be carried out to it, it is very suitable to the requirement of hospital and prevention and control unit, to solve rapid screening and the diagnosis of norovirus infected patient when clinical Sporadic cases or Epidemic outbreak of disease.
Description
Technical field
The present invention relates to norovirus detection technique field, specially a kind of norovirus GI type GII type joint-detection examination
Agent and preparation method thereof.
Background technique
Norovirus, also known as norwalk virus are a kind of virus that norovirus belongs in mankind's Caliciviridae, are one group
Form is similar, the antigenic virion being slightly different, and norovirus infectious diarrhea has prevalence in worldwide, entirely
It can infect every year, infection object is mainly adult and school-ager, and cold season presentation is high-incidence, and developed country is every year in institute
During some Non-bacterial diarrheas are broken out, 60-90% is caused by norovirus, and also there are similar results, In in many developed countries
In China five years old or less diarrhea children, norovirus recall rate is 15% or so, and antibody level of serum investigation shows population of China
The infection of middle norovirus is also very universal, and norovirus infectious diarrhea belongs to self-limited disease, without vaccine and specific drug
Object, the public pays attention to personal hygiene, food hygiene and drinking water hygiene, to avoid cross contamination be the key that prevent this disease, and scientist exists
A kind of virus causing disease is isolated in the patient's excrement for the acute diarrhea that developed country breaks out, hereafter, all over the world successively certainly
The virus-like particle that variform is similar but antigenicity is slightly different is isolated in gastroenteritis patient's excrement, first referred to as roundlet knot
Structure virus, referred to as norwalk group viruses, China report the first norovirus infection afterwards, and all parts of the country successively occur a lot of later
Norovirus infectious diarrhea epidemic outbreaks, it is norovirus that the 8th International Virus Nomenclature Committee, which ratifies the Virus Name,
Six genomes (I-GVI of G) of norovirus point, wherein only G I, G II and G IV can infect people, IV .1 of the G infection in G IV
People, IV .2 of G infects cat and dog, and G III, G V, GVI distinguish infected cattle, mouse and dog, and the most common norovirus is G at present in China
II, I type of G, norovirus route of transmission include human-to-human transmission, through food and water-borne transmission, human-to-human transmission can by fecal-oral route (including
Intake excrement or vomitus generate aerosol) or mediate contact be drained object pollution environment and propagate, food source, which spreads through sex intercourse, is
It is propagated by edible by the food that norovirus pollutes, pollution section may alternatively appear in the food and drink working people of infection norovirus
Member's contaminated food in preparing for a meal and serving the meals, also may occur in which food in production, transport and distribution procedure by containing norovirus
Human excrement or other materials (such as water etc.) pollute.
The technology that predominantly detects of norovirus has three classes at present: electron microscopy, and detection of nucleic acids (PCR) and antigen detect three classes side
Method is wherein fast and convenient direct the advantages of electron microscopy, but the disadvantage is that the typical calicivirus feature of norovirus shortage, is not easy
Observation, and need virus concentration reache a certain level just it is observed that, and Electronic Speculum is expensive, and technical requirements are high, is not suitable for
Extensive epidemiological survey, detection of nucleic acids program is cumbersome, and Pretreated process is more, by design of primers, nucleic acid purification, reaction
The limitation such as condition, is only limitted to laboratory operation, occurs false sun vulnerable to laboratory pollution, it is necessary to carry out sample in independent space
Product processing and inspection, antigen detection method, atopic is strong, fast and easy, and not yet discovery lid antiviral antibody is sick with other at present
There are cross reactions between poison, but since norovirus variability is strong, and a kind of heredity group type antibody cannot identify other hereditary groups
Type antigen.
Summary of the invention
(1) the technical issues of solving
In view of the deficiencies of the prior art, the present invention provides a kind of norovirus GI type GII type combined detection reagent and its
Preparation method, has that detection speed is fast, and sample process process is simple, the detection examination of easy to operate and high sensitivity norovirus
Agent item, the advantages of norovirus for two oligogene groups for causing acute human enterogastritis can be detected simultaneously, to solve background
The problem of proposed in technology.
