CN106841604A - A kind of Respirovirus quick detection kit and preparation method thereof - Google Patents
A kind of Respirovirus quick detection kit and preparation method thereof Download PDFInfo
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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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
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- 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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Abstract
The invention provides a kind of colloidal gold method Test paper for detecting Respirovirus.Test paper of the present invention includes sample pad, the glass fibre membrane containing colloid gold particle label, nitrocellulose filter and blotting paper, and wherein nitrocellulose filter includes the control zone that the detection zone formed by influenza A virus antibody, influenza B virus antibody, Respiratory Syncytial Virus(RSV) antibody, adenovirus antibody and anti-rat immune globulin antibody are formed.If there is in Respiratory Syncytial Virus(RSV), adenovirus, influenza A virus (FluA) and influenza B virus (FluB) any one or its any combination in sample, the antigen will be caught by the corresponding antibodies of immobilization in nitrocellulose filter detection zone, because the effect of collaurum forms aubergine band.Quick detection Respiratory Syncytial Virus(RSV), adenovirus, influenza A virus and influenza B virus can be realized by once-through operation using test paper of the invention, so as to simplify operating process, the purpose of quick detection is realized.
Description
Technical field
The present invention relates to area of medical diagnostics, and in particular to a kind of multiduty Respirovirus quick detection kit and
Its preparation method.
Background of invention
At present it was demonstrated that 95% acute upper respiratory tract infection and most lower respiratory illness be by bacterium outside disease
Original causes, wherein it is most common with Respirovirus, including Respiratory Syncytial Virus(RSV) (RSV) A types, Type B, adenovirus (ADV), influenza
Viral A (FluA), influenza virus B (FluB), parainfluenza virus 1 (PIV1), parainfluenza virus 2 (PIV2), parainfluenza virus 3
(PIV3), change of coxsackie b virus etc..Wherein, various viruses have the spies such as appeal is strong, propagation is fast, incubation period is short, morbidity is anxious
Point, and new pathogenic virus are also found constantly recently, such as human metapneumovirus, SARS virus, highly pathogenic fowl
Influenza virus and nowadays worldwide H1N1 influenza A virus etc., cause greatly tired to clinical disease diagnosis and treatment
It is difficult.
Respiratory tract infection be underage child in world wide (<5 years old) dead the first reason, about 5,000,000 can be caused every year
Death of child.Ill 3-8 times/year of the number of times average out to of upper respiratory tract infection of children, ALRI rate is lower than URTI, 1
Year children in infect ratio about 1~3%, school-ager to 5%~10%.Respirovirus refers to traditionally infringement respiratory tract
Virus, refer at least to 8 sections, more than the 200 kinds of virus of type.The virus of clinical common caused respiratory tract infection has gland
Viral (AdV), A type/influenza B virus (FluA and FluB) and Respiratory Syncytial Virus(RSV) (RSV) etc..
Adenovirus (AdV) is a kind of particle for not having tunicary a diameter of 70~90nm, wire double-stranded DNA virus.It is divided into
Two category, 6 subgroups (A-F), including 51 serotypes.Adenovirus can be propagated by people, water, medium and apparatus, room temperature bar
Under part, adenovirus has a cycle in dirt can extend to 3 weeks.Adenovirus be more easy in children and military camp personnel occur infection and
Extensive popular, most of infants at least infected a kind of adenopathy strain in after birth 5 years.The virus can cause pneumonia,
Febrile upper respiratory disease, conjunctivitis, cystitis, enterogastritis etc., the difference of the immune and cardio-pulmonary function according to the infected can be led
Cause serious respiratory distress even dead.ADV infection whole years distribute, and winter-spring season is relatively more, and positive rate is reached as high as
15%.Can be wiped from respiratory secretions sample, eye, pharynx wipe or excrement in be separated to virus, can also use IFT, RIA or ELISA
Quick diagnosis are made etc. method measure viral antigen, is 60%~65% in the positive positives rate of patient of virus purification, in sense
May occur in which neutralizing antibody, hemagglutination inhibition antibody and complement fixation antibody within 7 days after dye.Determining antibody with serological method can clearly examine
It is disconnected.
