CN106501513A - A kind of Potyviruses Rapid detection test strip and its preparation method and application - Google Patents
A kind of Potyviruses Rapid detection test strip and its preparation method and application Download PDFInfo
- Publication number
- CN106501513A CN106501513A CN201611111887.7A CN201611111887A CN106501513A CN 106501513 A CN106501513 A CN 106501513A CN 201611111887 A CN201611111887 A CN 201611111887A CN 106501513 A CN106501513 A CN 106501513A
- Authority
- CN
- China
- Prior art keywords
- line
- potyviruses
- rabbit
- antibody
- rabbit polyclonal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Sampling And Sample Adjustment (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of Potyviruses Rapid detection test strip and its preparation method and application, Potyviruses Rapid detection test strip, including sample pad, gold standard pad, NC films, detection line T line, nature controlling line C line, adsorptive pads and PVC base plates；Its preparation method includes the preparation of gold standard pad, the preparation of NC films, the preparation of sample pad, four steps of the assembling of test strips；Concrete application detecting step is：The steps such as sample treatment, Deca sample, result interpretation；Potyviruses include PVX, PVYNTN、PVYo, PLRV, PVS, PVM, PVA and tomato black ring virus.Easy to use, strong applicability of the invention, without the need for laboratory, do not need any specific apparatus, without the need for professional's training on operation, field, potato storing cellar, customs etc. any have where Rhizoma Solani tuber osi can test, each potato planting person can use.
Description
Technical field
The present invention relates to the use of immunochromatography technique detection plant viruses technical field, and in particular to a kind of Potyviruses
Rapid detection test strip and its preparation method and application.
Background technology
Rhizoma Solani tuber osi is the higher industrial crops of China's using value, and oneself is listed in one of 7 big crop of China, and Western China
The most important crop varieties in portion and main column support type industry, growth momentum are good, but current China Rhizoma Solani tuber osi per unit area yield is much
It is less than developed country, Potyviruses are to cause that potato yield is low, ropy main cause.In Chinese medical test such as
Early pregnancy, hepatitis B, parasite, the inspection of sexually transmitted disease (STD) and the inspection of animal epidemic for example Poultry diseases, disease of domestic animals, Mouse hepatitis virus,
There is the application of immuno-chromatographic test paper strip in pigs trichina disease, the diagnosis of dog disease virus；In Nicotiana tabacum L., Fructus Cucurbitae moschatae, Caulis et Folium Lactucae sativae, cucumber mosaic virus
Poison, annulus zonatus, nepovirus, Tobacco Vein Banding Caused by Potato Virus Y poison, citrus processing, calabash coe virus, Caulis et Folium Lactucae sativae clump branch are planted
The application of existing immuno-chromatographic test paper strip in the detection of substance, but also without field rapid detection test paper in potato crop
Application.
The technology of conventional detection Potyviruses is double antibody enzyme-linked immunosorbent assays method, and the method needs special
The technical staff of door is tested using special equipment in special laboratory, and operating process is loaded down with trivial details, and experimental period is long：1.5-
2 days.Field investigation and sampling is unable to, scene goes out result.The danger of virosiss is generally prevented and treated using virus-free seed potato in production at present
Evil, for guaranteeing the detoxification quality of potato primary stock, original seed, sets up quick, accurate, sensitive, efficient Potyviruses class disease
Malicious detection technique is the key technique of seed potato production.
Patent publication us related to this patent as follows are found by retrieval：
1st, the patent of Publication No. CN 103757138B disclose the beautiful potato of a kind of Yunnan Potato main breed 6, cloud potato 401,
Marmor upsilon, potato virus X, the 4 kinds of viral detections of marmor solani and corium solani in a surname No. 2 diseased plants of potato
Method.The present invention by the beautiful potato of Yunnan Potato main breed 6, cloud potato 401, a surname No. 2 diseased plants of potato in marmor upsilon,
Virus in the sick sample of potato virus X, marmor solani and 4 species specificity mixed antibody of corium solani trapping, it is not necessary to carry
Taking sample total serum IgE carries out random primer reverse transcription and specificity virus primer composite PCR reaction.This method will be quick, special
Immunoprecipitation is combined with sensitive multiplex RT -PCR, while arranging the corresponding virus PCR of healthy Rhizoma Solani tuber osi random primer cDNA products
Specific fragment positive plasmid as positive and negative compare, so as to quick, accurate, sensitively be applied to detect field potato plant
The aspects such as sample, antiviral material and the doubtful source of infection of field conditions.
2nd, the patent of Publication No. CN 103757138B is related to dual mono- PCR Simultaneous Detection of Potato Y of the RT diseases of two-step method
Poison, corium solani, mono- PCR Simultaneous Detection of Potato X viruses of triple RT, marmor solani, the side of potato virus S
Method and detection kit, the one kind for being specific to 5 kinds of larger Potyviruses of Potato Industry impact and setting up is synchronous to be examined
Survey 2 or 3 kind of viral fast method, detection time only uses 4h, and sensitivity height, high specificity, reliable and stable.The method overcomes
Existing to potato virus disease according to semeiologic routine diagnostic method False Rate is higher, serological method is difficult to detect dormancy
In Rhizoma Solani tuber osi virus and viral level less phloem virus corium solani the shortcomings of;The method is applied to horse
Bell potato seedling, the early diagnosiss of the multiple virosiss of potato seed, have to seed potato quality-monitoring and virosiss prevention important
Effect.
By contrast, there are the different of essence from above-mentioned patent publication us in present patent application.
Content of the invention
The technical problem to be solved is in order to overcome the shortcomings of existing method, by Potyviruses quick detection
Test strips are applied in Potato viruses detection, can achieve to Potyviruses high specific, high sensitivity, high precision in sample
Property, the detection of high stability, quick detection goes out potato virus disease, and a situation arises, is determining for potato detoxicating potato potato seed rank
The prevention and control of level and Potyviruses provide theoretical foundation.
An object of the present invention is to provide a kind of Potyviruses Rapid detection test strip, is by following technical side
Case is realized：
A kind of Potyviruses Rapid detection test strip, including sample pad 1, gold standard pad 2, NC films 3, detection line T line 4, nature controlling line
C lines 5, adsorptive pads 6 and PVC base plates 7, the top of the PVC base plates 7 are fitted with NC films 3, and 3 both upper ends thereof of NC films is taken respectively
Gold standard pad 2 and adsorptive pads 6 are connected to, 2 top of the gold standard pad is overlapped with sample pad 1, and the detection line T line 4 is positioned close to sample
On 3 surface of NC films of product pad 1, the nature controlling line C line 5 is positioned close on the NC films 3 of adsorptive pads 6, on the detection line T line 4
Potyviruses rabbit polyclonal antibody is coated with, the Potyviruses rabbit polyclonal antibody being coated on detection line T line 4 is molten
It is 0.5-1.5 L/cm that liquid draws film concentration, and the concentration for carrying out the Potyviruses rabbit polyclonal antibody solution for drawing film is
0.5-1.5mg/mL；It is coated with Potyviruses goat anti-rabbit antibody in the nature controlling line C line 5, described is coated on nature controlling line C line 5
On Potyviruses goat anti-rabbit antibody solution draw film concentration be 0.5-1.5 L/cm, described for carry out draw film potato disease
The concentration of malicious goat anti-rabbit antibody solution is 0.5-1.5mg/mL；Potyviruses gold colloidal mark is combined with described gold standard pad 2
Note thing, Potyviruses colloid gold label thing solution spraying concentration of the combination in gold standard pad 2 are 4-6 L/cm.
And, it is additionally provided with outside Potyviruses Rapid detection test strip and gets stuck 8,8 upper faces and the sample of getting stuck
1 corresponding position of pad is provided with well 9, and 8 upper faces that get stuck are provided with observation groove with 3 corresponding position of the NC films
10.
And, the gold standard pad 2 is 2-3mm with the length of the overlap joint of sample pad 1.
And, the Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line 5, it is coated on detection line T line 4
Potyviruses rabbit polyclonal antibody and combine Potyviruses colloid gold label thing in gold standard pad 2, can be following
Any one in combination：
Combination 1：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line 5 is PVX goat anti-rabbit antibodies, is coated on
Potyviruses rabbit polyclonal antibody on detection line T line 4 is PVX rabbit polyclonal antibodies, horse of the combination in gold standard pad 2
Bell potato virus colloidal gold label is PVX colloid gold label things；
Combination 2：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line 5 is PVYNTNGoat anti-rabbit antibody, described
The Potyviruses rabbit polyclonal antibody being coated on detection line T line 4 is PVYNTNRabbit polyclonal antibody, the combination is in gold mark
Potyviruses colloid gold label thing on pad 2 is PVYNTNColloid gold label thing；
Combination 3：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line 5 is PVYoGoat anti-rabbit antibody, the bag
It is PVY by the Potyviruses rabbit polyclonal antibody on detection line T line 4oRabbit polyclonal antibody, the combination is in gold standard pad 2
On Potyviruses colloid gold label thing be PVYoColloid gold label thing；
Combination 4：The Potyviruses goat anti-rabbit antibody that is coated in nature controlling line C line 5 is PLRV goat anti-rabbit antibodies, the bag
It is PLRV rabbit polyclonal antibodies by the Potyviruses rabbit polyclonal antibody on detection line T line 4, the combination is in gold standard pad 2
On Potyviruses colloid gold label thing be PLRV colloid gold label things；
Combination 5：The Potyviruses goat anti-rabbit antibody that is coated in nature controlling line C line 5 is PVS goat anti-rabbit antibodies, the bag
It is PVS rabbit polyclonal antibodies by the Potyviruses rabbit polyclonal antibody on detection line T line 4, the combination is in gold standard pad 2
Potyviruses colloid gold label thing be PVS colloid gold label things；
Combination 6：The Potyviruses goat anti-rabbit antibody that is coated in nature controlling line C line 5 is PVM goat anti-rabbit antibodies, the bag
It is PVM rabbit polyclonal antibodies by the Potyviruses rabbit polyclonal antibody on detection line T line 4, the combination is in gold standard pad 2
Potyviruses colloid gold label thing be PVM colloid gold label things；
Combination 7：The Potyviruses goat anti-rabbit antibody that is coated in nature controlling line C line 5 is PVA goat anti-rabbit antibodies, the bag
It is PVA rabbit polyclonal antibodies by the Potyviruses rabbit polyclonal antibody on detection line T line 4, the combination is in gold standard pad 2
Potyviruses colloid gold label thing be PVA colloid gold label things；
Combination 8：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line 5 is tomato black ring virus goat-anti rabbit-anti
Body, the Potyviruses rabbit polyclonal antibody being coated on detection line T line 4 is tomato black ring virus rabbit polyclonal antibody,
Potyviruses colloid gold label thing of the combination in gold standard pad 2 is tomato black ring virus colloid gold label thing.