(2) technical solution
To achieve the above object, the invention provides the following technical scheme: a kind of norovirus GI type GII type joint-detection fills
It sets, including PVC bottom plate, the left side bonding at the top of the PVC bottom plate is connected with sample pad, and the left side at the top of the PVC bottom plate is simultaneously
Positioned at the right side of sample pad, bonding is connected with colloid gold label pad, the left side at the top of the colloid gold label pad and sample pad bottom
Right side bond connection, bonding is connected with NC film, left side and colloid at the top of the NC film at the center at the top of the PVC bottom plate
The right side of golden label pad bottom bonds connection, and the top of the NC film is disposed with GI detection line, GII detection line from left to right
And nature controlling line, the left side of the GII detection line are located at the right side of GI detection line, the left side of the nature controlling line is located at GII detection line
Right side, the right side bonding at the top of the PVC bottom plate is connected with water absorption pad, at the top of the left side of the water absorption pad bottom and NC film
Right side bonding connection.
Preferably, label has anti-norovirus GI type monoclonal antibody 1 and the anti-promise of mouse such as on the colloid gold label pad
Viral GII type monoclonal antibody 2.
Preferably, the right side of colloid gold label pad, the Quality Control are close on the left of the GI detection line and GII detection line
The right side of line is close to the left side of water absorption pad.
Preferably, the material of the NC film is nitrocellulose filter, and the material of the colloid gold label pad is polyester film, institute
The material for stating sample pad is polyester film or glass fibre element film.
Preferably, a kind of norovirus GI type GII type combined detection reagent preparation method, preparation method includes following step
It is rapid:
A, it prepares sample pad: the sample pad being placed in sample pad treatment fluid and is impregnated, then will be handled containing sample pad
The sample pad of liquid is placed in temperature and is 18-24 DEG C, dries 16-for 24 hours in environment of the relative humidity less than 40%, spare；Its
In, sample pad treatment fluid is the 1mol/L phosphate buffer or Tris-that the pH containing 0.5% Tween-20,1%BSA is 8.0
HCl buffer；
B, it prepares colloid gold label pad: a, colloidal gold solution preparation: measuring 980mL process water into flask with graduated cylinder,
1% chlorogold solution of preparation amount is added, side stirring while being heated to boiling, stirs on one side, is rapidly added 1% citric acid of preparation amount
Three sodium solutions after color becomes orange red, continue to boil 15 minutes, close blender, allow colloidal gold solution natural cooling, recruitment
Colloidal gold solution is settled to 1000mL with water by skill, b, antibody label and the preparation of colloid gold label pad: the determination of optimal pH: is taken
The above-mentioned colloidal gold solution of 320ml takes the pH value of solution of potassium carbonate adjustment colloidal gold solution of 0.1M to suitable ph, by solution
It is put on magnetic stirring apparatus, adjustment revolving speed is 250rpm, and under stirring, the anti-norovirus GI type monoclonal of aequum mouse is added
Antibody 1 and the anti-norovirus GII type monoclonal antibody 2 of mouse, the concentration in solution is controlled 8-both antibody at this moment
10%BSA is added after static 15 minutes in 10ug/ml, makes the 1%GEG20000 of BSA final concentration of 0.1% and 0.32ml, continues
Stirring 10 minutes, centrifugation: the solution marked is transferred in centrifuge tube, balance up-regulation balance, and 2-8 DEG C, 10000rpm,
Supernatant is sucked out in centrifugation 30 minutes, the heart, collects precipitating, with gold mark conjugate dilution constant volume to former colloidal gold solution volume
10%, coating: taking polyester non-woven fabric, sets coating weight as 6-8ul/cm, 37 DEG C of drying temperature, relative humidity≤40% rotates
1.5 ms/min of speed, coated colloidal gold is transferred to drying room, drying temperature is 37 DEG C, and dry humidity is≤40%, when
Between 16-24 hours, after colloidal gold pad is dry, aluminium foil bag is sealed up for safekeeping spare；
C, it prepares detecting pad: having configured nature controlling line C and GI detection line according to recipe requirements and the antibody of GII detection line is molten
Liquid takes NC film (specification width 25mm), and the norovirus GI type GI detection line, the promise are disposably marked according to distance interval
Such as viral GII type GII detection line and the nature controlling line C line, the norovirus GI type GI detection line is coated with the anti-promise of mouse such as disease
Malicious GI type monoclonal antibody 3, the norovirus GII type GII detection line are coated with the anti-norovirus GII type monoclonal antibody of mouse