Influenza A virus and influenza B virus (FluA and FluB) mainly include internal core and outside virus
Cyst membrane, core is made up of nucleoprotein curling wrapping spirality RNA, and the antigen stabilization of nucleoprotein seldom morphs, with type
Specificity.According to the difference of nucleoprotein antigen, by influenza virus point first (A), second (B), third (C) three type, three type viruses have
Similar biochemistry and biological property.Pig and birds can carry Flu-A, and second, influenza C are relayed only between people.A type
Clinical symptoms with influenza B are similar, but admission rate is four times in influenza B virus caused by influenza A virus.It is B-mode
Influenza generally has myositis and gastrointestinal symptom.The few lower respiratory tract symptoms of influenza C, but occasional causes upper respiratory tract disease
Disease.Can cause it is anxious play hyperpyrexia, significantly weak, muscular soreness of whole body, and have a stuffy nose, upper to breathe catarrh symptom relative for runny nose and sneeze etc.
It is lighter.This disease has self limiting, but in infant, the elderly and there are easy Complicating Pneumonia In Patients of patient of cardiopulmonary underlying diseases etc. sternly
Weigh complication and cause death.Typically come across winter, the Northern Hemisphere is generally peaked in 1,2 months, the Southern Hemisphere it is popular when
Between it is later, torrid areas can fall ill throughout the year.Worldwide being very popular often has 2~3 popular ripples, and 3~4 weekly assemblies of general prevalence stop naturally
Only.The World Health Organization estimates the annual 100000000 people infection influenza in the Northern Hemisphere, and 50,000,000 times medical, and 300,000 times in hospital, mainly high-risk
Crowd and the elderly, annual 10000 people die from influenza, mainly weak the elderly.
Respiratory Syncytial Virus(RSV) (RSV) is a kind of RNA virus, and respiratory tract infection is suffered from from laboratory first in nineteen fifty
It is isolated in orangutan, is tunicary single-stranded RNA virus, a diameter of 120~200nm belongs to the Pneumovirinae of Paramyxoviridae
Category, only 1 serotype.Neonate and the baby within 6 months are more common in, can be propagated by the air spittle and close contact, dived
3~7 days volt phases, infectiousness is strong.It is two hypotypes of A, B that can be divided to RSV using monoclonal antibody, there is document report RSV A hypotypes
Infection onset urgency, state of an illness weight；The subtype B infection state of an illness is relatively light.RSV is Infant Viral Pneumonia and capillary branch all over the world
One of main pathogens of tracheitis, seriously threaten infantile health, especially premature, children with congenital heart disease, immune
The baby of defect and children's Yi Fa seriousness rsv infections.Any age group can all infect syncytial virus from neonate to adult.
Infant's symptom is heavier, can there is hyperpyrexia, rhinitis, pharyngitis and laryngitis, and capillary bronchitis and pneumonia are shown as later.A small number of sick children can
Concurrent tympanitis, pleurisy and myocarditis etc..After adult and older children infection, the infection of the upper respiratory tract is mainly shown as.Grind
Study carefully and show that RSV epidemic seasons concentrate on annual winter-spring season (in March in November to next year, peak appears in January in December to next year), I
State Beijing ground, Hangzhou, Suzhou and other temperate countries (such as Japan, the U.S.) are consistent, and medical child patient positive rate is reachable
42.50%.
Respiratory virus infection is one of clinical most common disease, and aetology is complicated, and accurate Etiology analysis are not
Only it is to make a definite diagnosis foundation, is also the basis of reasonable selection therapeutic scheme.Therefore understand Respirovirus detection means and carry out quick
Viral diagnosis, it has also become the task of top priority.It is clinical that the correlation that virus is produced is resisted for virus infection mainly detection body at present
Body, clinical diagnosis needs can not be met due to being influenceed by many factors such as immunity of organisms.Detection respiratory tract infection is main
Virus is more using virus purification cultivation, IIF (IF), DIF, alkaline phosphorus both at home and abroad at present
Sour enzyme alkali resistant acid phosphatase bridging enzyme linked immunosorbent assay (APAAP), Biotin-streptavidin-peroxidasemethod, Enzyme-linked Immunosorbent Assay
Method (ELISA), viral fast detection method, such as molecular biology method, multiple reverse transcription polymerase chain reaction (mRT-PCR), nido
Various detection means such as PCR and nucleic acid hybridization, Multiplex real-time PCR, biochip technology, suspension array technology.