The second object of the present invention is to provide a kind of preparation method of Potyviruses Rapid detection test strip, be by with
Lower technical scheme is realized：
A kind of preparation method of Potyviruses Rapid detection test strip, comprises the steps：
Step 1：The preparation of gold standard pad 2：
1) preparation of Potyviruses colloid gold label thing：
With colloidal gold solution labelling Potyviruses rabbit polyclonal antibody, the concentration of Potyviruses rabbit polyclonal antibody is 0.5-
1.5mg/mL；
Redissolve formula of liquid：PH8.0-8.8,8-12% sucrose, 3-6% trehaloses, 0.5-2% bovine serum albumin, solvent are 0.1-
The Tris-HcL solution of 0.2moL/L；
The preparation of colloidal gold solution：297-300 mL ultra-pure waters are taken in round-bottomed flask, it is 1-2% chlorine gold to add 2-4 mL concentration
Acid solution, mixes；Round-bottomed flask is placed in thermostat, clean stirrer, stirring at low speed ebuillition of heated is added；Disposably add
Enter 2-4mL, 1-2% citric acid three sodium solution, continue to boil；Moment observation solution colour change, by light yellow → black → purple
→ aubergine, when claret is changed into, after continuing to boil 4-6min, stops heating, is cooled to room temperature；Ultra-pure water does blank right
According to scanning absorption value of the colloidal gold solution between 500-550nm, if absorption peak occurs in 515-525nm, is prepared
Colloidal gold solution qualified；The gold colloidal outward appearance for preparing should pure, bright, without precipitation and floating thing, there is grease in liquid level
Abandon during with a large amount of black particle shape precipitate impurity；The color of optimal gold colloidal is red, and shape is circular；
The preparation technology of Potyviruses colloid gold label thing：2 centrifuge tubes are taken, 1-2mL colloidal gold solutions are added, 3-5 is added
The K of L 0.1mol/L2CO3, 4-6uL Potyviruses rabbit polyclonal antibodies are added, 25-35min are stood, is added 80-
120 L sealers, stand 25-35min, be centrifuged 25-35min, abandon supernatant under the conditions of 8000-10000rpm, 4 DEG C, add
The multiple solution suspension precipitations of 80-120 L, are centrifuged 10-20min under the conditions of 400-600rpm, 4 DEG C, and Aspirate supernatant is preserved standby
With；
2) combination of gold standard pad 2 and Potyviruses colloid gold label thing：From glass fibre membrane as gold standard pad fid
Material, by step 1) the Potyviruses colloid gold label thing for preparing, film instrument is drawn with three-dimensional large platform metal spraying be sprayed on glass fibre
On film, the optimal spraying concentration for spraying Potyviruses colloid gold label thing on glass fibre membrane is 4-6 L/cm, will spray
Glass fibre membrane be placed in 35-40 DEG C of drying baker, drying time 2-4h, take out, be put in aluminium foil bag, 180-230 DEG C of heat
Sealing is closed, is saved backup；
Step 2：The preparation of NC films 3：
Detection line T line and the preparation of nature controlling line C line：With buffer dilute the how grand antibody of Potyviruses rabbit to be coated to 0.5 ~
1.5mg/mL concentration, draws film gold spraying instrument with three-dimensional large platform and how grand for Potyviruses rabbit antibody is drawn film to apart from gold standard pad 1-
At 2cm on NC films, film is drawn on NC films with the concentration of 0.5 ~ 1.5 L/cm, detection line T line is constituted；Diluted with buffer and wait to be coated
Potyviruses goat anti-rabbit antibody draws film gold spraying instrument by Potyviruses goat-anti to 0.5 ~ 1.5mg/mL concentration with three-dimensional large platform
Rabbit antibody draws film on NC films at adsorptive pads 2-3cm, draws film on NC films with the film concentration of drawing of 0.5 ~ 1.5 L/cm, constitutes
Nature controlling line C line；Detection line T line and nature controlling line C line are apart from 4-6mm；There are detection line T line and the NC films 3 of nature controlling line C line to put by drawing
Dry under the conditions of 35-40 DEG C, encapsulate standby；
Buffer：The effect of buffer is to provide certain pH and ionic strength and makes Potyviruses rabbit polyclonal antibody and Rhizoma Solani tuber osi
Viral goat anti-rabbit antibody is firmly coated on NC films 3, and the pH value of buffer is 7.0 ~ 7.4；
Step 3：The preparation of sample pad 1：
From glass fibre membrane as the material for preparing sample pad 1, glass fibre membrane can be used after process；
The process step of glass fibre membrane：Glove are worn, glass fibre membrane is put in treatment fluid, is completely soaked, soak 5-
15min, takes out dehydration 3-5min afterwards, and horizontal on screen cloth is put into 35-40 DEG C of drying baker, dries to moisture constant in 20-30%,
Glass fibre membrane after by drying loads aluminium foil bag heat-sealing and preserves；
The formula for the treatment of fluid：PH7.8-8.0 phosphate buffers, 0.5-1.5%S9 surfactant solutions, 0.4-0.5% polysorbas20s,
0.5-1.5% bovine serum albumin, 0.1-0.3% polyvinylpyrrolidonesolution solution, solvent are water；
Step 4：The assembling of test strips：
Material is completed made for step 1 ~ 3, the specification according to PVC base plates 7 cuts into certain length and width, pastes
On PVC base plates 7 and installed in getting stuck 8 li；
The number of assembling steps of test strips：
1）NC films 3 are pasted onto on PVC base plates 7；
2）The top that gold standard pad 2 is overlapped on 3 one end of NC films, the lap of splice are 1-2mm；
3）The top that adsorptive pads 6 are overlapped on 3 other end of NC films, the lap of splice are 2-3mm；
4）The top that sample pad 1 is overlapped on gold standard pad 2, the lap of splice are 2-3mm；
5）The PVC base plates 7 for pasting are cut into bar in cutting machine；
6）Shell is filled after cutting into bar and loads aluminium foil bag in the lump with desiccant, labelled after sealing.
And, in Potyviruses colloid gold label thing preparation method step, the Potyviruses rabbit polyclonal of addition
Antibody can be PVX rabbit polyclonal antibodies, PVYNTNRabbit polyclonal antibody, PVYoRabbit polyclonal antibody, PLRV rabbit polyclonals resist
Body, PVS rabbit polyclonal antibodies, PVM rabbit polyclonal antibodies, PVA rabbit polyclonal antibodies or tomato black ring virus rabbit polyclonal antibody
In one kind；
If in Potyviruses colloid gold label thing preparation method step, addition is PVX rabbit polyclonal antibodies, can prepare
Into PVX colloid gold label things；If that added is PVYNTNRabbit polyclonal antibody, can be prepared into PVYNTNColloid gold label thing；Such as
That fruit adds is PVYoRabbit polyclonal antibody, can be prepared into PVYoColloid gold label thing；If added is that PLRV rabbit polyclonals resist
Body, can be prepared into PLRV colloid gold label things;If added is PVS rabbit polyclonal antibodies, PVS colloid gold labels can be prepared into
Thing；If added is PVM rabbit polyclonal antibodies, PVM colloid gold label things can be prepared into；If added is many grams of PVA rabbits
Grand antibody, can be prepared into PVA colloid gold label things；If added is tomato black ring virus rabbit polyclonal antibody, can be prepared into
Tomato black ring virus colloid gold label thing；In addition to adding Potyviruses rabbit polyclonal antibody species difference, other preparation works
Skill parameter is all consistent.
And, the preparation method tool of the Potyviruses rabbit polyclonal antibody and Rhizoma Solani tuber osi bell potato virus goat anti-rabbit antibody
Body is：
The preparation of Potyviruses rabbit polyclonal antibody：1-2mL Potyviruses rabbit anti-serums are taken, 1-2mL phosphoric acid buffers are added
Liquid vibrates in an oscillator, adds 2-4mL saturated ammonium sulphate Potyviruses antibody after mixing；Then in 8000-
Be centrifuged under the conditions of 10000rpm, 10-15min, 4 DEG C or so, precipitate Potyviruses antibody, then horse is dissolved with phosphate buffer
Bell potato antiviral antibody, is then removed by filtration unnecessary ammonium sulfate by PD-10coLum fibre columns, obtains final product Potyviruses rabbit many
Clonal antibody；
The preparation of Potyviruses goat anti-rabbit antibody：1-2mL Potyviruses goat-anti rabbit anti-serums are taken, adds 1-2mL phosphoric acid to delay
Rush liquid to vibrate in an oscillator, after mixing, add 2-4mL saturated ammonium sulphates virus；Then in 8000-10000rpm, 10-
15min, is centrifuged under the conditions of 4 DEG C or so, precipitates Potyviruses antibody, is then resisted with phosphate buffer dissolving Potyviruses
Body, is then removed by filtration unnecessary ammonium sulfate by PD-10coLum fibre columns, obtains final product Potyviruses goat anti-rabbit antibody.
Potyviruses rabbit anti-serum preparation method：1-2ml Potyviruses are taken, by subcutaneous 4 points injection health males
Rabbit, injects once week about, total co-injection three times；After third time injection one week, taken a blood sample by ear vein, that is, obtain Ma Ling
The antiserum of potato virus.
Potyviruses goat-anti rabbit anti-serum preparation method：Take 1-2ml Potyviruses and exempt from serum antibody, by subcutaneous 4
The healthy male goat of point injection, injects once, total co-injection three times week about;After third time injection one week, by ear vein
Blood sampling, that is, obtain goat-anti rabbit anti-serum；
And, the Potyviruses goat anti-rabbit antibody and Potyviruses rabbit polyclonal antibody can be selected in following combination
Any one combination is prepared；
Combination 1：PVX goat anti-rabbit antibodies, PVX rabbit polyclonal antibodies；
Combination 2： PVYoGoat anti-rabbit antibody, PVYoRabbit polyclonal antibody；
Combination 3： PVYNTNGoat anti-rabbit antibody, PVYNTNRabbit polyclonal antibody；
Combination 4：PLRV goat anti-rabbit antibodies, PLRV rabbit polyclonal antibodies；
Combination 5：PVS goat anti-rabbit antibodies, PVS rabbit polyclonal antibodies；
Combination 6：PVM goat anti-rabbit antibodies, PVM rabbit polyclonal antibodies
Combination 7：PVA goat anti-rabbit antibodies, PVA rabbit polyclonal antibodies；
Combination 8：Tomato black ring virus goat anti-rabbit antibody, tomato black ring virus rabbit polyclonal antibody；
And, step 2：In the preparation of NC films 3：It is used for drawing the Potyviruses goat anti-rabbit antibody of film nature controlling line C line and is used for drawing
The how grand antibody of the Potyviruses rabbit of film detection line T line can select any one in following combination：
Combination 1：PVX goat anti-rabbit antibodies, PVX rabbit polyclonal antibodies；
Combination 2： PVYoGoat anti-rabbit antibody, PVYoRabbit polyclonal antibody；
Combination 3： PVYNTNGoat anti-rabbit antibody, PVYNTNRabbit polyclonal antibody；
Combination 4：PLRV goat anti-rabbit antibodies, PLRV rabbit polyclonal antibodies；
Combination 5：PVS goat anti-rabbit antibodies, PVS rabbit polyclonal antibodies；
Combination 6：PVM goat anti-rabbit antibodies, PVM rabbit polyclonal antibodies；
Combination 7：PVA goat anti-rabbit antibodies, PVA rabbit polyclonal antibodies；
Combination 8：Tomato black ring virus goat anti-rabbit antibody, tomato black ring virus rabbit polyclonal antibody；
The third object of the present invention is to provide a kind of application process of Potyviruses Rapid detection test strip, is by following
Technical scheme is realized：
A kind of application of Rhizoma Solani tuber osi Rapid detection test strip detects that to potato samples to be checked concrete detecting step is：
(1) sample treatment：Potato leaf is taken, the ratio by leaf weight g and buffering liquid product V is 1:8-1:10 ratio adds
Enter phosphate buffer, green juice is ground, the PH of phosphate buffer is 7.4-7.8；
(2) the above-mentioned sample for processing is added drop-wise in the sample pad of Rhizoma Solani tuber osi Rapid detection test strip, stands 0.5 ~ 3 minute,
Observation detection line T line and nature controlling line C line；
(3) result interpretation：Detection line T line and nature controlling line C line manifest aubergine for the positive；Detection line T line does not manifest purplish red
Color, nature controlling line C line manifest aubergine, are as a result feminine gender；Detection line T line and nature controlling line C line do not manifest aubergine or other are existing
As pointing out test strips failure.
In the present invention：PVX represents potato virus X, PVYNTNRepresent the NTN strains of marmor upsilon, PVYoRepresent horse
The O strains of bell potato Y virus, PLRV represent corium solani, and PVS represents potato virus S, and PVM represents Rhizoma Solani tuber osi M diseases
Poison, PVA represent marmor solani.
The Cleaning Principle of the present invention：Potyviruses are obtained from colloid gold label Potyviruses rabbit polyclonal antibody
Colloid gold label thing, is sprayed in gold standard pad；On detection line T line, coating can occur immunoreation with purpose thing albumen composition
Potyviruses rabbit polyclonal antibody, coated Potyviruses goat anti-rabbit antibody in nature controlling line C line, used as indicatrix.When
Potyviruses gold colloidal mark after measuring samples are loaded onto in sample pad, in sample on potato disease toxalbumin and gold standard pad
Note thing formed immune complex, due to capillary effect, the complex to the swimming of adsorptive pads direction, this complex be coated on inspection
There is specific immunity association reaction in the Potyviruses rabbit polyclonal antibody on survey line T lines, form Potyviruses gold colloidal
Label, antigen（Antigen refers to Potyviruses）, Potyviruses rabbit polyclonal antibody triplet complex and be trapped within
On detection line T line, it is progressively enriched with to form deeper purplish red colo(u)r streak；As capillary effect continues swimming forward, Potyviruses glue
There is specific immunoreation with the Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line and cut in body gold label
Staying, being progressively enriched with and deeper purplish red colo(u)r streak is formed in nature controlling line C line, unnecessary unconjugated material continues chromatography and arrives adsorptive pads
On, therefore positive findingses are judged to what purplish red colo(u)r streak all occurred in detection line T line and nature controlling line C line；If not containing Ma Ling in sample
Potato virus, when Potyviruses colloid gold label thing reaches detection line T line, not with the potato disease being coated on detection line T line
There is immunoreation in malicious rabbit polyclonal antibody, therefore do not occur chromogenic line at detection line T line, and Potyviruses gold colloidal
Label continues swimming forward, with the Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line, special immunoreation occurs
And be trapped, be progressively enriched with and purplish red colo(u)r streak is formed on nature controlling line, therefore only occur purplish red line in Quality Control and be judged to negative findings.
Detection line T line and nature controlling line C line do not manifest purplish red colo(u)r streak or other phenomenons occur, point out test strips failure, detection failure.
Beneficial effects of the present invention：
（1）Immunochromatography technique is applied to potato virus disease detection by the present invention first, it is achieved that high specific, high-sensitive
Detection performance.
（1）Easy to use, strong applicability of the invention：Without the need for laboratory, do not need any specific apparatus, without the need for professional
Training on operation, field, potato storing cellar, customs etc. be any have where Rhizoma Solani tuber osi by test, each potato seed
Plant person can use.
（2）Detection time of the present invention is short, it is only necessary to can draw testing result within 0.5 3 minutes.
（3）Sensitivity of the present invention is high：A piece of leaf of potato subsample can be used for detecting, ELISA test strip potato samples
Potyviruses minimal detectable concentration be 1ng/mL.
（4）Accuracy of the present invention is high：The accuracy rate of the ELISA test strip potato samples and DASELISA immunity
The accuracy rate of adsorption experiment method fits like a glove, and coincidence rate is up to 100%.
（5）High specificity of the present invention：Only this kind virus-positive material tests line of test strips of one virus of Rhizoma Solani tuber osi and Quality Control
There is obvious band in line, and purplish red colo(u)r streak occurs in other positive materials only nature controlling line, and purplish red colo(u)r streak does not occur in detection line；Rhizoma Solani tuber osi
The test strip of virus, to healthy plant and the testing result of buffer control, there is purplish red colo(u)r streak in only C lines, and T lines do not occur
Purplish red colo(u)r streak color, is as a result negative, and therefore this test strips has specificity.