4, sheep anti-mouse igg polyclonal antibody is coated at nature controlling line (C line), distance interval setting is as follows: bottom of the GI detection line from NC film
End distance is 7mm, is 12mm with a distance from bottom end of the GII detection line from NC film, is 17mm, setting with a distance from bottom end of the nature controlling line from NC film
Drying temperature is 37 DEG C, and dry in the environment of relative humidity≤40%, the time 16-24 hours, the NC film being coated with was through drying tower
After drying, spool spare；
D, pasting board and cutting: by the sample pad, the label pad, the detecting pad, water absorption pad sequence overlap joint institute
It states on PVC bottom plate, and is cut into width 3.3mm (stripe shape) and 4.0mm (card-type) according to specification requirement, check that interior parlor is warm and humid
Degree, it is ensured that temperature is 18-24 DEG C, humidity :≤40% could start to work, and PVC board is unfolded on table top, and both ends are sticked to operation
On platform, the absciss layer paper that middle section width is 25mm is torn, NC film alignment straight line of crossing is attached to the place for tearing absciss layer paper, then
Tear the following protection sheet of PVC board, colloidal gold pasted in PVC board, allow the top edge of colloidal gold and cross NC film it is following
Edge be overlapped 1.0-2.0 millimeters, sample pad is pasted onto the lower section of colloidal gold, allow sample pad top edge and colloidal gold it is following
Edge be overlapped 1.0-2.0 millimeters, tear the absciss layer paper of PVC board top, blotting paper straight line pasted in PVC board, allow blotting paper with
The top edge of scribing line NC film is overlapped 2 millimeters or so, pastes adhesive tape, allows adhesive tape that colloidal gold pad is completely covered, and operation is automatic
Cutting machine, width cutting as required.
(3) beneficial effect
Compared with prior art, the present invention provides a kind of norovirus GI type GII type combined detection reagent and its preparations
Method, have it is following the utility model has the advantages that
1, present invention simultaneously provides a kind of simple process, reproducible, finished product structures to stablize, the good promise of service performance such as disease
The preparation method of malicious GI type/GII type combined detection reagent, due to that can check the promise of two kinds of genotype (GI type/GII type) simultaneously
Such as virus, recall rate can be greatly improved, and parting can be carried out to it, is very suitable to the requirement of hospital and prevention and control unit, is come
The rapid screening of norovirus infected patient when the clinical Sporadic cases of solution or Epidemic outbreak of disease and diagnosis.
2, pass through epidemiological survey: there are many reason of leading to diarrhea, in epidemiological survey, it usually needs first really
It surely is that bacterium or virus infection cause, test strips of the invention can be sub- with the norovirus GI and GII in joint-detection excrement
Type prevents classification error, provides diagnosis basis to treatment and medication, while norovirus infection is in distribute and break out two kinds of shapes
Formula, this test strips is on the basis of can quickly be detected and analyzed sample, comprehensive analysis, and differentiation is distributed and broken out.
3, pass through epidemic monitoring: discovery epidemic situation is to take the basis for the measure of effectively preventing in time, and pathogeny detection can be epidemic disease
Feelings prevention and control provide important scientific basis, this kit can provide a kind of rapid detection method, for distributing or the sieve of Outbreak
It looks into.
4, primary to be loaded by norovirus Main Subtype GI and the GII joint inspection to human infection is caused, it can obtain simultaneously
Two are obtained as a result, recall rate can be improved in joint-detection.
5, the norovirus GI type/GII type joint-detection test paper of the invention, high sensitivity, high specificity, and speed
Spend fast (5-10 minutes), (Sample pretreatment process is simple) easy to operate, detection that is reproducible, and being prepared with this method
Test strips structure is stablized, and service performance is not necessarily to specialized facilities well, is suitable for a variety of occasions such as clinical detection or generaI investigation.
Detailed description of the invention
Fig. 1 is structure of the invention perspective view.
In figure: 1, PVC bottom plate；2, sample pad；3, colloid gold label pad；4, NC film；5, GI detection line；6, GII detection line；
7, nature controlling line；8, water absorption pad.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
All components of the invention are general standarized component or component as known to those skilled in the art, structure and
Principle is all that those skilled in the art can be learnt by technical manual or be known by routine experiment method.