But above-mentioned detection method is required to special instrument and equipment and professional training personnel, and can just enter in laboratory
Row detection, high cost, complex operation, time-consuming, is unsuitable for Clinical screening, is more unsuitable for live detection immediately.Such as disease
Poison culture is the goldstandard of Viral diagnosis, but this detection method cycle is very long, typically takes the time of 3~7 days, and sensitivity is not
It is high.
RT-PCR can realize detecting in 4-8 hours to sample, and sensitivity and specificity are very high, but be not suitable for
The density of population such as airport, port, railway station, hospital are big, flow of the people is big, be difficult to allow the public place of crowd's aggregation for a long time.
Therefore, clinically urgent need sets up a kind of quick, easy, efficient at present, once can simultaneously identify various viruses simultaneously
The quick respiratory diseases virus detection method that can be widely popularized.
The content of the invention
For the deficiency of existing detection technique, it is an object of the invention to provide a kind of glue for detecting Respirovirus
Body gold method Test paper and preparation method thereof.Using test strips of the invention or kit, can be realized by once-through operation quick
Detection Respiratory Syncytial Virus(RSV), adenovirus, influenza A virus (FluA) and influenza B virus (FluB), so as to simplify operation
Process, realizes the purpose of quick detection.
Colloidal gold method Test paper for detecting Respirovirus of the present invention, the test paper includes sample pad, contains
Have glass fibre membrane, nitrocellulose filter and the blotting paper of colloid gold particle label, wherein sample pad, contain colloid gold particle
The glass fibre membrane of label, nitrocellulose filter and blotting paper are sequentially overlapped and are pasted onto on base plate, the nitrocellulose filter
Including be coated with influenza A virus antibody detection zone, be coated with influenza B virus antibody detection zone, be coated with breathing
The detection zone of road syncytial virus antibody, the detection zone for being coated with adenovirus antibody and it is coated with the control of anti-rat immune globulin antibody
Area processed, the colloid gold particle label includes that colloid gold label Anti-adenovirus antibody, colloid gold label preventing respiratory close born of the same parents' disease
Malicious antibody, colloid gold label influenza A virus antibody and colloid gold label influenza B poison antibody.
The principle of colloidal gold method Test paper of the present invention is：Testing sample is instilled the sample pad of detection reagent plate
Afterwards, on glass fibre membrane colloid gold particle label is dissolved.If there is Respiratory Syncytial Virus(RSV), adenovirus, first in sample
Any one or its any combination in type influenza virus (FluA) and influenza B virus (FluB), then colloid gold particle label point
Immunocomplex is not formed to the corresponding viral antigen in sample.The immunocomplex is because capillarity is in nitrocellulose filter
Middle movement, by the influenza A virus antibody of immobilization, influenza B virus antibody in the corresponding detection zone of nitrocellulose filter, exhales
Inhale road syncytial virus antibody or adenovirus antibody catches, because the effect of collaurum forms aubergine band.The aubergine of this test paper
Band can estimate confirmation, and adenovirus, Respiratory Syncytial Virus(RSV), influenza A virus or B-mode stream are whether there is in judgement sample
Any one in Influenza Virus or its any combination.It should be noted that antibody and corresponding same viral glue that detection zone is fixed
The antibody of body gold mark is different antibody, for example from different companies, with different method prepare, kind it is different or
For same virus but the different antibody of binding site, preferably both are to resist for same viral but binding site is different
Body, can so ensure colloid gold label immunocomplex because accordingly being examined when capillarity is moved in nitrocellulose filter
The antibody for surveying immobilization in area is recognized.
On the other hand, no matter whether there is adenovirus, respiratory syncytial viral antigens, influenza A virus or second in sample
Any one in type influenza virus or its any combination, remaining colloidal gold labeled monoclonal antibody continue to be moved in nitrocellulose filter,
In the anti-rat immune globulin antibody capture that control zone is immobilized, aubergine band is formed because of collaurum.Which show glue
The normal movement of body gold labelled antibody.