（6）Stability of the present invention is strong：By test strips be placed in be allowed to dry in the sealing aluminium foil bag of drying prescription, protect at room temperature and 37 DEG C
After depositing long-time, color slightly weakens, and other different temperatures, the test result of different time are illustrated in felicity condition without significant change
Lower preservation, this test strips have preferable stability.
（7）Testing cost of the present invention is low；To temperature without specific demand, need not freeze, store convenient transportation, room temperature can be preserved
24 months.
Description of the drawings
Fig. 1 front views of the present invention（Do not draw and get stuck）；
Fig. 2 is Fig. 1 left views；
Fig. 3 present invention gets stuck structural representation；
It is positive findingses schematic diagram in Fig. 4；
It is negative findings schematic diagram in Fig. 5；
It is test strips failure schematic diagram in Fig. 6.
In figure：1 sample pad, 2 gold standard pads, 3NC films, 4 detection line T lines, 5 nature controlling line C lines, 6 adsorptive pads, 7PVC base plates, 8 cards
Shell, 9 wells, 10 observation grooves.
Specific embodiment
With reference to embodiment, the present invention is described in further detail, but embodiments of the present invention not limited to this,
Protection scope of the present invention can not be limited with following embodiments.
Experimental technique used in following embodiments if no special instructions, is conventional method；The material that used,
Reagent etc., if no special instructions, commercially obtains.
Embodiment 1
A kind of Potyviruses Rapid detection test strip, as shown in Figure 1 and Figure 2, including sample pad 1, gold standard pad 2, NC films 3, inspection
Survey line T lines 4, nature controlling line C line 5, adsorptive pads 6 and PVC base plates 7, the top of the PVC base plates 7 are fitted with NC films 3, the NC films 3
Both upper ends thereof is overlapped with gold standard pad 2 and adsorptive pads 6 respectively, and 2 top of the gold standard pad is overlapped with sample pad 1, the detection line T line
4 are positioned close on 3 surface of NC films of sample pad 1, and the nature controlling line C line 5 is positioned close on the NC films 3 of adsorptive pads 6, described
Potyviruses rabbit polyclonal antibody, the Potyviruses rabbit being coated on detection line T line 4 is coated with detection line T line 4
It is 0.5 L/cm that Anti-TNF-α liquid solution draws film concentration, the Potyviruses rabbit polyclonal antibody solution for carrying out stroke film
Concentration be 0.5mg/mL；It is coated with Potyviruses goat anti-rabbit antibody in the nature controlling line C line 5, described is coated on nature controlling line C
It is 0.5 L/cm that Potyviruses goat anti-rabbit antibody solution on line 5 draws film concentration, the potato disease for carrying out stroke film
The concentration of malicious goat anti-rabbit antibody solution is 0.5mg/mL；Potyviruses colloid gold label thing is combined with described gold standard pad 2,
Potyviruses colloid gold label thing solution spraying concentration of the combination in gold standard pad 2 is 4 L/cm.
In the present embodiment, as shown in figure 3, be additionally provided with outside Potyviruses Rapid detection test strip get stuck 8, described get stuck
8 upper faces are provided with well 9 with 1 corresponding position of sample pad, and 8 upper faces that get stuck are corresponding with the NC films 3
Position is provided with observation groove 10.
In the present embodiment, the length of NC films 3 and the overlap joint of gold standard pad 2 is 1mm, and the NC films 3 are overlapped with adsorptive pads 6
Length be 2mm, the gold standard pad 2 is 2mm with the length of the overlap joint of sample pad 1.
In the present embodiment, the Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line 5, detection line T is coated on
Potyviruses rabbit polyclonal antibody and the Potyviruses colloid gold label thing being coated in gold standard pad 2 on line 4, Ke Yishi
Any one in below combining：
Combination 1：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line 5 is PVX goat anti-rabbit antibodies, is coated on
Potyviruses rabbit polyclonal antibody on detection line T line 4 is PVX rabbit polyclonal antibodies, horse of the combination in gold standard pad 2
Bell potato virus colloidal gold label is PVX colloid gold label things；
Combination 2：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line 5 is PVYoGoat anti-rabbit antibody, the bag
It is PVY by the Potyviruses rabbit polyclonal antibody on detection line T line 4oRabbit polyclonal antibody, the combination is in gold standard pad 2
On Potyviruses colloid gold label thing be PVYoColloid gold label thing；
Group and 3：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line 5 is PVYNTNGoat anti-rabbit antibody, described
The Potyviruses rabbit polyclonal antibody being coated on detection line T line 4 is PVYNTNRabbit polyclonal antibody, the combination is in gold mark
Potyviruses colloid gold label thing on pad 2 is PVYNTNColloid gold label thing；
Combination 4：The Potyviruses goat anti-rabbit antibody that is coated in nature controlling line C line 5 is PLRV goat anti-rabbit antibodies, the bag
It is PLRV rabbit polyclonal antibodies by the Potyviruses rabbit polyclonal antibody on detection line T line 4, the combination is in gold standard pad 2
On Potyviruses colloid gold label thing be PLRV colloid gold label things；
Combination 5：The Potyviruses goat anti-rabbit antibody that is coated in nature controlling line C line 5 is PVS goat anti-rabbit antibodies, the bag
It is PVS rabbit polyclonal antibodies by the Potyviruses rabbit polyclonal antibody on detection line T line 4, the combination is in gold standard pad 2
Potyviruses colloid gold label thing be PVS colloid gold label things；
Combination 6：The Potyviruses goat anti-rabbit antibody that is coated in nature controlling line C line 5 is PVM goat anti-rabbit antibodies, the bag
It is PVM rabbit polyclonal antibodies by the Potyviruses rabbit polyclonal antibody on detection line T line 4, the combination is in gold standard pad (2)
On Potyviruses colloid gold label thing be PVM colloid gold label things；
Combination 7：The Potyviruses goat anti-rabbit antibody that is coated in nature controlling line C line 5 is PVA goat anti-rabbit antibodies, the bag
It is PVA rabbit polyclonal antibodies by the Potyviruses rabbit polyclonal antibody on detection line T line 4, the combination is in gold standard pad 2
Potyviruses colloid gold label thing be PVA colloid gold label things；
Combination 8：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line 5 is tomato black ring virus goat-anti rabbit-anti
Body, the Potyviruses rabbit polyclonal antibody being coated on detection line T line 4 is tomato black ring virus rabbit polyclonal antibody,
Potyviruses colloid gold label thing of the combination in gold standard pad 2 is tomato black ring virus colloid gold label thing.
Embodiment 2
A kind of Potyviruses Rapid detection test strip, as shown in Figure 1 and Figure 2, including sample pad 1, gold standard pad 2, NC films 3, inspection
Survey line T lines 4, nature controlling line C line 5, adsorptive pads 6 and PVC base plates 7, the top of the PVC base plates 7 are fitted with NC films 3, the NC films 3
Both upper ends thereof is overlapped with gold standard pad 2 and adsorptive pads 6 respectively, and 2 top of the gold standard pad is overlapped with sample pad 1, the detection line T line
4 are positioned close on 3 surface of NC films of sample pad 1, and the nature controlling line C line 5 is positioned close on the NC films 3 of adsorptive pads 6, described
Potyviruses rabbit polyclonal antibody, the Potyviruses rabbit being coated on detection line T line 4 is coated with detection line T line 4
It is 1 L/cm that Anti-TNF-α liquid solution draws film concentration, described for carrying out the Potyviruses rabbit polyclonal antibody solution for drawing film
Concentration is 1mg/mL；It is coated with Potyviruses goat anti-rabbit antibody in the nature controlling line C line 5, described is coated on nature controlling line C line 5
On Potyviruses goat anti-rabbit antibody solution draw film concentration be 1 L/cm, described for carry out draw film Potyviruses goat-anti
The concentration of rabbit-anti liquid solution is 1mg/mL；Potyviruses colloid gold label thing, the combination is combined with described gold standard pad 2
Potyviruses colloid gold label thing solution spraying concentration in gold standard pad 2 is 5 L/cm.
In the present embodiment, as shown in figure 3, be additionally provided with outside Potyviruses Rapid detection test strip get stuck 8, described get stuck
8 upper faces are provided with well 9 with 1 corresponding position of sample pad, and 8 upper faces that get stuck are corresponding with the NC films 3
Position is provided with observation groove 10.
In the present embodiment, the length of NC films 3 and the overlap joint of gold standard pad 2 is 1.5mm, and the NC films 3 are taken with adsorptive pads 6
The length for connecing is 2.5mm, and the gold standard pad 2 is 2.5mm with the length of the overlap joint of sample pad 1.
In the present embodiment, the Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line 5, detection line T is coated on
Potyviruses rabbit polyclonal antibody and the Potyviruses colloid gold label thing being coated in gold standard pad 2 on line 4, Ke Yishi
Any one in embodiment 1 in 8 kinds of combinations.
Embodiment 3
A kind of Potyviruses Rapid detection test strip, as shown in Figure 1 and Figure 2, including sample pad 1, gold standard pad 2, NC films 3, inspection
Survey line T lines 4, nature controlling line C line 5, adsorptive pads 6 and PVC base plates 7, the top of the PVC base plates 7 are fitted with NC films 3, the NC films 3
Both upper ends thereof is overlapped with gold standard pad 2 and adsorptive pads 6 respectively, and 2 top of the gold standard pad is overlapped with sample pad 1, the detection line T line
4 are positioned close on 3 surface of NC films of sample pad 1, and the nature controlling line C line 5 is positioned close on the NC films 3 of adsorptive pads 6, described
Potyviruses rabbit polyclonal antibody, the Potyviruses rabbit being coated on detection line T line 4 is coated with detection line T line 4
It is 1.5 L/cm that Anti-TNF-α liquid solution draws film concentration, the Potyviruses rabbit polyclonal antibody solution for carrying out stroke film
Concentration be 1.5mg/mL；It is coated with Potyviruses goat anti-rabbit antibody in the nature controlling line C line 5, described is coated on nature controlling line C
It is 1.5 L/cm that Potyviruses goat anti-rabbit antibody solution on line 5 draws film concentration, the potato disease for carrying out stroke film
The concentration of malicious goat anti-rabbit antibody solution is 1.5mg/mL；Potyviruses colloid gold label thing is combined with described gold standard pad 2,
Potyviruses colloid gold label thing solution spraying concentration of the combination in gold standard pad 2 is 6 L/cm.
In the present embodiment, as shown in figure 3, be additionally provided with outside Potyviruses Rapid detection test strip get stuck 8, described get stuck
8 upper faces are provided with well 9 with 1 corresponding position of sample pad, and 8 upper faces that get stuck are corresponding with the NC films 3
Position is provided with observation groove 10.
In the present embodiment, the length of NC films 3 and the overlap joint of gold standard pad 2 is 2mm, and the NC films 3 are overlapped with adsorptive pads 6
Length be 3mm, the gold standard pad 2 is 3mm with the length of the overlap joint of sample pad 1.
In the present embodiment, the Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line 5, detection line T is coated on
Potyviruses rabbit polyclonal antibody and the Potyviruses colloid gold label thing being coated in gold standard pad 2 on line 4, Ke Yishi
Any one in embodiment 1 in 8 kinds of combinations.