Referring to Fig. 1, a kind of norovirus GI type GII type joint-detection device, including PVC bottom plate 1,1 top of PVC bottom plate
Left side bonding be connected with sample pad 2, the left side at 1 top of the PVC bottom plate and right side bonding for being located at sample pad 2 is connected with colloidal gold
Label pad 3, the left side at 3 top of colloid gold label pad and the right side of 2 bottom of sample pad bond and connect, the center at 1 top of PVC bottom plate
Place's bonding is connected with NC film 4, and the right side that the left side at 4 top of NC film and colloid gold label pad 3 bottoms, which bonds, to be connected, the top of NC film 4
Portion is disposed with GI detection line 5, GII detection line 6 and nature controlling line 7 from left to right, and the left side of GII detection line 6 is located at GI detection
The right side of line 5, the left side of nature controlling line 7 are located at the right side of GII detection line 6, and the right side bonding at 1 top of PVC bottom plate is connected with water suction
Pad 8, the right side at 4 top of left side and NC film of 8 bottom of water absorption pad, which bonds, to be connected, and label has anti-promise such as on colloid gold label pad 3
Viral GI type monoclonal antibody 1 and the anti-norovirus GII type monoclonal antibody 2 of mouse, the left side of GI detection line 5 and GII detection line 6
It is close to the right side of colloid gold label pad 3, close to the left side of water absorption pad 8, the material of NC film 4 is that nitric acid is fine on the right side of nature controlling line 7
Plain film is tieed up, the material of colloid gold label pad 3 is polyester film, and the material of sample pad 2 is polyester film or glass fibre element film, the present invention
A kind of simple process is provided simultaneously, reproducible, finished product structure is stablized, the good norovirus GI type of service performance/GII type joint
The preparation method of detection reagent can be mentioned significantly due to that can check two kinds of genotype GI type/GII type norovirus simultaneously
High detection rate, and can carry out parting to it, is very suitable to the requirement of hospital and prevention and control unit, come solve clinical Sporadic cases or
The rapid screening of norovirus infected patient when Epidemic outbreak of disease and diagnosis.
A kind of norovirus GI type GII type combined detection reagent preparation method, preparation method includes the following steps:
A, it prepares sample pad 2: sample pad 2 being placed in 2 treatment fluid of sample pad and is impregnated, then will be handled containing sample pad 2
The sample pad 2 of liquid is placed in temperature and is 18-24 DEG C, dries 16-for 24 hours in environment of the relative humidity less than 40%, spare；Wherein,
2 treatment fluid of sample pad is the 1mol/L phosphate buffer or Tris-HCl that the pH containing 0.5% Tween-20,1%BSA is 8.0
Buffer；
B, colloid gold label pad 3:a, colloidal gold solution preparation are prepared: measuring 980mL process water into flask with graduated cylinder,
1% chlorogold solution of preparation amount is added, side stirring while being heated to boiling, stirs on one side, is rapidly added 1% citric acid of preparation amount
Three sodium solutions after color becomes orange red, continue to boil 15 minutes, close blender, allow colloidal gold solution natural cooling, recruitment
Colloidal gold solution is settled to 1000mL with water by skill, prepared by b, antibody label and colloid gold label pad 3: the determination of optimal pH:
Take the above-mentioned colloidal gold solution of 320ml, take 0.1M solution of potassium carbonate adjustment colloidal gold solution pH value to suitable ph, will be molten
Liquid is put on magnetic stirring apparatus, and adjustment revolving speed is 250rpm, and under stirring, the anti-norovirus GI type Dan Ke of aequum mouse is added
Grand antibody 1 and the anti-norovirus GII type monoclonal antibody 2 of mouse, the concentration in solution is controlled 8-both antibody at this moment
10%BSA is added after static 15 minutes in 10ug/ml, makes the 1%GEG20000 of BSA final concentration of 0.1% and 0.32ml, continues
Stirring 10 minutes, centrifugation: the solution marked is transferred in centrifuge tube, balance up-regulation balance, and 2-8 DEG C, 10000rpm,
Supernatant is sucked out in centrifugation 30 minutes, the heart, collects precipitating, with gold mark conjugate dilution constant volume to former colloidal gold solution volume
10%, coating: taking polyester non-woven fabric, sets coating weight as 6-8ul/cm, 37 DEG C of drying temperature, relative humidity≤40% rotates
1.5 ms/min of speed, coated colloidal gold is transferred to drying room, drying temperature is 37 DEG C, and dry humidity is≤40%, when
Between 16-24 hours, after colloidal gold pad is dry, aluminium foil bag is sealed up for safekeeping spare；