Preferably, in Test paper on glass fibre membrane colloid gold label Anti-adenovirus antibody, colloid gold label are anti-to exhale
Inhale the consumption point of road syncytial virus antibody, colloid gold label influenza A virus antibody and colloid gold label influenza B poison antibody
Wei not 25-45 μ g/cm2, more preferably 30-36 μ g/cm2。
Preferably, detection zone contains on nitrocellulose filter in Test paper influenza A virus antibody, influenza B
The consumption of antiviral antibody, Respiratory Syncytial Virus(RSV) antibody and adenovirus antibody is 0.1-0.5 μ g/mm, more preferably 0.2-0.3 μ
g/mm。
Preferably, the consumption of the anti-rat immune globulin antibody that control zone is contained is on nitrocellulose filter in Test paper
0.1-0.5 μ g/mm, more preferably 0.3-0.4 μ g/mm.
Influenza A virus antibody, influenza B virus antibody, respiratory syncystial disease described in Test paper of the present invention
Malicious antibody and adenovirus antibody are mouse source antibody, more preferably mouse resource monoclonal antibody.
Anti- rat immune globulin antibody described in Test paper of the present invention can be rabbit-anti rat immune globulin antibody, sheep
Anti- rat immune globulin antibody or hunchbacked goat anti-mouse immunoglobulin antibody, preferably goat anti-mouse immunoglobulin antibody.
Present invention also offers a kind of preparation method for detecting the colloidal gold method Test paper of Respirovirus, including
Following steps：
(1) colloidal gold solution is prepared using chemical reduction method, by Anti-adenovirus antibody, anti-influenza A virus antibody and anti-
Influenza B virus antibody and anti respiratory syncitial virus antibodies are added separately in colloidal gold solution, will prepare colloid
Gold-antibody particulate labels are uniformly combined on glass fibre element film, freeze-drying；
(2) influenza A virus antibody, influenza B virus antibody, Respiratory Syncytial Virus(RSV) antibody, adenopathy will be coated with
The buffer solution of malicious antibody and anti-rat immune globulin antibody is sprayed on formation detection line and nature controlling line on nitrocellulose filter respectively,
Fully dry；
(3) one side in base plate adheres to sample pad, glass fibre membrane, nitrocellulose filter and water suction with sequentially mutually overlapping
Paper, obtains final product Test paper.
In the above method, colloid gold label Anti-adenovirus antibody, colloid gold label in Test paper on glass fibre membrane
The use of anti respiratory syncitial virus antibodies, colloid gold label influenza A virus antibody and colloid gold label influenza B poison antibody
Amount is respectively 25-45 μ g/cm2, more preferably 30-36 μ g/cm2。
It is detection zone contains on nitrocellulose filter in Test paper influenza A virus antibody, B-mode in the above method
The consumption of Antibody of Influenza, Respiratory Syncytial Virus(RSV) antibody and adenovirus antibody is 0.1-0.5 μ g/mm, more preferably 0.2-
0.3μg/mm。
In the above method, in Test paper on nitrocellulose filter the anti-rat immune globulin antibody that control zone is contained use
Measure is 0.1-0.5 μ g/mm, more preferably 0.3-0.4 μ g/mm.
In the above method, influenza A virus antibody, influenza B virus antibody, respiratory tract described in Test paper are closed
Cellular virus antibody and adenovirus antibody are mouse source antibody, more preferably mouse resource monoclonal antibody.Wherein, it should be noted that collaurum mark
The note antibody antibody corresponding with detection zone should be different antibody.
In the above method, the anti-rat immune globulin antibody described in Test paper can be anti-for rabbit-anti rat immune globulin
Body, goat anti-mouse immunoglobulin antibody or hunchbacked goat anti-mouse immunoglobulin antibody, preferably goat anti-mouse immunoglobulin antibody.
In the above method, it is ensured that push down 0.5~1mm of glass fibre membrane one end, the glass fibre membrane other end in sample pad one end
0.5~1mm of nitrocellulose filter one end is pushed down, one end of blotting paper is pushed down nitrocellulose filter 0.5~1mm of the other end, must be examined
Test paper.
The reagent strip that Test paper after assembling is cut into 0.5cm moneys on cutting machine is stand-by, after In Aluminium Foil Packing, you can make
For finished product is put in storage.