Embodiment 4
A kind of preparation method of Potyviruses Rapid detection test strip, comprises the steps：
Step 1：The preparation of gold standard pad 2：
1) preparation of PVX colloid gold labels thing：
With colloidal gold solution labelling PVX rabbit polyclonal antibodies, the concentration of PVX rabbit polyclonal antibodies is 0.5-1.5mg/mL；
Redissolve formula of liquid：PH8.0-8.8,8-12% sucrose, 3-6% trehaloses, 0.5-2% bovine serum albumin, solvent are 0.1-
The Tris-Hcl solution of 0.2mol/L；
The preparation of colloidal gold solution：297-300 mL ultra-pure waters are taken in round-bottomed flask, it is 1-2% chlorine gold to add 2-4 mL concentration
Acid solution, mixes；Round-bottomed flask is placed in thermostat, clean stirrer, stirring at low speed ebuillition of heated is added；Disposably add
Enter 2-4mL, 1-2% citric acid three sodium solution, continue to boil；Moment observation solution colour change, by light yellow → black → purple
→ aubergine, when claret is changed into, after continuing to boil 4-6min, stops heating, is cooled to room temperature；Ultra-pure water does blank right
According to scanning absorption value of the colloidal gold solution between 500-550nm, if absorption peak occurs in 515-525nm, is prepared
Colloidal gold solution qualified；The gold colloidal outward appearance for preparing should pure, bright, without precipitation and floating thing, there is grease in liquid level
Abandon during with a large amount of black particle shape precipitate impurity；The color of optimal gold colloidal is red, and shape is circular；
The preparation technology of PVX colloid gold label things：2 centrifuge tubes are taken, 1-2mL colloidal gold solutions are added, 3-5 L are added
The K of 0.1mol/L2CO3, 4-6 LPVX rabbit polyclonal antibodies are added, 25-35min is stood, 80-120 l closings are added
Agent, stands 25-35min, under the conditions of 8000-10000rpm, 4 DEG C is centrifuged 25-35min, abandons supernatant, adds 80-120 L multiple
Solution suspension is precipitated, and is centrifuged 10-20min under the conditions of 400-600rpm, 4 DEG C, and Aspirate supernatant is saved backup；
2) combination of gold standard pad 2 and PVX colloid gold label things：From glass fibre membrane as the backing material of gold standard pad, will walk
The rapid PVX colloid gold label things for 1) preparing, draw film instrument with three-dimensional large platform metal spraying and are sprayed on glass fibre membrane, in glass fibre
The spraying concentration for spraying colloid gold label thing on film is 4-6 L/cm, and the glass fibre membrane for having sprayed is placed in 35-40 DEG C of drying
In case, 2-4h, took out, was put in aluminium foil bag drying time, and 180-230 DEG C of heat sealing is saved backup；
Step 2：The preparation of NC films 3：
Detection line T line and the preparation of nature controlling line C line：The how grand antibody of PVX rabbits to be coated is diluted to 0.5 ~ 1.5mg/mL with buffer
Concentration, draws film gold spraying instrument with three-dimensional large platform and how grand for PVX rabbits antibody is drawn film on NC films at gold standard pad 1-2cm, with 0.5
The concentration of ~ 1.5 L/cm draws film on NC films, constitutes detection line T line；PVX goat anti-rabbit antibodies to be coated are diluted extremely with buffer
0.5 ~ 1.5mg/mL concentration, draws film gold spraying instrument with three-dimensional large platform and PVX goat anti-rabbit antibodies is drawn film at adsorptive pads 2-3cm
On NC films, film is drawn on NC films with the film concentration of drawing of 0.5 ~ 1.5 L/cm, nature controlling line C line is constituted；Detection line T line and nature controlling line C
Linear distance 4-6mm；To draw under the conditions of 3) the NC films that have detection line T line and nature controlling line C line are placed in 35-40 DEG C and dry, encapsulate standby；
Buffer：The effect of buffer is to provide certain pH and ionic strength and makes PVX rabbit polyclonal antibodies and PVX goat anti-rabbit antibodies
Firmly it is coated on NC films, the pH value of buffer is 7.0 ~ 7.4；
Step 3：The preparation of sample pad 1：
From glass fibre membrane as the material for preparing sample pad 1, glass fibre membrane can be used after process；
The process step of glass fibre membrane：Glove are worn, glass fibre membrane is put in treatment fluid, is completely soaked, soak 5-
15min, takes out dehydration 3-5min afterwards, and horizontal is put into 35-40 DEG C of drying baker on screen cloth, dries left in 20-30 to moisture constant
The right side, by drying after glass fibre membrane load aluminium foil bag heat-sealing preserve；
The formula for the treatment of fluid：PH7.8-8.0 phosphate buffers, 0.5-1.5%S9 surfactant solutions, 0.4-0.5% polysorbas20s,
0.5-1.5% bovine serum albumin, 0.1-0.3% polyvinylpyrrolidonesolution solution, solvent are water；
Step 4：The assembling of test strips
Material is completed made for step 1 ~ 3, the specification according to PVC base plates 7 cuts into certain length and width, pastes
On PVC base plates 7 and installed in getting stuck 8 li；
The number of assembling steps of test strips：
1）NC films 3 are pasted onto on PVC base plates 7；
2）The top that gold standard pad 2 is overlapped on 3 one end of NC films, the lap of splice are 1-2mm；
3）The top that adsorptive pads 6 are overlapped on 3 other end of NC films, the lap of splice are 2-3mm；
4）The top that sample pad 1 is overlapped on gold standard pad 2, the lap of splice are 2-3mm；
5）The PVC base plates 7 for pasting are cut into bar in cutting machine；
6）Shell is filled after cutting into bar and loads aluminium foil bag in the lump with desiccant, labelled after sealing.
In the present embodiment, the preparation method of the PVX rabbit polyclonal antibodies and PVX goat anti-rabbit antibodies is specially：
The preparation of PVX polyclonal antibodies：1-2mLPVX rabbit anti-serums are taken, add 1-2mL phosphate buffers to vibrate in an oscillator,
2-4mL saturated ammonium sulphate PVX antibody is added after mixing；Then in 8000-10000rpm, 10-15min, 4 DEG C or so condition
Lower centrifugation, is precipitated PVX antibody, then dissolves PVX antibody with phosphate buffer, be then removed by filtration by PD-10coLum fibre columns
Unnecessary ammonium sulfate, obtains final product PVX rabbit polyclonal antibodies.
The preparation of PVX goat anti-rabbit antibodies：1-2mLPVX goat-anti rabbit anti-serums are taken, and 1-2mL phosphate buffers are added in vibration
Vibrate in device, after mixing, add 2-4mL saturated ammonium sulphate PVX antibody；Then in 8000-10000rpm, 10-15min, 4
DEG C or so under the conditions of be centrifuged, precipitate PVX antibody, then with phosphate buffer dissolve PVX antibody, then by PD-10coLum fibre
Dimension post is removed by filtration unnecessary ammonium sulfate, obtains final product PVX goat anti-rabbit antibodies.
The preparation method of PVX rabbit anti-serums：2mLPVX is taken, by subcutaneous 4 points injection health male rabbits, is noted week about
Penetrate once, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain the antiserum of PVX.
The preparation method of PVX goat-anti rabbit anti-serums：2mLPVX rabbit anteserum antibody is taken, by subcutaneous 4 points injection health males
Goat, injects once week about, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain PVX
Goat-anti rabbit anti-serum.
Embodiment 5
A kind of preparation method of Potyviruses Rapid detection test strip, comprises the steps：
Step 1：The preparation of gold standard pad 2：
1) PVYoThe preparation of colloid gold label thing：
With colloidal gold solution labelling PVYoRabbit polyclonal antibody, PVYoThe concentration of rabbit polyclonal antibody is 0.5-1.5mg/mL；
The preparation of formula of liquid and colloidal gold solution is redissolved with embodiment 4；
PVYoThe preparation technology of colloid gold label thing：2 centrifuge tubes are taken, 1-2mL colloidal gold solutions are added, 3-5 L are added
The K of 0.1mol/L2CO3, add 4-6 LPVYoRabbit polyclonal antibody, stands 25-35min, adds 80-120 L closings
Agent, stands 25-35min, under the conditions of 8000-10000rpm, 4 DEG C is centrifuged 25-35min, abandons supernatant, adds 80-120 L multiple
Solution suspension is precipitated, and is centrifuged 10-20min under the conditions of 400-600rpm, 4 DEG C, and Aspirate supernatant is saved backup；
2) gold standard pad 2 and PVYoThe combination of colloid gold label thing：From glass fibre membrane as the backing material of gold standard pad, incite somebody to action
Step 1) PVY for preparingoColloid gold label thing, draws film instrument with three-dimensional large platform metal spraying and is sprayed on glass fibre membrane, in glass
PVY is sprayed on fibrous membraneoThe optimal spraying concentration of colloid gold label thing is 4-6 L/cm, and the glass fibre membrane for having sprayed is placed in
In 35-40 DEG C of drying baker, 2-4h, took out, was put in aluminium foil bag drying time, and 180-230 DEG C of heat sealing is saved backup；
Step 2：The preparation of NC films 3：
Detection line T line and the preparation of nature controlling line C line：PVY to be coated is diluted with bufferoThe how grand antibody of rabbit is to 0.5 ~ 1.5mg/mL
Concentration, draws film gold spraying instrument by PVY with three-dimensional large platformoThe how grand antibody of rabbit draws film on NC films at gold standard pad 1-2cm, with
The concentration of 0.5 ~ 1.5 L/cm draws film on NC films, constitutes detection line T line；PVY to be coated is diluted with bufferoGoat anti-rabbit antibody
To 0.5 ~ 1.5mg/mL concentration, film gold spraying instrument is drawn by PVY with three-dimensional large platformoGoat anti-rabbit antibody draws film to apart from adsorptive pads 2-
At 3cm on NC films, film is drawn on NC films with the film concentration of drawing of 0.5 ~ 1.5 L/cm, nature controlling line C line is constituted；Detection line T line and matter
Control line C linear distance 4-6mm；To draw under the conditions of the NC films 3 that have detection line T line and nature controlling line C line are placed in 35-40 DEG C and dry, encapsulation
Standby；
Step 3：The preparation of sample pad 1：With embodiment 4
Step 4：The assembling of test strips：With embodiment 4
In the present embodiment, the PVYoRabbit polyclonal antibody and PVYoThe preparation method of goat anti-rabbit antibody is specially：
PVYoThe preparation of polyclonal antibody：Take 1-2mLPVYoRabbit anti-serum, adds 1-2mL phosphate buffers to shake in an oscillator
Swing, after mixing, add 2-4mL saturated ammonium sulphate PVYoAntibody；Then at 8000-10000rpm, 10-15min, 4 DEG C or so
Under the conditions of be centrifuged, precipitate PVYoAntibody, then PVY is dissolved with phosphate bufferoAntibody, then passes through PD-10coLum fibre columns mistakes
Unnecessary ammonium sulfate is filtered out, PVY is obtained final productoRabbit polyclonal antibody；
PVYoThe preparation of goat anti-rabbit antibody：Take 1-2mLPVYoGoat-anti rabbit anti-serum, adds 1-2mL phosphate buffers in agitator
Middle vibration, adds 2-4mL saturated ammonium sulphate PVY after mixingoAntibody；Then in 8000-10000rpm, 10-15min, 4 DEG C
It is centrifuged under the conditions of left and right, precipitates PVYoAntibody, then dissolves PVY with phosphate bufferoAntibody, then fine by PD-10coLum
Dimension post is removed by filtration unnecessary ammonium sulfate, obtains final product PVYoGoat anti-rabbit antibody；
PVYoThe preparation method of rabbit anti-serum：Take 2mLPVYo, by subcutaneous 4 points injection health male rabbits, inject week about
Once, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain PVYoAntiserum；
PVYoThe preparation method of goat-anti rabbit anti-serum：Take 2mLPVYoRabbit anteserum antibody, by subcutaneous 4 points injection health male mountains
Sheep, injects once week about, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain PVYoSheep
Anti-rabbit antiserum.
Embodiment 6
A kind of preparation method of Potyviruses Rapid detection test strip, comprises the steps：
Step 1：The preparation of gold standard pad 2：
1)PVYNTNThe preparation of colloid gold label thing：
With colloidal gold solution labelling PVYNTNRabbit polyclonal antibody, PVYNTNThe concentration of rabbit polyclonal antibody is 0.5-1.5mg/mL；
The preparation of formula of liquid and colloidal gold solution is redissolved with embodiment 4；
PVYNTNThe preparation technology of colloid gold label thing：2 centrifuge tubes are taken, 1-2mL colloidal gold solutions are added, 3-5 L are added
The K of 0.1mol/L2CO3, add 4-6 L PVYNTNRabbit polyclonal antibody, 25-35min add 80-120 L sealers,
25-35min is stood, under the conditions of 8000-10000rpm, 4 DEG C, is centrifuged 25-35min, abandon supernatant, add 80-120 L to redissolve liquid
Suspend precipitation, is centrifuged 10-20min under the conditions of 400-600rpm, 4 DEG C, and Aspirate supernatant is saved backup；
2) gold standard pad 2 and PVYNTNThe combination of colloid gold label thing：From glass fibre membrane as the backing material of gold standard pad, incite somebody to action
Step 1) PVY for preparingNTNColloid gold label thing, draws film instrument with three-dimensional large platform metal spraying and is sprayed on glass fibre membrane, in glass
PVY is sprayed on fibrous membraneNTNThe optimal spraying concentration of colloid gold label thing is 4-6 L/cm, and the glass fibre membrane for having sprayed is placed in
In 35-40 DEG C of drying baker, 2-4h, took out, was put in aluminium foil bag drying time, and 180-230 DEG C of heat sealing is saved backup；
Step 2：The preparation of NC films 3：
Detection line T line and the preparation of nature controlling line C line：PVY to be coated is diluted with bufferNTNThe how grand antibody of rabbit is to 0.5 ~ 1.5mg/
ML concentration, draws film gold spraying instrument by PVY with three-dimensional large platformNTNThe how grand antibody of rabbit draws film on NC films at gold standard pad 1-2cm,
Film is drawn on NC films with the concentration of 0.5 ~ 1.5 L/cm, detection line T line is constituted；PVY to be coated is diluted with bufferNTNGoat-anti rabbit
Antibody draws film gold spraying instrument by PVY to 0.5 ~ 1.5mg/mL concentration with three-dimensional large platformNTNGoat anti-rabbit antibody draws film to distance water suction
At pad 2-3cm on NC films, film is drawn on NC films with the film concentration of drawing of 0.5 ~ 1.5 L/cm, nature controlling line C line is constituted；Detection line T line
With nature controlling line C line apart from 4-6mm；To draw under the conditions of the NC films 3 that have detection line T line and nature controlling line C line are placed in 35-40 DEG C and dry,
Encapsulation is standby；
Step 3：The preparation of sample pad 1：With embodiment 4
Step 4：The assembling of test strips：With embodiment 4
In the present embodiment, the PVYNTNRabbit polyclonal antibody and PVYNTNThe preparation method of goat anti-rabbit antibody is specially：
PVYNTNThe preparation of polyclonal antibody：Take 1-2mL PVYNTNRabbit anti-serum, adds 1-2mL phosphate buffers in an oscillator
Vibration, adds 2-4mL saturated ammonium sulphate PVY after mixingNTNAntibody；Then in 8000-10000rpm, 10-15min, 4 DEG C
It is centrifuged under the conditions of left and right, precipitates PVYNTNAntibody, then PVY is dissolved with phosphate bufferNTNAntibody, then fine by PD-10coLum
Dimension post is removed by filtration unnecessary ammonium sulfate, obtains final product PVYNTNRabbit polyclonal antibody；
PVYNTNThe preparation of goat anti-rabbit antibody：Take 1-2mL PVYNTNGoat-anti rabbit anti-serum, adds 1-2mL phosphate buffers in vibration
Vibrate in device, after mixing, add 2-4mL saturated ammonium sulphate PVYNTNAntibody；Then in 8000-10000rpm, 10-15min,
It is centrifuged under the conditions of 4 DEG C or so, precipitates PVYNTNAntibody, then dissolves PVY with phosphate bufferNTNAntibody, then passes through PD-
10coLum fibre columns are removed by filtration unnecessary ammonium sulfate, obtain final product PVYNTNGoat anti-rabbit antibody；
PVYNTNThe preparation method of rabbit anti-serum：Take 2mLPVYNTN, by subcutaneous 4 points injection health male rabbits, note week about
Penetrate once, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain PVYNTNAntiserum；
PVYNTNThe preparation method of goat-anti rabbit anti-serum：Take 2mLPVYNTNRabbit anteserum antibody, by subcutaneous 4 points injection health males
Goat, injects once week about, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtained
PVYNTNGoat-anti rabbit anti-serum.