C, it prepares detecting pad: having configured the antibody of nature controlling line 7C and GI detection line 5 Yu GII detection line 6 according to recipe requirements
Solution takes 4 specification width 25mm of NC film, and norovirus GI type GI detection line 5, norovirus are disposably marked according to distance interval
GII type GII detection line 6 and nature controlling line 7C line, norovirus GI type GI detection line 5 are coated with the anti-norovirus GI type monoclonal of mouse
Antibody 3, norovirus GII type GII detection line 6 are coated with the anti-norovirus GII type monoclonal antibody 4 of mouse, at nature controlling line 7C line
It is coated with sheep anti-mouse igg polyclonal antibody, distance interval setting is as follows: being 7mm with a distance from bottom end of the GI detection line 5 from NC film 4,
It is 12mm with a distance from bottom end of the GII detection line 6 from NC film 4, is 17mm with a distance from bottom end of the nature controlling line 7 from NC film 4, sets drying temperature
Be 37 DEG C, it is dry in the environment of relative humidity≤40%, the time 16-24 hours, the NC film 4 being coated with after drying tower is dried,
It spools spare；
D, pasting board and cutting: sample pad 2, label pad, detecting pad, water absorption pad 8 are sequentially overlapped on PVC bottom plate 1, and according to
Specification requirement is cut into width 3.3mm stripe shape and 4.0mm card-type, checks interior parlor temperature and humidity, it is ensured that temperature is 18-24 DEG C, wet
Degree :≤40% could start to work, and PVC board is unfolded on table top, and both ends are sticked on station, tear middle section width and be
The alignment straight line of NC film 4 of crossing is attached to the place for tearing absciss layer paper, then tears the following protection sheet of PVC board by the absciss layer paper of 25mm,
Colloidal gold is pasted in PVC board, makes the top edge of colloidal gold and the scribing line lower edge of NC film 4 1.0-2.0 millimeters Chong Die, it will
Sample pad 2 is pasted onto the lower section of colloidal gold, makes the top edge of sample pad 2 1.0-2.0 millimeters Chong Die with the lower edge of colloidal gold,
The absciss layer paper for tearing PVC board top, blotting paper straight line is pasted in PVC board, makes the top edge of blotting paper and NC film 4 of crossing heavy
It is 2 millimeters or so folded, adhesive tape is pasted, allows adhesive tape that colloidal gold pad is completely covered, operates automatic cutting machine, width as required
Cutting.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (5)
1. a kind of norovirus GI type GII type joint-detection device, including PVC bottom plate (1), it is characterised in that: the PVC bottom plate
(1) the left side bonding at the top of is connected with sample pad (2), the left side at the top of the PVC bottom plate (1) and the right side for being located at sample pad (2)
Side bonding is connected with colloid gold label pad (3), the right side in the left side at the top of the colloid gold label pad (3) and sample pad (2) bottom
Side bonding connects, and bonds and is connected with NC film (4) at the center at the top of the PVC bottom plate (1), the left side at the top of the NC film (4)
It bonds and connects with the right side of colloid gold label pad (3) bottom, the top of the NC film (4) is disposed with GI detection from left to right
The left side of line (5), GII detection line (6) and nature controlling line (7), the GII detection line (6) is located at the right side of GI detection line (5), institute
The left side for stating nature controlling line (7) is located at the right side of GII detection line (6), and the right side bonding at the top of the PVC bottom plate (1) is connected with suction
Water cushion (8), the left side of water absorption pad (8) bottom and the right side at the top of NC film (4) bond and connect.
2. a kind of norovirus GI type GII type joint-detection device according to claim 1, it is characterised in that: the glue
Label has anti-norovirus GI type monoclonal antibody 1 and the anti-norovirus GII type monoclonal antibody of mouse in body gold label pad (3)
2。
3. a kind of norovirus GI type GII type joint-detection device according to claim 1, it is characterised in that: the GI
The right side of colloid gold label pad (3), the right side of the nature controlling line (7) are close on the left of detection line (5) and GII detection line (6)
Close to the left side of water absorption pad (8).
4. a kind of norovirus GI type GII type joint-detection device according to claim 1, it is characterised in that: the NC
The material of film (4) is nitrocellulose filter, and the material of the colloid gold label pad (3) is polyester film, the material of the sample pad (2)
Matter is polyester film or glass fibre element film.