It is a further object of the present invention to provide a kind of detection kit, the kit includes above-mentioned Test paper, makes
With specification and packing box.
The Test paper that the present invention is prepared have sensitivity higher, with safe operation (without radioactive waste),
Easy (completions of the step of simple operations one), be adapted to one/part detect (put exempt from, enzyme exempt to be not suitable for one/part or a small amount of sample detection)
With quick (there can be result within 15 minutes or so), it is capable of achieving to Respiratory Syncytial Virus(RSV), adenovirus, influenza A virus
(FluA) and influenza B virus (FluB) antigen scene and family's self-inspection, make up at this stage chromatography kit sensitivity it is not high and
The defect of parting can not be realized, is possible to reduce the risk of crowd massing infection to greatest extent, lowered disease at epidemic situation scene
The harm for causing.
Test paper of the invention can realize on a paper slip detecting 4 kinds of viruses simultaneously, shorten detection cycle, reduce because
The phenomenon of missing inspection for single certain parting of detection, and medical disposable material has been saved, thus have relative to existing detection kit
There is obvious technical advantage.
Brief description of the drawings
Fig. 1：The structural representation of Test paper.Wherein, 1 is sample pad, and 2 is glass fibre membrane, and 3 is nitrocellulose
Film, 4 is blotting paper, and 5 is base plate, and 6 is detection zone (T lines), and 7 is control zone (C lines), and 8 is antigen to be checked, and 9 is colloid gold label
Antibody, 10 is the immunocomplex that colloidal gold labeled monoclonal antibody is formed with viral antigen, and 11 is detection domain antibodies, and 12 is anti-for control zone
Body.
Fig. 2：Glass fibre film preparation schematic diagram containing colloid gold particle label
Specific embodiment
The following is specific embodiment of the invention, technical scheme is done and is further described, but it is of the invention
Protection domain be not limited to these embodiments.It is every to be included in this hair without departing substantially from the change of present inventive concept or equivalent substitute
Within bright protection domain.
The preparation of the Respirovirus quick detection test paper of embodiment 1
(1) preparation of collaurum
Using chemical reduction method, to trisodium citrate reducing agent is added in aqueous solution of chloraurate, required glue is prepared into
Body gold grain.Method is heated to boiling first by 0.01%HAuCL4 solution 500ml, is rapidly added the trisodium citrate water of 8.0ml
Solution, is heated to continuing to boil 10-15min after there is red.Recover volume to 500ml.
(2) prepared by collaurum-antibody conjugates
A. colloidal gold solution 500ml is taken, in the state of magnetic stirrer, with the K of 0.2M2CO3 solution adjusts PH
System 8.0.
B. the mouse monoclonal antibody (Japanese tuno companies) of anti respiratory syncytial virus is diluted to 0.01MPBS
1.5mg/ml, takes the colloidal gold solution that 5 centrifuge tubes respectively add 1ml, respectively in preceding 4 pipe add 30ul, 40ul, 45ul,
50ul antibody to be marked, used as control, room temperature places 5min to last pipe after being well mixed.Then respectively added in preceding 4 pipe
100ul concentration is 10% NaCl solution.Room temperature places 10min after mixing.With the antibody added without the pipe for becoming blueness
Amount is used as optimum protein labelled amount.
The mouse monoclonal antibody of anti-adenovirus, the mouse monoclonal antibody and anti-influenza B of anti-influenza A virus (FluA)
The preparation of the mouse monoclonal antibody (being purchased from Japanese tuno companies) of viral (FluB) is ibid.
C. the amount of antibody needed for calculating is measured according to volume and mark practicality, above-mentioned 4 kinds of antibody of 1.5mg/ml is delayed respectively
Slowly it is added in colloidal gold solution, 20min is stirred at room temperature.
D. plus 10%BSA, to final concentration 2%, 5min is stirred at room temperature.
E. plus 10%PEG, to final concentration 1%, 5min is stirred at room temperature.
F.10000rpm/min 30min is centrifuged, supernatant is carefully drawn, liquid suspension precipitation is preserved with the collaurum of 100ml.Put
4 DEG C save backup.
(3) treatment of gold standard pad
The glass fibre element film for taking 15mm*300mm is placed on stainless steel disc, and colloidal gold antibody combination liquid is uniformly contained
It is immersed on glass fibre element film, freeze overnight on freeze dryer is then put into, to being completely dried.