Embodiment 7
A kind of preparation method of Potyviruses Rapid detection test strip, comprises the steps：
Step 1：The preparation of gold standard pad 2：
1) preparation of PLRV colloid gold labels thing：
With colloidal gold solution labelling PLRV rabbit polyclonal antibodies, the concentration of PLRV rabbit polyclonal antibodies is 0.5-1.5mg/mL；
The preparation of formula of liquid and colloidal gold solution is redissolved with embodiment 4；
The preparation technology of PLRV colloid gold label things：2 centrifuge tubes are taken, 1-2mL colloidal gold solutions are added, 3-5 L are added
The K of 0.1mol/L2CO3, 4-6 L PLRV rabbit polyclonal antibodies are added, 25-35min is stood, 80-120 L closings are added
Agent, stands 25-35min, under the conditions of 8000-10000rpm, 4 DEG C is centrifuged 25-35min, abandons supernatant, adds 80-120 L multiple
Solution suspension is precipitated, and is centrifuged 10-20min under the conditions of 400-600rpm, 4 DEG C, and Aspirate supernatant is saved backup；
2) combination of gold standard pad 2 and PLRV colloid gold label things：From glass fibre membrane as the backing material of gold standard pad, incite somebody to action
Step 1) the PLRV colloid gold label things that prepare, film instrument is drawn with three-dimensional large platform metal spraying and be sprayed on glass fibre membrane, in glass
The optimal spraying concentration for spraying PLRV colloid gold label things on fibrous membrane is 4-6 L/cm, and the glass fibre membrane for having sprayed is placed in
In 35-40 DEG C of drying baker, 2-4h, took out, was put in aluminium foil bag drying time, and 180-230 DEG C of heat sealing is saved backup；
Step 2：The preparation of NC films 3：
Detection line T line and the preparation of nature controlling line C line：The how grand antibody of PLRV rabbits to be coated is diluted to 0.5 ~ 1.5mg/mL with buffer
Concentration, with three-dimensional large platform draw film gold spraying instrument by how grand for PLRV rabbits antibody draw film on NC films at gold standard pad 1-2cm, with
The concentration of 0.5 ~ 1.5 L/cm draws film on NC films, constitutes detection line T line；PLRV goat anti-rabbit antibodies to be coated are diluted with buffer
To 0.5 ~ 1.5mg/mL concentration, film gold spraying instrument is drawn with three-dimensional large platform and PLRV goat anti-rabbit antibodies are drawn film to apart from adsorptive pads 2-
At 3cm on NC films, film is drawn on NC films with the film concentration of drawing of 0.5 ~ 1.5 L/cm, nature controlling line C line is constituted；Detection line T line and matter
Control line C linear distance 4-6mm；To draw under the conditions of the NC films 3 that have detection line T line and nature controlling line C line are placed in 35-40 DEG C and dry, encapsulation
Standby；
Step 3：The preparation of sample pad 1：With embodiment 4
Step 4：The assembling of test strips：With embodiment 4
In the present embodiment, the preparation method of the PLRV rabbit polyclonal antibodies and PLRV goat anti-rabbit antibodies is specially：
The preparation of PLRV polyclonal antibodies：1-2mL PLRV rabbit anti-serums are taken, adds 1-2mL phosphate buffers to shake in an oscillator
Swing, after mixing, add 2-4mL saturated ammonium sulphate PLRV antibody；Then at 8000-10000rpm, 10-15min, 4 DEG C or so
Under the conditions of be centrifuged, precipitate PLRV antibody, then with phosphate buffer dissolve PLRV antibody, then pass through PD-10coLum fibre columns mistakes
Unnecessary ammonium sulfate is filtered out, PLRV rabbit polyclonal antibodies are obtained final product；
The preparation of PLRV goat anti-rabbit antibodies：1-2mL PLRV goat-anti rabbit anti-serums are taken, and 1-2mL phosphate buffers are added in agitator
Middle vibration, adds 2-4mL saturated ammonium sulphate PLRV antibody after mixing；Then in 8000-10000rpm, 10-15min, 4 DEG C
Be centrifuged under the conditions of left and right, precipitate PLRV antibody, then PLRV antibody is dissolved with phosphate buffer, then fine by PD-10coLum
Dimension post is removed by filtration unnecessary ammonium sulfate, obtains final product PLRV goat anti-rabbit antibodies；
The preparation method of PLRV rabbit anti-serums：2mLPLRV is taken, by subcutaneous 4 points injection health male rabbits, is injected week about
Once, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain the antiserum of PLRV；
The preparation method of PLRV goat-anti rabbit anti-serums：2mLPLRV rabbit anteserum antibody is taken, by subcutaneous 4 points injection health male mountains
Sheep, injects once week about, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain PLRV sheep
Anti-rabbit antiserum.
Embodiment 8
A kind of preparation method of Potyviruses Rapid detection test strip, comprises the steps：
Step 1：The preparation of gold standard pad 2：
1) preparation of PVS colloid gold labels thing：
With colloidal gold solution labelling PVS rabbit polyclonal antibodies, the concentration of PVS rabbit polyclonal antibodies is 0.5-1.5mg/mL；
The preparation of formula of liquid and colloidal gold solution is redissolved with embodiment 4；
The preparation technology of PVS colloid gold label things：2 centrifuge tubes are taken, 1-2mL colloidal gold solutions are added, 3-5 l are added
The K of 0.1mol/L2CO3, 4-6 L PVS rabbit polyclonal antibodies are added, 25-35min is put, 80-120 L sealers are added,
25-35min is stood, under the conditions of 8000-10000rpm, 4 DEG C, is centrifuged 25-35min, abandon supernatant, add 80-120 L to redissolve liquid
Suspend precipitation, is centrifuged 10-20min under the conditions of 400-600rpm, 4 DEG C, and Aspirate supernatant is saved backup；
2) combination of gold standard pad 2 and PVS colloid gold label things：From glass fibre membrane as the backing material of gold standard pad, will walk
The rapid PVS colloid gold label things for 1) preparing, draw film instrument with three-dimensional large platform metal spraying and are sprayed on glass fibre membrane, in glass fibre
The optimal spraying concentration for spraying PVS colloid gold label things on film is 4-6 L/cm, and the glass fibre membrane for having sprayed is placed in 35-40
In DEG C drying baker, 2-4h, took out, was put in aluminium foil bag drying time, and 180-230 DEG C of heat sealing is saved backup；
Step 2：The preparation of NC films 3：
Detection line T line and the preparation of nature controlling line C line：The how grand antibody of PVS rabbits to be coated is diluted to 0.5 ~ 1.5mg/mL with buffer
Concentration, draws film gold spraying instrument with three-dimensional large platform and how grand for PVS rabbits antibody is drawn film on NC films at gold standard pad 1-2cm, with 0.5
The concentration of ~ 1.5 L/cm draws film on NC films, constitutes detection line T line；PVS goat anti-rabbit antibodies to be coated are diluted extremely with buffer
0.5 ~ 1.5mg/mL concentration, draws film gold spraying instrument with three-dimensional large platform and PVS goat anti-rabbit antibodies is drawn film at adsorptive pads 2-3cm
On NC films, film is drawn on NC films with the film concentration of drawing of 0.5 ~ 1.5 L/cm, nature controlling line C line is constituted；Detection line T line and nature controlling line C
Linear distance 4-6mm；To draw under the conditions of the NC films 3 that have detection line T line and nature controlling line C line are placed in 35-40 DEG C and dry, encapsulate standby；
Step 3：The preparation of sample pad 1：With embodiment 4
Step 4：The assembling of test strips：With embodiment 4
In the present embodiment, the preparation method of the PVS rabbit polyclonal antibodies and PVS goat anti-rabbit antibodies is specially：
The preparation of PVS polyclonal antibodies：1-2mL PVS rabbit anti-serums are taken, adds 1-2mL phosphate buffers to shake in an oscillator
Swing, after mixing, add 2-4mL saturated ammonium sulphate PVS antibody；Then at 8000-10000rpm, 10-15min, 4 DEG C or so
Under the conditions of be centrifuged, precipitate PVS antibody, then with phosphate buffer dissolve PVS antibody, then filtered by PD-10coLum fibre columns
Unnecessary ammonium sulfate is removed, PVS rabbit polyclonal antibodies are obtained final product；
The preparation of PVS goat anti-rabbit antibodies：1-2mL PVS goat-anti rabbit anti-serums are taken, 1-2mL phosphate buffers is added in an oscillator
Vibration, adds 2-4mL saturated ammonium sulphate PVS antibody after mixing；Then on 8000-10000rpm, 10-15min, a 4 DEG C of left side
Be centrifuged under the conditions of the right side, precipitate PVS antibody, then PVS antibody is dissolved with phosphate buffer, then pass through PD-10coLum fibre columns
Unnecessary ammonium sulfate is removed by filtration, PVS goat anti-rabbit antibodies are obtained final product；
The preparation method of PVS rabbit anti-serums：2mLPVS is taken, by subcutaneous 4 points injection health male rabbits, one is injected week about
Secondary, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain the antiserum of PVS；
The preparation method of PVS goat-anti rabbit anti-serums：2mLPVS rabbit anteserum antibody is taken, by subcutaneous 4 points injection health male goats,
Inject week about once, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain PVS goat-anti rabbits
Antiserum.
Embodiment 9
A kind of preparation method of Potyviruses Rapid detection test strip, comprises the steps：
Step 1：The preparation of gold standard pad 2：
1) preparation of PVM colloid gold labels thing：
With colloidal gold solution labelling PVM rabbit polyclonal antibodies, the concentration of PVM rabbit polyclonal antibodies is 0.5-1.5mg/mL；
The preparation of formula of liquid and colloidal gold solution is redissolved with embodiment 4；
The preparation technology of PVM colloid gold label things：2 centrifuge tubes are taken, 1-2mL colloidal gold solutions are added, 3-5 L are added
The K of 0.1mol/L2CO3, 4-6 LPVM rabbit polyclonal antibodies are added, 25-35min is put, 80-120 L sealers are added,
25-35min is stood, under the conditions of 8000-10000rpm, 4 DEG C, is centrifuged 25-35min, abandon supernatant, add 80-120 L to redissolve liquid
Suspend precipitation, is centrifuged 10-20min under the conditions of 400-600rpm, 4 DEG C, and Aspirate supernatant is saved backup；
2) combination of gold standard pad 2 and PVM colloid gold label things：From glass fibre membrane as the backing material of gold standard pad, will walk
The rapid PVM colloid gold label things for 1) preparing, draw film instrument with three-dimensional large platform metal spraying and are sprayed on glass fibre membrane, in glass fibre
The optimal spraying concentration for spraying PVM colloid gold label things on film is 4-6 L/cm, and the glass fibre membrane for having sprayed is placed in 35-40
In DEG C drying baker, 2-4h, took out, was put in aluminium foil bag drying time, and 180-230 DEG C of heat sealing is saved backup；
Step 2：The preparation of NC films 3：
Detection line T line and the preparation of nature controlling line C line：The how grand antibody of PVM rabbits to be coated is diluted to 0.5 ~ 1.5mg/mL with buffer
Concentration, draws film gold spraying instrument with three-dimensional large platform and how grand for PVM rabbits antibody is drawn film on NC films at gold standard pad 1-2cm, with 0.5
The concentration of ~ 1.5 L/cm draws film on NC films, constitutes detection line T line；PVM goat anti-rabbit antibodies to be coated are diluted extremely with buffer
0.5 ~ 1.5mg/mL concentration, draws film gold spraying instrument with three-dimensional large platform and PVM goat anti-rabbit antibodies is drawn film at adsorptive pads 2-3cm
On NC films, film is drawn on NC films with the film concentration of drawing of 0.5 ~ 1.5 L/cm, nature controlling line C line is constituted；Detection line T line and nature controlling line C
Linear distance 4-6mm；To draw under the conditions of the NC films 3 that have detection line T line and nature controlling line C line are placed in 35-40 DEG C and dry, encapsulate standby；
Step 3：The preparation of sample pad 1：With embodiment 4
Step 4：The assembling of test strips：With embodiment 4
In the present embodiment, the preparation method of the PVM rabbit polyclonal antibodies and PVM goat anti-rabbit antibodies is specially：
The preparation of PVM polyclonal antibodies：1-2mL PVM rabbit anti-serums are taken, adds 1-2mL phosphate buffers to shake in an oscillator
Swing, after mixing, add 2-4mL saturated ammonium sulphate PVM antibody；Then at 8000-10000rpm, 10-15min, 4 DEG C or so
Under the conditions of be centrifuged, precipitate PVM antibody, then with phosphate buffer dissolve PVM antibody, then filtered by PD-10coLum fibre columns
Unnecessary ammonium sulfate is removed, PVM rabbit polyclonal antibodies are obtained final product；
The preparation of PVM goat anti-rabbit antibodies：1-2mL PVM goat-anti rabbit anti-serums are taken, 1-2mL phosphate buffers is added in an oscillator
Vibration, adds 2-4mL saturated ammonium sulphate PVM antibody after mixing；Then on 8000-10000rpm, 10-15min, a 4 DEG C of left side
Be centrifuged under the conditions of the right side, precipitate PVM antibody, then PVM antibody is dissolved with phosphate buffer, then pass through PD-10coLum fibre columns
Unnecessary ammonium sulfate is removed by filtration, PVM goat anti-rabbit antibodies are obtained final product；
The preparation method of PVM rabbit anti-serums：2mLPVM is taken, by subcutaneous 4 points injection health male rabbits, one is injected week about
Secondary, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain the antiserum of PVM；
The preparation method of PVM goat-anti rabbit anti-serums：2mLPVM rabbit anteserum antibody is taken, by subcutaneous 4 points injection health male goats,
Inject week about once, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain PVM goat-anti rabbits
Antiserum.