5. a kind of norovirus GI type GII type combined detection reagent preparation method, it is characterised in that: preparation method includes following
Step:
A, sample pad (2) are prepared: the sample pad (2) is placed in sample pad (2) treatment fluid and is impregnated, sample pad then will be contained
(2) sample pad (2) for the treatment of fluid is placed in temperature and is 18-24 DEG C, dries 16-in environment of the relative humidity less than 40%
For 24 hours, spare；Wherein, sample pad (2) treatment fluid is that the 1mol/L phosphate that the pH containing 0.5% Tween-20,1%BSA is 8.0 is slow
Fliud flushing or Tris-HCl buffer；
B, it prepares colloid gold label pad (3): a, colloidal gold solution preparation: measuring 980mL process water into flask with graduated cylinder, add
Enter 1% chlorogold solution of preparation amount, side stirring while being heated to boiling, stirs on one side, is rapidly added 1% citric acid three of preparation amount
Sodium solution after color becomes orange red, continues to boil 15 minutes, closes blender, allows colloidal gold solution natural cooling, uses technique
Colloidal gold solution is settled to 1000mL with water, b, antibody label and colloid gold label pad (3) preparation: the determination of optimal pH:
Take the above-mentioned colloidal gold solution of 320ml, take 0.1M solution of potassium carbonate adjustment colloidal gold solution pH value to suitable ph, will be molten
Liquid is put on magnetic stirring apparatus, and adjustment revolving speed is 250rpm, and under stirring, the anti-norovirus GI type Dan Ke of aequum mouse is added
Grand antibody 1 and the anti-norovirus GII type monoclonal antibody 2 of mouse, the concentration in solution is controlled 8-both antibody at this moment
10%BSA is added after static 15 minutes in 10ug/ml, makes the 1%GEG20000 of BSA final concentration of 0.1% and 0.32ml, continues
Stirring 10 minutes, centrifugation: the solution marked is transferred in centrifuge tube, balance up-regulation balance, and 2-8 DEG C, 10000rpm,
Supernatant is sucked out in centrifugation 30 minutes, the heart, collects precipitating, with gold mark conjugate dilution constant volume to former colloidal gold solution volume
10%, coating: taking polyester non-woven fabric, sets coating weight as 6-8ul/cm, 37 DEG C of drying temperature, relative humidity≤40% rotates
1.5 ms/min of speed, coated colloidal gold is transferred to drying room, drying temperature is 37 DEG C, and dry humidity is≤40%, when
Between 16-24 hours, after colloidal gold pad is dry, aluminium foil bag is sealed up for safekeeping spare；
C, it prepares detecting pad: having configured the anti-of nature controlling line (7) C and GI detection line (5) and GII detection line (6) according to recipe requirements
Liquid solution takes NC film (4) (specification width 25mm), and the norovirus GI type GI detection line is disposably marked according to distance interval
(5), the norovirus GII type GII detection line (6) and the nature controlling line (7) C line, the norovirus GI type GI detection line
(5) it is coated with the anti-norovirus GI type monoclonal antibody 3 of mouse, it is anti-that the norovirus GII type GII detection line (6) is coated with mouse
Norovirus GII type monoclonal antibody 4 is coated with sheep anti-mouse igg polyclonal antibody at nature controlling line (7) (C line), and distance interval is set
It is fixed as follows: to be 7mm with a distance from bottom end of the GI detection line (5) from NC film (4), GII detection line (6) is with a distance from the bottom end from NC film (4)
12mm is 17mm with a distance from bottom end of the nature controlling line (7) from NC film (4), sets drying temperature as 37 DEG C, the ring of relative humidity≤40%
Dry under border, the time 16-24 hours, the NC film (4) being coated with was spooled spare after drying tower is dry；
D, pasting board and cutting: the sample pad (2), the label pad, the detecting pad, the water absorption pad (8) sequence are overlapped
On the PVC bottom plate (1), and it is cut into width 3.3mm (stripe shape) and 4.0mm (card-type) according to specification requirement, checks interior parlor
Temperature and humidity, it is ensured that temperature is 18-24 DEG C, humidity :≤40% could start to work, and PVC board is unfolded on table top, and both ends are sticked to
On station, the absciss layer paper that middle section width is 25mm is torn, scribing line NC film (4) alignment straight line is attached to and tears absciss layer paper
Place, then tear the following protection sheet of PVC board, colloidal gold is pasted in PVC board, allow colloidal gold top edge and scribing line NC film
(4) lower edge is overlapped 1.0-2.0 millimeters, and sample pad (2) is pasted onto the lower section of colloidal gold, allows the top edges of sample pad (2)
It is 1.0-2.0 millimeters Chong Die with the lower edge of colloidal gold, the absciss layer paper of PVC board top is torn, blotting paper straight line is pasted into PVC
On plate, allow blotting paper and cross NC film (4) Chong Die 2 millimeters or so of top edge, patch adhesive tape, allow adhesive tape to be completely covered
Colloidal gold pad operates automatic cutting machine, width cutting as required.
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