(4) preparation of antibody solid phase nitrocellulose filter
A. the monoclonal antibody (being purchased from Abcam companies) of anti respiratory syncytial virus is diluted to concentration with the PBS of 0.01M
The antibody-solutions of 2.8 ± 0.1mg/ml, are sprayed on nitrocellulose filter with point film instrument with the amount of 1 μ l/cm, form detection line.
It is similar, by the monoclonal antibody of anti-adenovirus, the monoclonal antibody of anti-influenza A virus (FluA) and anti-
The monoclonal antibody (be purchased from Abcam companies) of influenza B virus (FluB) PBS of 0.01M be diluted to concentration 2.8 ±
The antibody-solutions of 0.1mg/ml, are sprayed on nitrocellulose filter with point film instrument with the amount of 1 μ l/cm, are spaced 0.5-0.8cm.
B. sheep anti-mouse igg polyclonal antibody (being purchased from Abcam companies) is diluted to 2.0 ± 0.1mg/ml's with 0.01MPBS
Antibody-solutions, are sprayed on nitrocellulose filter with point film instrument with the amount of 1 μ l/cm, form the spacing between nature controlling line, and detection line
It is 0.8cm.
C. will be fixed with during the nitrocellulose filter of antibody puts 37 DEG C of baking ovens and fully dry.
D. put and save backup in dry environment.
(5) assemble
A. antibody solid phase nitrocellulose filter is pasted in plastics liner centre position.
B. the top of nitrocellulose film location is fixed on plastics liner, blotting paper is pasted；Paste gold standard pad in lower end；Inhale
Water cushion and gold standard pad are with nitrocellulose filter edge about 2mm.
C. sample pad, sample pad covering gold standard pad about 2mm are pasted in the lower section of label pad.
D. the liner after assembling, the reagent strip that 0.5-1cm moneys are cut on cutting machine is stand-by.The finished product structure of Test paper
As shown in Figure 1.Wherein, 1 is sample pad, and 2 is glass fibre membrane, and 3 is nitrocellulose filter, and 4 is blotting paper, and 5 is base plate, and 6 are
Detection zone (T lines), 7 is control zone (C lines), and 8 is antigen to be checked, and 9 is colloidal gold labeled monoclonal antibody, 10 be colloidal gold labeled monoclonal antibody with
The immunocomplex that viral antigen is formed, 11 is detection domain antibodies, and 12 is control domain antibodies.
(6) pack
(7) finished product storage.Final finished kit includes Test paper, operation instructions and the packing box of above-mentioned preparation.
The detection application of the kit of embodiment 2
This kit can qualitatively detect the Virus Sample in nasal cavity wiping solution, throat wiping solution, nasal cavity aspirated liquid sample；
By the detection to clinical sample, positive, negative coincidence rate is high, with sensitivity and specificity higher.
Unknown 1180 parts of source of infection patients clinical sample, its positives 410 parts, negative 770 are detected in three Different hospitals
Part.
Sensitivity：399 parts of the positive is detected in 410 parts of positive samples, sensitivity is 97.3%.Wherein detect respiratory tract conjunction
97 parts of cellular virus sample, 114 parts of adenovirus, 85 parts of influenza A virus (FluA), 103 parts of influenza B virus (FluB).
Specificity：760 parts of feminine gender is detected in 770 parts of negative samples, specificity is 98.7%.
Embodiment 3：Adenovirus detects comparative study
Using immunochromatography principle, the Adenovirus Antigen in qualitative detection throat wiping solution sample.To be sterilized swab stick from mouth
In the fully-inserted throat in chamber, centered on swallowing the rubescent position of rear wall, maxilla almond, friction for several times, gathers mucodermis.Collection
When, it should be noted that saliva should not be encountered.
0.5ml samples extract (to the datum line of lower section) are added in attached extracting pipe in advance, is placed on
In stand.Stirred in the sample extract that sterilizing swab stick after collecting sample is immersed in extracting pipe.From extracting pipe
Outside, with finger extrude sterilizing swab stick for several times, make sample extract be thoroughly impregnated sterilizing swab stick, then extract sterilizing swab stick,
The liquid for twisting out is to be measured as sample.Extruding extracting pipe, drop 3 drips to the sample drop bottom of detection plate, and knot is read after 15min
Really.
This sample mode immunofluorescence technique confirms：Throat wiping solution 204, including 70 confirmed through immunofluorescence technique
ADV positive samples and 134 ADV negative samples；By the detection of examination reagent it is positive is 70, the two detection is positive consistent to be
69；By the detection of examination reagent it is negative is 134, the two detection it is negative it is consistent be 133, refer to following table：
Sensitivity=69/70 × 100%=98.6%
Specificity=133/134 × 100%=99.2%
Total uniformity=(69+133)/(70+134) × 100%=99.0%
Embodiment 4：Influenza A virus (FluA) and influenza B virus (FluB) detection comparative study
The modulator approach of 4.1 samples
0.5ml sample extracts are added in sampling pipe in advance, is placed in stand.
1. nasal cavity aspirated liquid
During nasal cavity aspirated liquid 0.5ml is suspended in into transport medium or 2ml physiological saline.Having been added in sample extracting
0.5ml suspensions are added in the sampling pipe of liquid, it is to be measured as sample after being sufficiently mixed.
2. Nasopharyngeal swabs (nasal cavity wiping solution), oropharynx swab (throat wiping solution)
Stirred in the sample extract that swab stick after collecting sample is immersed in sampling pipe.From the outer of sampling pipe
Side, swab stick is extruded for several times with finger, sample extract is thoroughly impregnated swab stick, then extracts swab stick, and the liquid for twisting out is used as sample
Product are to be measured.
4.2. operating method is detected
1. by 100 μ L under the sample drop of sampling pipe (about 3 drop) to the sample drop bottom of detection plate.
2. result is observed after 15 minutes.
4.3rd, the performance of Test paper
The minimum detectable activity of this product：The sensitivity of influenza virus type minimum detection is 3.0 × 104TCID50/ every time
Detection.The sensitivity of Influenza B minimum detection is 1.5 × 105TCID50/ detection every time.
The reactivity of popularity common cold virus：
1. influenza A virus：The H1N1 of hypotype：5 plants, H2N2：3 plants, H3N2：7 plants, reaction has been identified entirely.
2. influenza B is viral：9 plants of influenza B viruses, have been identified reaction entirely.
Embodiment 5：Respiratory Syncytial Virus(RSV) detects comparative study
Respiratory Syncytial Virus(RSV) propagates extensive, big to infant's harm, even results in death, and harm has what is be gradually increasing
Trend.And the means of existing detection, such as classical virus purification of laboratory diagnostic technique one and culture is time-consuming oversize can not to be done
To detection in time, and serology such as needs could detect the meaning for losing detection completely after reaching to a certain degree at the state of an illness.And show
Practise medicine and use large-scale instrument more institute, not only instrument is sufficiently expensive, even if after disposable input buys instrument, subsequently needing to use
To reagent be also sufficiently expensive.
Test paper prepared by the present invention is convenient for the fast and accurately detection of Respiratory Syncytial Virus(RSV) is provided, and is as early as possible
Diagnosis, reduction transmission of disease and the correct therapeutic scheme of determination, prevent abuse of antibiotics from providing important evidence.
From methodology angle, colloidal gold immunity chromatography is compared as follows with other method：
Product of the present invention can be contrasted with single collaurum Product checking respiratory syncytial virus infected similar on the market：
Claims (10)
1. a kind of colloidal gold method Test paper for detecting Respirovirus, the test paper includes sample pad, contains collaurum
The glass fibre membrane of particulate labels, nitrocellulose filter and blotting paper, wherein sample pad, contain colloid gold particle label
Glass fibre membrane, nitrocellulose filter and blotting paper are sequentially overlapped and are pasted onto on base plate, and the nitrocellulose filter includes coating
The detection zone that has influenza A virus antibody, the detection zone for being coated with influenza B virus antibody, it is coated with respiratory syncystial disease
The detection zone of malicious antibody, the detection zone for being coated with adenovirus antibody and the control zone of anti-rat immune globulin antibody is coated with, institute
State colloid gold particle label including colloid gold label Anti-adenovirus antibody, colloid gold label anti respiratory syncitial virus antibodies,
Colloid gold label influenza A virus antibody and colloid gold label influenza B poison antibody.
2. Test paper according to claim 1, it is characterised in that：Antibody and corresponding same virus that detection zone is fixed
The antibody of colloid gold label be different antibody.
3. Test paper according to claim 1, it is characterised in that：Collaurum mark in Test paper on glass fibre membrane
Note Anti-adenovirus antibody, colloid gold label anti respiratory syncitial virus antibodies, colloid gold label influenza A virus antibody and glue
The consumption of body gold mark influenza B poison antibody is respectively 25-45 μ g/cm2。
4. Test paper according to claim 1, it is characterised in that：Detection zone contains on nitrocellulose filter in Test paper
The consumption of some influenza A virus antibody, influenza B virus antibody, Respiratory Syncytial Virus(RSV) antibody and adenovirus antibody is
0.1-0.5μg/mm。
5. Test paper according to claim 1, it is characterised in that：Control zone contains on nitrocellulose filter in Test paper
The consumption of the anti-rat immune globulin antibody having is 0.1-0.5 μ g/mm.
6. Test paper according to claim 1, it is characterised in that：Influenza A virus described in Test paper resists
Body, influenza B virus antibody, Respiratory Syncytial Virus(RSV) antibody and adenovirus antibody are mouse source antibody.
7. Test paper according to claim 1, it is characterised in that：Anti- rat immune globulin described in Test paper resists
Body is rabbit-anti rat immune globulin antibody, goat anti-mouse immunoglobulin antibody or hunchbacked goat anti-mouse immunoglobulin antibody.
8. Test paper according to claim 7, it is characterised in that：Anti- rat immune globulin described in Test paper resists
Body is goat anti-mouse immunoglobulin antibody.
9. a kind of preparation method for detecting the colloidal gold method Test paper of Respirovirus, comprises the following steps：
(1) colloidal gold solution is prepared using chemical reduction method, by Anti-adenovirus antibody, anti-influenza A virus antibody and anti-B-mode
Antibody of Influenza and anti respiratory syncitial virus antibodies are added separately in colloidal gold solution, will prepare collaurum-anti-
Body particulate labels are uniformly combined on glass fibre element film, freeze-drying；
(2) influenza A virus antibody, influenza B virus antibody, Respiratory Syncytial Virus(RSV) antibody, adenovirus will be coated with to resist
The buffer solution of body and anti-rat immune globulin antibody is sprayed on formation detection line and nature controlling line on nitrocellulose filter respectively, fully
Dry；
(3) one side in base plate adheres to sample pad, glass fibre membrane, nitrocellulose filter and blotting paper with sequentially mutually overlapping,
Obtain final product Test paper.
10. a kind of detection kit, the detection kit includes Test paper described in claim any one of 1-9, uses
Specification and packing box.
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| CN108037290A (en) * | 2017-11-13 | 2018-05-15 | 深圳市博卡生物技术有限公司 | A kind of kit and its detection method of the detection of respiratory pathogen antigen |
| CN109900913A (en) * | 2019-04-26 | 2019-06-18 | 江苏硕世生物科技股份有限公司 | A kind of A type and influenza B virus antigen colloidal gold method combined detection kit and preparation method thereof |
| CN111474353A (en) * | 2020-06-01 | 2020-07-31 | 石家庄洹众生物科技有限公司 | Electronic detection device and kit for influenza virus and rhinovirus |
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| CN114527272A (en) * | 2022-01-21 | 2022-05-24 | 北京泰格科信生物科技有限公司 | Respiratory tract infection virus antigen combined detection kit and preparation method thereof |
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| WO2022115570A1 (en) * | 2020-11-25 | 2022-06-02 | Maxim Biomedical, Inc. | Test kits, devices and methods for detecting infection |
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| WO2022115570A1 (en) * | 2020-11-25 | 2022-06-02 | Maxim Biomedical, Inc. | Test kits, devices and methods for detecting infection |
| CN114527272A (en) * | 2022-01-21 | 2022-05-24 | 北京泰格科信生物科技有限公司 | Respiratory tract infection virus antigen combined detection kit and preparation method thereof |
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