Embodiment 10
A kind of preparation method of Potyviruses Rapid detection test strip, comprises the steps：
Step 1：The preparation of gold standard pad 2：
1) preparation of PVA colloid gold labels thing：
With colloidal gold solution labelling PVA rabbit polyclonal antibodies, the concentration of PVA rabbit polyclonal antibodies is 0.5-1.5mg/mL；
The preparation of formula of liquid and colloidal gold solution is redissolved with embodiment 4；
The preparation technology of PVA colloid gold label things：2 centrifuge tubes are taken, 1-2mL colloidal gold solutions are added, 3-5 L are added
The K of 0.1mol/L2CO3, 4-6 L PVA rabbit polyclonal antibodies are added, 25-35min is put, 80-120 L sealers are added,
25-35min is stood, under the conditions of 8000-10000rpm, 4 DEG C, is centrifuged 25-35min, abandon supernatant, add 80-120 L to redissolve liquid
Suspend precipitation, is centrifuged 10-20min under the conditions of 400-600rpm, 4 DEG C, and Aspirate supernatant is saved backup；
2) combination of gold standard pad 2 and PVA colloid gold label things：From glass fibre membrane as the backing material of gold standard pad, will walk
The rapid PVA colloid gold label things for 1) preparing, draw film instrument with three-dimensional large platform metal spraying and are sprayed on glass fibre membrane, in glass fibre
The optimal spraying concentration for spraying PVA colloid gold label things on film is 4-6 L/cm, and the glass fibre membrane for having sprayed is placed in 35-40
In DEG C drying baker, 2-4h, took out, was put in aluminium foil bag drying time, and 180-230 DEG C of heat sealing is saved backup；
Step 2：The preparation of NC films 3：
Detection line T line and the preparation of nature controlling line C line：The how grand antibody of PVA rabbits to be coated is diluted to 0.5 ~ 1.5mg/mL with buffer
Concentration, draws film gold spraying instrument with three-dimensional large platform and how grand for PVA rabbits antibody is drawn film on NC films at gold standard pad 1-2cm, with 0.5
The concentration of ~ 1.5 L/cm draws film on NC films, constitutes detection line T line；PVA goat anti-rabbit antibodies to be coated are diluted extremely with buffer
0.5 ~ 1.5mg/mL concentration, draws film gold spraying instrument with three-dimensional large platform and PVA goat anti-rabbit antibodies is drawn film at adsorptive pads 2-3cm
On NC films, film is drawn on NC films with the film concentration of drawing of 0.5 ~ 1.5 L/cm, nature controlling line C line is constituted；Detection line T line and nature controlling line C
Linear distance 4-6mm；To draw under the conditions of the NC films 3 that have detection line T line and nature controlling line C line are placed in 35-40 DEG C and dry, encapsulate standby；
Step 3：The preparation of sample pad 1：With embodiment 4
Step 4：The assembling of test strips：With embodiment 4
In the present embodiment, the PVA rabbit polyclonal antibodies and the anti-sheep rabbit-anti preparations of PVA are specially：
The preparation of PVA polyclonal antibodies：1-2mL PVA rabbit anti-serums are taken, adds 1-2mL phosphate buffers to shake in an oscillator
Swing, after mixing, add 2-4mL saturated ammonium sulphate PVA antibody；Then at 8000-10000rpm, 10-15min, 4 DEG C or so
Under the conditions of be centrifuged, precipitate PVA antibody, then with phosphate buffer dissolving PVA antibody, then filtered by PD-10coLum fibre columns
Unnecessary ammonium sulfate is removed, PVA rabbit polyclonal antibodies are obtained final product；
The preparation of PVA goat anti-rabbit antibodies：1-2mL PVA goat-anti rabbit anti-serums are taken, 1-2mL phosphate buffers is added in an oscillator
Vibration, adds 2-4mL saturated ammonium sulphate PVA antibody after mixing；Then on 8000-10000rpm, 10-15min, a 4 DEG C of left side
It is centrifuged under the conditions of the right side, precipitates PVA antibody, then use phosphate buffer dissolving PVA antibody, then passes through PD-10coLum fibre columns
Unnecessary ammonium sulfate is removed by filtration, PVA goat anti-rabbit antibodies are obtained final product；
The preparation method of PVA rabbit anti-serums：2mLPVA is taken, by subcutaneous 4 points injection health male rabbits, one is injected week about
Secondary, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain the antiserum of PVA；
The preparation method of PVA goat-anti rabbit anti-serums：2mLPVA rabbit anteserum antibody is taken, by subcutaneous 4 points injection health male goats,
Inject week about once, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain PVA goat-anti rabbits
Antiserum.
Embodiment 11
A kind of preparation method of Potyviruses Rapid detection test strip, comprises the steps：
Step 1：The preparation of gold standard pad 2：
1) preparation of tomato black ring virus colloid gold label thing：
With colloidal gold solution labelling tomato black ring virus rabbit polyclonal antibody, the concentration of tomato black ring virus rabbit polyclonal antibody
For 0.5-1.5mg/mL；
The preparation of formula of liquid and colloidal gold solution is redissolved with embodiment 4；
The preparation technology of tomato black ring virus colloid gold label thing：2 centrifuge tubes are taken, 1-2mL colloidal gold solutions are added, is added
The K of 3-5 L 0.1mol/L2CO3, 4-6 L tomato black ring virus polyclonal antibodies are added, 25-35min are stood, is added
80-120 L sealers, stand 25-35min, be centrifuged 25-35min, abandon supernatant under the conditions of 8000-10000rpm, 4 DEG C, plus
Entering the multiple solution suspension precipitations of 80-120 L, 10-20min being centrifuged under the conditions of 400-600rpm, 4 DEG C, Aspirate supernatant is preserved
Standby；
2) combination of gold standard pad 2 and tomato black ring virus colloid gold label thing：From glass fibre membrane as gold standard pad support
Material, by step 1) the tomato black ring virus colloid gold label thing for preparing, film instrument is drawn with three-dimensional large platform metal spraying be sprayed on glass
On fibrous membrane, the optimal spraying concentration for spraying tomato black ring virus colloid gold label thing on glass fibre membrane is 4-6 L/cm,
The glass fibre membrane for having sprayed is placed in 35-40 DEG C of drying baker, 2-4h, took out, be put in aluminium foil bag, 180-230 drying time
DEG C heat sealing, saves backup；
Step 2：The preparation of NC films 3：
Detection line T line and the preparation of nature controlling line C line：With buffer dilute the how grand antibody of tomato black ring virus rabbit to be coated to 0.5 ~
1.5mg/mL concentration, draws film gold spraying instrument with three-dimensional large platform and how grand for tomato black ring virus rabbit antibody is drawn film to apart from gold standard pad 1-
At 2cm on NC films, film is drawn on NC films with the concentration of 0.5 ~ 1.5 L/cm, detection line T line is constituted；Diluted with buffer and wait to be coated
Tomato black ring virus goat anti-rabbit antibody draws film gold spraying instrument by tomato black ring virus to 0.5 ~ 1.5mg/mL concentration with three-dimensional large platform
Goat anti-rabbit antibody draws film on NC films apart from adsorptive pads 2-3cm at, draws film concentration stroke film on NC films with 0.5 ~ 1.5 L/cm,
Constitute nature controlling line C line；Detection line T line and nature controlling line C line are apart from 4-6mm；The NC films 3 for having detection line T line and nature controlling line C line will be drawn
Dry under the conditions of being placed in 35-40 DEG C, encapsulate standby；
Step 3：The preparation of sample pad 1：With embodiment 4
Step 4：The assembling of test strips：With embodiment 4
In the present embodiment, the tomato black ring virus rabbit polyclonal antibody and tomato black ring virus goat anti-rabbit antibody preparation method
Specially：
The preparation of tomato black ring virus polyclonal antibody：1-2mL tomato black ring virus rabbit anti-serums are taken, adds 1-2mL phosphoric acid to delay
Rush liquid to vibrate in an oscillator, after mixing, add 2-4mL saturated ammonium sulphate tomato black ring virus antibody；Then in 8000-
It is centrifuged under the conditions of 10000rpm, 10-15min, 4 DEG C or so, precipitates tomato black ring virus antibody, then dissolved with phosphate buffer
Tomato black ring virus antibody, is then removed by filtration unnecessary ammonium sulfate by PD-10coLum fibre columns, obtains final product Fructus Lycopersici esculenti black ring
Malicious rabbit polyclonal antibody；
The preparation of tomato black ring virus goat anti-rabbit antibody：1-2mL tomato black ring virus goat-anti rabbit anti-serums are taken, 1-2mL phosphorus is added
Acid buffer vibrates in an oscillator, adds 2-4mL saturated ammonium sulphate tomato black ring virus antibody after mixing；Then exist
It is centrifuged under the conditions of 8000-10000rpm, 10-15min, 4 DEG C or so, precipitates tomato black ring virus antibody, then use phosphoric acid buffer
Liquid dissolves tomato black ring virus antibody, is then removed by filtration unnecessary ammonium sulfate by PD-10coLum fibre columns, obtains final product Fructus Lycopersici esculenti
Black ring virus goat anti-rabbit antibody；
The preparation method of tomato black ring virus rabbit anti-serum：2mL tomato black ring viruses are taken, by subcutaneous 4 points injection health males
Rabbit, injects once week about, total co-injection three times;After third time injection one week, taken a blood sample by ear vein, that is, obtain Fructus Lycopersici esculenti
The antiserum of black ring virus；
The preparation method of tomato black ring virus goat-anti rabbit anti-serum：Take 2mL tomato black ring viruses and exempt from serum antibody, by subcutaneous 4
The healthy male goat of point injection, injects once, total co-injection three times week about;After third time injection one week, by ear vein
Blood sampling, that is, obtain tomato black ring virus goat-anti rabbit anti-serum.
Embodiment 12
A kind of application of Rhizoma Solani tuber osi Rapid detection test strip, concrete detecting step is：
(1) sample treatment：Potato leaf is taken, the ratio by leaf weight g and buffering liquid product V is 1:8-1:10 ratio adds
Enter phosphate buffer, green juice is ground, the PH of phosphate buffer is 7.4-7.8；
(2) the above-mentioned sample for processing is added drop-wise in the sample pad of Rhizoma Solani tuber osi Rapid detection test strip, stands 0.5 ~ 3 point
Clock, observation detection line T line and nature controlling line C line；
(3) result interpretation：As shown in figure 4, detection line T line and nature controlling line C line manifest aubergine for the positive；As shown in figure 5,
Detection line T line does not manifest aubergine, and nature controlling line C line manifests aubergine, is as a result feminine gender；As shown in fig. 6, detection line T line and matter
Control line C lines do not manifest aubergine or other phenomenons, point out test strips failure.
Experiment content
1st, the measure of Potyviruses Rapid detection test strip sensitivity
Detection sample is taken, 10 are diluted respectively1Again, 102Again, 103Again, 104Again, 105Again, 106Again, 107Again, 108Times.Carry out spirit
Sensitivity compares, by Potyviruses concentration be set as 1ng/mL, 10ng/mL, 30ng/mL, 100ng/mL, 300ng/mL,
1000ng/mL, 3000ng/mL, the Cmin of test strips detection Potyviruses is 1ng/mL.Potyviruses include PVX,
PVYNTN、PVYo, PLRV, PVS, PVM, PVA and tomato black ring virus.
2nd, the measure of Potyviruses Rapid detection test strip stability
Test strips are sealed with aluminium foil bag, desiccant is added, be respectively placed in 4 DEG C, 37 DEG C, under room temperature condition, be wherein placed in 4 DEG C and
Room temperature condition test strips took out 3 every 7 days, were placed in 37 DEG C of daily taking-up 3, respectively detection negative sample and positive sample
This.In observation the presence or absence of detection line T line and nature controlling line C line, shade and gold standard pad 2 on Rhizoma Solani tuber osi virus colloidal gold label
Releasing degree.From the point of view of test strips test result, by test strips be placed in be allowed to dry in the sealing aluminium foil bag of drying prescription, room temperature and 37
After long-time is preserved at DEG C, color slightly weakens, and other different temperatures, the test result of different time are illustrated suitable without significant change
Preserve under the conditions of, this test strips has preferable stability.Potyviruses include PVX, PVYNTN、PVYo、PLRV、PVS、
PVM, PVA and tomato black ring virus.
3rd, the specific measure of Potyviruses Rapid detection test strip
The test strip of PVX is passed through and PVX, PVS, PVA, PLRV, PVM, PVYo、PVYNTNThe positive material test of virus
Relatively, only there is substantially purplish red colo(u)r streak, other positive materials only matter in PVX virus-positives material tests line T lines and nature controlling line C line
There is purplish red colo(u)r streak in control C lines, and purplish red colo(u)r streak does not occur in detection line T line；The test strip of Potyviruses, to healthy plant and
There is aubergine in the testing result of buffer control, only C lines, and aubergine do not occur in T lines, are as a result feminine genders, therefore this test strips
There is specificity.
4th, the measure of Potyviruses Rapid detection test strip accuracy
The plant of 96 Rhizoma Solani tuber osis is cooked whether containing the viral detections of X of the test strip of PVX；Double-antibody sandwich is used simultaneously
Enzyme-linked immunosorbent assay method detects that to above-mentioned sample the testing result of two kinds of detection methods is completely the same, and coincidence rate reaches
100%；Use PVY0Test strip do the detection of the whether virus of the O strains containing Y virus to the plant of 100 Rhizoma Solani tuber osis；With
When detect that to above-mentioned sample the testing result of two kinds of detection methods is complete with DASELISA immunoadsorption assay method
Unanimously, coincidence rate is up to 100%；The plant of 100 Rhizoma Solani tuber osis is cooked whether containing the viral inspections of A of the test strip of PVA viruses
Survey；Above-mentioned sample is detected with DASELISA immunoadsorption assay method simultaneously, the detection knot of two kinds of detection methods
Really completely the same, coincidence rate is up to 100%；The plant of 100 Rhizoma Solani tuber osis is cooked whether containing S viruses of the test strip of PVS
Detection；Above-mentioned sample is detected with DASELISA immunoadsorption assay method simultaneously, the detection of two kinds of detection methods
As a result completely the same, coincidence rate is up to 100%；The plant of 100 Rhizoma Solani tuber osis is cooked whether containing M viruses of the test strip of PVM
Detection；Above-mentioned sample is detected with DASELISA immunoadsorption assay method simultaneously, the inspection of two kinds of detection methods
Survey result completely the same, coincidence rate is up to 100%;The plant of 100 Rhizoma Solani tuber osis is cooked of the test strip of tomato black ring virus is
The no detection containing tomato black ring virus virus；Above-mentioned sample is carried out with DASELISA immunoadsorption assay method simultaneously
Detection, the testing result of two kinds of detection methods is completely the same, and coincidence rate is up to 100%.Use PVYNTNTest strip to 98 horses
The plant of bell potato is cooked the detection of the whether virus of the NTN strains containing Y virus；DASELISA immunoadsorption assay is used simultaneously
Method detects that to above-mentioned sample the testing result of two kinds of detection methods is completely the same, and coincidence rate is up to 100%;Detection with PLRV
Whether test strips are done to the plant of 100 Rhizoma Solani tuber osis containing the viral detections of V；DASELISA immunoadsorption reality is used simultaneously
Test method to detect above-mentioned sample, the testing result of two kinds of detection methods is completely the same, coincidence rate is up to 100%.This experiment be enough to
Illustrate that the accuracy of Potyviruses Rapid detection test strip is high.
Claims (10)
1. a kind of Potyviruses Rapid detection test strip, including sample pad (1), gold standard pad (2), NC films (3), detection line T
Line (4), nature controlling line C line (5), adsorptive pads (6) and PVC base plates (7), the top of PVC base plates (7) are fitted with NC films (3), institute
State NC films (3) both upper ends thereof and be overlapped with gold standard pad respectively（2）And adsorptive pads（6）, gold standard pad (2) top is overlapped with sample pad
(1), detection line T line (4) are positioned close on NC films (3) surface of sample pad (1), and described nature controlling line C line (5) are arranged
On the NC films (3) of adsorptive pads (6), it is characterised in that：
It is coated with Potyviruses rabbit polyclonal antibody on detection line T line (4), described is coated on detection line T line (4)
It is 0.5-1.5 L/cm that Potyviruses rabbit polyclonal antibody solution draws film concentration, the Potyviruses for carrying out stroke film
The concentration of rabbit polyclonal antibody solution is 0.5-1.5mg/mL；
Potyviruses goat anti-rabbit antibody, the horse being coated in nature controlling line C line (5) is coated with nature controlling line C line (5)
It is 0.5-1.5 L/cm that bell potato virus goat anti-rabbit antibody solution draws film concentration, the Potyviruses goat-anti for carrying out stroke film
The concentration of rabbit-anti liquid solution is 0.5-1.5mg/mL；
Potyviruses colloid gold label thing, Rhizoma Solani tuber osi of the combination in gold standard pad (2) is combined with gold standard pad (2)
Virus colloidal gold label solution spraying concentration is 4-6 L/cm.
2. Potyviruses Rapid detection test strip according to claim 1, it is characterised in that：Potyviruses are quickly examined
It is additionally provided with outside test paper slip and gets stuck（8）, described get stuck（8）Upper face position corresponding with sample pad (1) is provided with sample-adding
Hole（9）, described get stuck（8）Upper face position corresponding with NC films (3) is provided with observation groove（10）.
3. Potyviruses Rapid detection test strip according to claim 1, it is characterised in that：NC films (3) and gold
Mark pad (2) length that overlaps is 1-2mm, and NC films (3) and the length that adsorptive pads (6) are overlapped are 2-3mm, the gold standard pad
(2) length overlapped with sample pad (1) is 2-3mm.
4. Potyviruses Rapid detection test strip according to claim 1, it is characterised in that：Described it is coated on nature controlling line
Potyviruses goat anti-rabbit antibody on C lines (5), the Potyviruses rabbit polyclonal antibody being coated on detection line T line (4) and
In conjunction with the Potyviruses colloid gold label thing in gold standard pad (2), can be any one in following combination：
Combination 1：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line (5) is PVX goat anti-rabbit antibodies, coated
Potyviruses rabbit polyclonal antibody on detection line T line (4) is PVX rabbit polyclonal antibodies, and the combination is in gold standard pad (2)
On Potyviruses colloid gold label thing be PVX colloid gold label things；
Combination 2：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line (5) is PVYNTNGoat anti-rabbit antibody, institute
It is PVY to state the Potyviruses rabbit polyclonal antibody being coated on detection line T line (4)NTNRabbit polyclonal antibody, the combination exist
Potyviruses colloid gold label thing in gold standard pad (2) is PVYNTNColloid gold label thing；
Combination 3：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line (5) is PVYoGoat anti-rabbit antibody, described
The Potyviruses rabbit polyclonal antibody being coated on detection line T line (4) is PVYoRabbit polyclonal antibody, the combination is in gold mark
Potyviruses colloid gold label thing on pad (2) is PVYoColloid gold label thing；
Combination 4：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line (5) is PLRV goat anti-rabbit antibodies, described
The Potyviruses rabbit polyclonal antibody being coated on detection line T line (4) is PLRV rabbit polyclonal antibodies, and the combination is in gold mark
Potyviruses colloid gold label thing on pad (2) is PLRV colloid gold label things；
Combination 5：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line (5) is PVS goat anti-rabbit antibodies, described
The Potyviruses rabbit polyclonal antibody being coated on detection line T line (4) is PVS rabbit polyclonal antibodies, and the combination is in gold mark
Potyviruses colloid gold label thing on pad (2) is PVS colloid gold label things；
Combination 6：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line (5) is PVM goat anti-rabbit antibodies, described
The Potyviruses rabbit polyclonal antibody being coated on detection line T line (4) is PVM rabbit polyclonal antibodies, and the combination is in gold mark
Potyviruses colloid gold label thing on pad (2) is PVM colloid gold label things；
Combination 7：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line (5) is PVA goat anti-rabbit antibodies, described
The Potyviruses rabbit polyclonal antibody being coated on detection line T line (4) is PVA rabbit polyclonal antibodies, and the combination is in gold mark
Potyviruses colloid gold label thing on pad (2) is PVA colloid gold label things；
Combination 8：The Potyviruses goat anti-rabbit antibody being coated in nature controlling line C line (5) is tomato black ring virus goat-anti rabbit
Antibody, the Potyviruses rabbit polyclonal antibody being coated on detection line T line (4) is that tomato black ring virus rabbit polyclonal resists
Body, Potyviruses colloid gold label thing of the combination in gold standard pad (2) is tomato black ring virus colloid gold label thing.
5. a kind of preparation method of the Potyviruses Rapid detection test strip based on described in claim 1, comprises the steps：
Step 1：The preparation of gold standard pad (2)：
1) preparation of Potyviruses colloid gold label thing：
With colloidal gold solution labelling Potyviruses rabbit polyclonal antibody, the concentration of Potyviruses rabbit polyclonal antibody is 0.5-
1.5mg/mL；
Redissolve formula of liquid：PH8.0-8.8,8-12% sucrose, 3-6% trehaloses, 0.5-2% bovine serum albumin, solvent are 0.1-
The Tris-HcL solution of 0.2mol/L；
The preparation of colloidal gold solution：297-300 mL ultra-pure waters are taken in round-bottomed flask, it is 1-2% chlorine gold to add 2-4 mL concentration
Acid solution, mixes；Round-bottomed flask is placed in thermostat, clean stirrer, stirring at low speed ebuillition of heated is added；Disposably add
Enter 2-4mL, 1-2% citric acid three sodium solution, continue to boil；Moment observation solution colour change, by light yellow → black → purple
→ aubergine, when claret is changed into, continues to boil 4-6min, stops heating, be cooled to room temperature；Ultra-pure water does blank,
Scanning absorption value of the colloidal gold solution between 500-550nm, if absorption peak occurs in 515-525nm, the glue for preparing
Body gold solution is qualified；
The preparation technology of Potyviruses colloid gold label thing：2 centrifuge tubes are taken, 1-2mL colloidal gold solutions are added, 3-5 is added
The K2CO3 of L 0.1mol/L, adds 4-6 L Potyviruses rabbit polyclonal antibodies, stands 25-35min, adds 80-
120 L sealers, stand 25-35min, be centrifuged 25-35min, abandon supernatant under the conditions of 8000-10000rpm, 4 DEG C, add
The multiple solution suspension precipitations of 80-120 L, are centrifuged 10-20min under the conditions of 400-600rpm, 4 DEG C, and Aspirate supernatant is preserved standby
With；
2) combination of gold standard pad (2) and Potyviruses colloid gold label thing：From glass fibre membrane as gold standard pad support
Material, by step 1) the Potyviruses colloid gold label thing for preparing, film instrument is drawn with three-dimensional large platform metal spraying be sprayed on glass fibers
On dimension film, the optimal spraying concentration for spraying Potyviruses colloid gold label thing on glass fibre membrane is 4-6 L/cm, will spray
Good glass fibre membrane is placed in 35-40 DEG C of drying baker, and 2-4h, took out, be put in aluminium foil bag, 180-230 DEG C drying time
Heat sealing, saves backup；
Step 2：The preparation of NC films (3)：
Detection line T line（4）And nature controlling line C line（5）Preparation：The how grand antibody of Potyviruses rabbit to be coated is diluted extremely with buffer
0.5 ~ 1.5mg/mL concentration, draws film gold spraying instrument with three-dimensional large platform and how grand for Potyviruses rabbit antibody is drawn film to apart from gold standard pad
At 1-2cm on NC films, film is drawn in NC films with the concentration of 0.5 ~ 1.5 L/cm（3）On, constitute detection line T line（4）；Dilute with buffer
Potyviruses goat anti-rabbit antibody to be coated is released to 0.5 ~ 1.5mg/mL concentration, film gold spraying instrument is drawn by Rhizoma Solani tuber osi with three-dimensional large platform
Viral goat anti-rabbit antibody draws film to NC films at adsorptive pads 2-3cm（3）On, with 0.5 ~ 1.5 L/cm draw film concentration draw film in
NC films（3）On, constitute nature controlling line C line（5）；Detection line T line（4）And nature controlling line C line（5）Apart from 4-6mm；To draw has detection line T
Line（4）And nature controlling line C line（5）NC films (3) be placed in 35-40 DEG C under the conditions of dry, encapsulate standby；
Buffer：The effect of buffer is to provide certain pH and ionic strength and makes Potyviruses rabbit polyclonal antibody and Rhizoma Solani tuber osi
Viral goat anti-rabbit antibody is firmly coated in NC films（3）On, the pH value of buffer is 7.0 ~ 7.4；
Step 3：The preparation of sample pad (1)：
From glass fibre membrane as the material for preparing sample pad (1), glass fibre membrane can be used after process；
The process step of glass fibre membrane：Glove are worn, glass fibre membrane is put in treatment fluid, is completely soaked, soak 5-
15min, takes out dehydration 3-5min afterwards, and horizontal on screen cloth is put into 35-40 DEG C of drying baker, dries to moisture constant in 20-30%,
Glass fibre membrane after by drying loads aluminium foil bag heat-sealing and preserves；
The formula for the treatment of fluid：PH7.8-8.0 phosphate buffers, 0.5-1.5%S9 surfactant solutions, 0.4-0.5% polysorbas20s,
0.5-1.5% bovine serum albumin, 0.1-0.3% polyvinylpyrrolidonesolution solution, solvent are water；
Step 4：The assembling of test strips：
Material is completed made for step 1 ~ 3, according to PVC base plates（7）Specification cut into certain length and width, paste
PVC base plates（7）Go up and be arranged on and get stuck（8）In；
The number of assembling steps of test strips：
1）By NC films（3）It is pasted onto PVC base plates（7）On；
2）By gold standard pad（2）It is overlapped on NC films（3）The top of one end, the lap of splice are 1-2mm；
3）By adsorptive pads（6）It is overlapped on NC films（3）The top of the other end, the lap of splice are 2-3mm；
4）By sample pad（1）It is overlapped on gold standard pad（2）Top, the lap of splice is 2-3mm；
5）By the PVC base plates for pasting in cutting machine（7）Cut into bar；
6）Shell is filled after cutting into bar and loads aluminium foil bag in the lump with desiccant, labelled after sealing.
6. the preparation method of Potyviruses Rapid detection test strip according to claim 5, it is characterised in that：
In Potyviruses colloid gold label thing preparation method step, the Potyviruses rabbit polyclonal antibody of addition can be
PVX rabbit polyclonal antibodies, PVYNTNRabbit polyclonal antibody, PVYoRabbit polyclonal antibody, PLRV rabbit polyclonal antibodies, many grams of PVS rabbits
One kind in grand antibody, PVM rabbit polyclonal antibodies, PVA rabbit polyclonal antibodies or tomato black ring virus rabbit polyclonal antibody；
If in Potyviruses colloid gold label thing preparation method step, addition is PVX rabbit polyclonal antibodies, can prepare
Into PVX colloid gold label things；If that added is PVYNTNRabbit polyclonal antibody, can be prepared into PVYNTNColloid gold label thing；Such as
That fruit adds is PVYoRabbit polyclonal antibody, can be prepared into PVYoColloid gold label thing；If added is that PLRV rabbit polyclonals resist
Body, can be prepared into PLRV colloid gold label things;If added is PVS rabbit polyclonal antibodies, PVS colloid gold labels can be prepared into
Thing；If added is PVM rabbit polyclonal antibodies, PVM colloid gold label things can be prepared into；If added is many grams of PVA rabbits
Grand antibody, can be prepared into PVA colloid gold label things；If added is tomato black ring virus rabbit polyclonal antibody, can be prepared into
Tomato black ring virus colloid gold label thing；In addition to adding Potyviruses rabbit polyclonal antibody species difference, other preparation works
Skill parameter is all consistent.
7. the preparation method of Potyviruses Rapid detection test strip according to claim 5, it is characterised in that：
The preparation method of the Potyviruses rabbit polyclonal antibody and Rhizoma Solani tuber osi bell potato virus goat anti-rabbit antibody is specially：
The preparation of Potyviruses rabbit polyclonal antibody：1-2mL Potyviruses rabbit anti-serums are taken, 1-2mL phosphoric acid buffers are added
Liquid vibrates in an oscillator, adds 2-4mL saturated ammonium sulphate Potyviruses antibody after mixing；Then in 8000-
Be centrifuged under the conditions of 10000rpm, 10-15min, 4 DEG C or so, precipitate Potyviruses antibody, then horse is dissolved with phosphate buffer
Bell potato antiviral antibody, is then removed by filtration unnecessary ammonium sulfate by PD-10coLum fibre columns, obtains final product Potyviruses rabbit many
Clonal antibody；
The preparation of Potyviruses goat anti-rabbit antibody：1-2mL Potyviruses goat-anti rabbit anti-serums are taken, adds 1-2mL phosphoric acid to delay
Rush liquid to vibrate in an oscillator, after mixing, add 2-4mL saturated ammonium sulphates virus；Then in 8000-10000rpm, 10-
15min, is centrifuged under the conditions of 4 DEG C or so, precipitates Potyviruses antibody, is then resisted with phosphate buffer dissolving Potyviruses
Body, is then removed by filtration unnecessary ammonium sulfate by PD-10coLum fibre columns, obtains final product Potyviruses goat anti-rabbit antibody.
8. the preparation method of Potyviruses Rapid detection test strip according to claim 7, it is characterised in that：
The Potyviruses goat anti-rabbit antibody and Potyviruses rabbit polyclonal antibody can select any in following combination
A kind of combination is prepared；
Combination 1：PVX goat anti-rabbit antibodies, PVX rabbit polyclonal antibodies；
Combination 2： PVYoGoat anti-rabbit antibody, PVYoRabbit polyclonal antibody；
Combination 3： PVYNTNGoat anti-rabbit antibody, PVYNTNRabbit polyclonal antibody；
Combination 4：PLRV goat anti-rabbit antibodies, PLRV rabbit polyclonal antibodies；
Combination 5：PVS goat anti-rabbit antibodies, PVS rabbit polyclonal antibodies；
Combination 6：PVM goat anti-rabbit antibodies, PVM rabbit polyclonal antibodies
Combination 7：PVA goat anti-rabbit antibodies, PVA rabbit polyclonal antibodies；
Combination 8：Tomato black ring virus goat anti-rabbit antibody, tomato black ring virus rabbit polyclonal antibody.
9. the preparation method of Potyviruses Rapid detection test strip according to claim 5, it is characterised in that：Step 2：
In the preparation of NC films (3)：It is used for drawing film nature controlling line C line（5）Potyviruses goat anti-rabbit antibody and be used for draw a film detection line T line
（4）The how grand antibody of Potyviruses rabbit can select in following combination any one：
Combination 1：PVX goat anti-rabbit antibodies, PVX rabbit polyclonal antibodies；
Combination 2： PVYoGoat anti-rabbit antibody, PVYoRabbit polyclonal antibody；
Combination 3： PVYNTNGoat anti-rabbit antibody, PVYNTNRabbit polyclonal antibody；
Combination 4：PLRV goat anti-rabbit antibodies, PLRV rabbit polyclonal antibodies；
Combination 5：PVS goat anti-rabbit antibodies, PVS rabbit polyclonal antibodies；
Combination 6：PVM goat anti-rabbit antibodies, PVM rabbit polyclonal antibodies
Combination 7：PVA goat anti-rabbit antibodies, PVA rabbit polyclonal antibodies；
Combination 8：Tomato black ring virus goat anti-rabbit antibody, tomato black ring virus rabbit polyclonal antibody.
10. a kind of application of Rhizoma Solani tuber osi Rapid detection test strip, it is characterised in that：With quick according to claim 1 ~ 4
Test strip detects that to potato samples to be checked concrete detecting step is：
(1) sample treatment：Potato leaf is taken, the ratio by leaf weight g and buffering liquid product V is 1:8-1:10 ratio adds
Enter phosphate buffer, green juice is ground, the PH of phosphate buffer is 7.4-7.8；
(2) the above-mentioned sample for processing is added drop-wise in the sample pad of Rhizoma Solani tuber osi Rapid detection test strip, stands 0.5 ~ 3 point
Clock, observation detection line T line and nature controlling line C line；
(3) result interpretation：Detection line T line and nature controlling line C line manifest aubergine for the positive；Detection line T line does not manifest purplish red
Color, nature controlling line C line manifest aubergine, are as a result feminine gender；Detection line T line and nature controlling line C line do not manifest aubergine or other are existing
As pointing out test strips failure.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611111887.7A CN106501513B (en) | 2016-12-06 | 2016-12-06 | A kind of Potyvirus Rapid detection test strip and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611111887.7A CN106501513B (en) | 2016-12-06 | 2016-12-06 | A kind of Potyvirus Rapid detection test strip and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106501513A true CN106501513A (en) | 2017-03-15 |
| CN106501513B CN106501513B (en) | 2019-06-04 |
Family
ID=58329354
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611111887.7A Active CN106501513B (en) | 2016-12-06 | 2016-12-06 | A kind of Potyvirus Rapid detection test strip and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106501513B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109164261A (en) * | 2018-08-24 | 2019-01-08 | 中国农业大学 | A kind of method and its special antibody detecting corium solani |
| CN111596063A (en) * | 2019-12-31 | 2020-08-28 | 贵州省烟草科学研究院 | Rapid test card for simultaneously detecting PVY and TVBMV and preparation and use methods thereof |
| CN112557656A (en) * | 2021-01-19 | 2021-03-26 | 黑龙江省农业科学院克山分院 | Test strip for synchronously detecting various potato viruses and preparation method and application thereof |
| CN114414807A (en) * | 2021-12-23 | 2022-04-29 | 南京珀尔泰生物技术有限公司 | Test strip for detecting hepatitis B surface antigen and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101210920A (en) * | 2006-12-30 | 2008-07-02 | 河南农业大学 | Tobacco potato Y virus field rapid detection test paper preparation method |
| CN103901216A (en) * | 2014-04-22 | 2014-07-02 | 上海科华生物工程股份有限公司 | Human H-FABP colloidal gold test paper and preparation method thereof |
| CN104678098A (en) * | 2015-03-13 | 2015-06-03 | 苏州万木春生物技术有限公司 | Preparation method of joint-inspection test paper for cardiac troponin I and heart-type fatty acid binding protein |
| CN105543261A (en) * | 2016-02-26 | 2016-05-04 | 山东鲁蔬种业有限责任公司 | Application of potato leaf roll virus recombination CP antibody in preparing test paper strip |
-
2016
- 2016-12-06 CN CN201611111887.7A patent/CN106501513B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101210920A (en) * | 2006-12-30 | 2008-07-02 | 河南农业大学 | Tobacco potato Y virus field rapid detection test paper preparation method |
| CN103901216A (en) * | 2014-04-22 | 2014-07-02 | 上海科华生物工程股份有限公司 | Human H-FABP colloidal gold test paper and preparation method thereof |
| CN104678098A (en) * | 2015-03-13 | 2015-06-03 | 苏州万木春生物技术有限公司 | Preparation method of joint-inspection test paper for cardiac troponin I and heart-type fatty acid binding protein |
| CN105543261A (en) * | 2016-02-26 | 2016-05-04 | 山东鲁蔬种业有限责任公司 | Application of potato leaf roll virus recombination CP antibody in preparing test paper strip |
Non-Patent Citations (2)
| Title |
|---|
| IRINA SAFENKOVA ET AL: "Factors influencing the detection limit of the lateral-flow", 《ANAL BIOANAL CHEM》 * |
| 魏梅生等: "马铃薯X病毒和马铃薯Y病毒胶体金免疫层析试纸条的研制", 《植物保护》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109164261A (en) * | 2018-08-24 | 2019-01-08 | 中国农业大学 | A kind of method and its special antibody detecting corium solani |
| CN111596063A (en) * | 2019-12-31 | 2020-08-28 | 贵州省烟草科学研究院 | Rapid test card for simultaneously detecting PVY and TVBMV and preparation and use methods thereof |
| CN112557656A (en) * | 2021-01-19 | 2021-03-26 | 黑龙江省农业科学院克山分院 | Test strip for synchronously detecting various potato viruses and preparation method and application thereof |
| CN112557656B (en) * | 2021-01-19 | 2023-09-05 | 黑龙江省农业科学院克山分院 | Test strip for synchronously detecting various potato viruses and preparation method and application thereof |
| CN114414807A (en) * | 2021-12-23 | 2022-04-29 | 南京珀尔泰生物技术有限公司 | Test strip for detecting hepatitis B surface antigen and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106501513B (en) | 2019-06-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106501513A (en) | A kind of Potyviruses Rapid detection test strip and its preparation method and application | |
| CN106918704A (en) | Synchronous detection aflatoxin and time-resolved fluoroimmunoassay chromatography kit, the preparation method and application of carbaryl composite pollution | |
| CN105092861A (en) | Immunofluorescence chromatography test paper for CRP (C-reaction protein)/SAA (Serum amyloid A protein) quantitative combined detection and preparation method of immunofluorescence chromatography test paper | |
| CN104459144B (en) | A kind of PRV velogen strain and vaccine strain differentiate Test paper | |
| CN103266088B (en) | A kind of H7 subtype avian influenza virus monoclonal antibody and test kit | |
| CN104407137A (en) | Test paper for identifying and detecting virulent strain and low virulent strain of hog cholera virus | |
| CN105044367A (en) | Colloidal gold immunochromatographic assay test strip for detecting dexamethasone residues in milk | |
| CN110007095A (en) | A kind of dengue virus NS 1 antigen test strip, kit and preparation method thereof | |
| CN105675874A (en) | Colloidal gold test strip for detection of imidacloprid and application thereof | |
| CN204964521U (en) | CRPSAA ration jointly detects immunofluorescence chromatography test paper | |
| CN108196054A (en) | A kind of test strips for detecting glycyrrhizic acid and its preparation method and application | |
| CN106771123A (en) | A kind of preparation method of dengue virus IgM/IgG antibody Test paper | |
| CN105424937B (en) | A kind of progesterone colloidal gold immuno-chromatography test paper strip of monkey early pregnancy detection | |
| CN102226811A (en) | Preparation method of diagnostic test paper for detecting H-FABP (heart-type fatty acid binding protein) | |
| CN202141725U (en) | Magnetic bead immunochromatographic kit for qualitative/ quantitative detection of surface antigen of hepatitis B virus | |
| CN103412121B (en) | Colloidal gold immune test strip for rapid detection of Papaya ringspot virus | |
| CN106771273B (en) | One kind detection Formoterol colloidal gold immuno-chromatography test paper strip and preparation method and application | |
| CN101666800B (en) | Fast test strip of vesicular stomatitis virus (VSV) and preparation method thereof | |
| CN105675875B (en) | A kind of colloid gold immune test paper bar of quick detection southern rice black-streaked dwarf virus and preparation method thereof | |
| CN108226514A (en) | A kind of newcastle disease virus antibody rapid quantitative detection reagent box and its application | |
| CN104360068A (en) | Semi-quantitative rapid detection test paper for white spot syndrome virus (WSSV), and preparation method of test paper | |
| CN102520177A (en) | Method for quantitative detection of zearalenone | |
| CN201397332Y (en) | Colloidal gold test strip for rapidly detecting swine influenza virus | |
| CN101520459B (en) | Rabbit hemorrhagic disease virus colloidal gold test strip | |
| CN202599960U (en) | Colloidal gold test strip for rapid detection of O-type foot-and-mouth disease virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |