CN206563749U - A kind of WSSV immune colloid gold quick detection kit - Google Patents

Abstract

The utility model is related to a kind of WSSV immune colloid gold quick detection kit, and the test strips in plastic casing and shell are constituted.Plastic casing upper shell is provided with adding mouth and observation window, and test strips include backing and the end to end sample pad sticked on backing, gold labeling antibody pad, chromatographic film and adsorptive pads.The detection line being parallel to each other and control line are coated with chromatographic film.The colloid gold label albumen of white spot syndrome virus resisting monoclonal antibody, the different another white spot syndrome virus resisting monoclonal antibody of detection line coating viral antigen binding site, control line coating secondary antibody are sprayed with gold labeling antibody pad.With white spot syndrome virus in double antibody sandwich method detection sample, testing result is reflected by the colour developing of detection line and control line.The kit sensitivity is high, and specificity is good, and detection time is short, and preparation method is simple, is adapted to the quick detection and monitoring of market, aquaculture base to white spot syndrome virus in the aquatic products such as shrimps, crab class.

Description

A kind of WSSV immune colloid gold quick detection kit

Technical field

The utility model is related to a kind of WSSV immune colloid gold quick detection kit.

Background technology

Prawn white spot syndrome (White Spot Syndrome, abbreviation WSS) is by white spot syndrome virus (White Spot Syndrome Virum, abbreviation WSSV) caused by a kind of prawn communicable disease, the sick pathogeneticing characteristic be the death rate Height, it is dead fast.Common symptom：There are white particle or hickie (some ill shrimps of circle at epidermis before this in dying shrimp Group reddens or pink), then ill shrimp occurs that drowsiness, body colour reddens or turned yellow, or is gathered in pond side, and food ration is drastically Reduce, and the higher death rate occur.Pass through following three kinds of modes when pathology judge：It can see hypertrophy with the gill and epidermis tabletting Core, or observe the abnormal aggegation of hemolymph with dark field, or inclusion body is seen in the histotomy of target tissue.The United Nations's grain with Agricultural organization (FAO) by the WSS and on shrimp cream head disease and prawn taura syndrome and is referred to as " the three of influence world's prawn culturing Major disease ".WSS has also been put into OIE (OIE) aquatic animal epidemic disease register.

WSSV is to endanger prawn virus disease the most serious in whole world cultured prawn.WSSV is most of right except that can infect Outside shrimp kind, moreover it is possible to infect other non-to species, such as amphipoda, Ostracode, crab class, Copepods, ephydrid class Crustachia Animal.These Crustaceanses may act as the intermediate host of WSSV propagation in the ecosystem, to cultured prawn and wild Crustachia Animal resources constitute a serious threat.For China, WSSV is the fast-developing main bottleneck of the domestic shrimp culture industry of influence One of.In No. 1125 bulletin of the Ministry of Agriculture of China in 2008《First, two, three class animal epidemic disease register》In, WSSV is divided into One class animal epidemic.In view of at present it is not yet found that effect treatment method, fast and accurately pathogen separation detection technique turned into The focus of scholars' research.

At present, WSSV detection method mainly has：Observation method of naked eye, traditional histological method, electron microscopic observation method, biochemistry inspection Survey, immunological method, cell culture method, molecular biology method.The technological means such as applied immunology, molecular biology are researched and developed Aquatic animal pathogenic conditions quick detection kit can be fast and accurately diagnosed to be within a few minutes to a few houres aquatic products disease Evil, is relatively adapted to basic unit's disease prevention and control librarian use.Currently, foreign countries have established the cause of diseases such as multiple aquatic animal virus, bacterium Quick determination method, the false knot nuclearity pasteurellosis detection kit of European biotech firm of Greece development and sale, utilize ELISA technologies, testing result is can determine that with visually observing；The U.S. biology research and development can detect simultaneously prawn BP, HPV, IHHNV, MBV, NHP, WSSV kit, and detect prawn TSV, YHV kit.Over nearly 10 years, China is great to some The Fast Detection Technique of disease pathogen compared with in-depth study, reach or connect in terms of species, technical merit, ease for use Nearly international most advanced level.Synchronous PCR etc. including setting up KHV, IHHNV, TSV and distinguish the different geographical malicious spiders of WSSV simultaneously is quick Detection technique, have developed detection kits such as Aeromonas hydrophila, prawn's virus WSSV, prawn TSV etc..China WSSV detection Test stone (SN/T 1151.2-2011) is examined in the entry and exit of standard Primary Reference China, and the standard specifies prawn white spot disease PCR, three kinds of detection methods of dot hybridization and in situ hybridization.But either kit is still entered and left the border inspection as defined in examination criteria Survey method, it is still excessively complicated for grass-roots work personnel, it is difficult to operate, often also need to deliver to the testing department of specialty Or laboratory is detected.Therefore developing a kind of detection WSSV colloidal gold fast detecting reagent kit becomes simplified WSSV detections New way.

Utility model content

The utility model provides the WSSV that a kind of sensitivity is high, detection time is short, easy to operate and exempted from Epidemic disease colloidal gold fast detecting reagent kit.

A kind of WSSV immune colloid gold quick detection kit, including：In plastic casing and shell Test strips, it is characterised in that the plastic casing upper shell be provided with adding mouth and observation window, the test strips include backing and The end to end sample pad sticked on backing, gold labeling antibody pad, chromatographic film and adsorptive pads, there is 0.2~0.3cm joint It is overlapping, the sample pad is located at the lower section of adding mouth, and the chromatographic film is located at the lower section of observation window, is coated with the chromatographic film The detection line and control line being parallel to each other.

The backing is made up of PVC board, and sample pad is made up of glass fibre, gold labeling antibody pad can by glass fibre or Polyester film is made, and chromatographic film can be made up of nitrocellulose filter, cellulose acetate film or poly tetrafluoroethylene, and adsorptive pads are by filter paper It is made.

The colloid gold label albumen of anti-WSSV monoclonal antibodies is sprayed with the gold labeling antibody pad.

The detection line is coated with anti-WSSV monoclonal antibodies, control line coating secondary antibody.

Anti- WSSV monoclonal antibodies on the gold labeling antibody pad exist with the anti-WSSV monoclonal antibodies in detection line There are different binding sites on WSSV.

The secondary antibody can be sheep anti-mouse igg, rabbit anti-mouse igg.

The utility model uses double-antibody sandwich immunochromatography principle.Anti- WSSV monoclonal antibodies (gold labeling antibody) are marked by gold With the antigen-reactive in analyte sample fluid, the anti-WSSV monoclonal antibodies-WSSV antigenic compounds of gold mark are formed.When the composite layer Analysis to another anti-WSSV monoclonal antibodies (coated antibody) being coated in chromatographic film during detection line, in advance in detection line can know Antigen not in the compound.The result of reaction just forms the interlayer structure of gold labeling antibody-WSSV antigens-coated antibody, finally Make the colloid gold particle in gold labeling antibody fixed herein and be accumulate to the macroscopic red line of appearance.Unreacted gold labeling antibody Then continue to chromatography to move ahead, be combined when reaching and being coated in advance at control line secondary antibody, so as to occur here by colloid Gold grain is fixed and accumulates and show macroscopic red line.Result is：Detection line colour developing represents that WSSV is positive；Detection line Do not develop the color, represent that WSSV is negative, that is, detect extremely low without WSSV or WSSV contents in sample；Control line develops the color, and represents reagent Box is effective；Control line does not develop the color, and illustrates that kit fails, i.e. gold labeling antibody or secondary antibody inactivation, and testing result is invalid.

The utility model has the advantages that：

Sensitivity is high, and the detection of this kit is limited to 1mg/kg；Specific high, false negative rate and false positive rate are below 5%； Detection time about 30min, testing result can with the naked eye judge；Preparation method is simple, stability is high, reproducible.

Brief description of the drawings

Fig. 1 is WSSV immune colloid gold quick detection kit sectional view, wherein 1 is outside plastics Shell, 2 be adding mouth, and 3 be observation window, and 4 be backing, and 5 be sample pad, and 6 be gold labeling antibody pad, and 7 be chromatographic film, and 8 be detection Line, 9 be control line, and 10 be adsorptive pads, and 11 be adhesive sticker.

Fig. 2 is WSSV immune colloid gold quick detection kit top view, wherein 2 be adding mouth, 3 It is detection line for observation window, 8,9 be control line.

Fig. 3 reads schematic diagram, wherein T for the result of WSSV immune colloid gold quick detection kit For detection line, C is control line；T lines develop the color with C lines in Fig. 3-a, are positive；There was only the colour developing of C lines in Fig. 3-b, be negative；Figure T lines disappear with C lines in 3-c, are invalid.

Embodiment

The preparation of the prawn white spot syndrome virus immunity colloidal gold fast detecting reagent kit of example one

The processing of 1 sample pad

By plain film 1%BSA (w/v), 1%Tween-20 (w/v) PEG4000 PBS (0.015M, pH of glass fibre =7.4) uniformly soak and dry 2h at 2h, 37 DEG C, it is positioned over dry environment and saves backup.

It is prepared by 2 gold labeling antibody pads

The preparation of 2.1 colloidal gold solutions

The mean size of colloid gold particle is 30nm, and its preparation method is that the lemons of 1mL 1% are added in 100mL deionized waters Lemon acid trisodium, is rapidly added the gold chlorides of 1mL 1%, continues to boil 10min, after cooling, saved backup at 4 DEG C after boiling.

The preparation of 2.2 gold labeling antibodies

Take the anti-WSSV monoclonal antibodies of 15 μ g to be added in 1mL colloidal gold solutions, be slowly stirred 1min, add 1% ox blood Pure albumen, 4 DEG C overnight；Then by it at 4 DEG C, 18000g centrifugations 110min；Centrifugation is taken, with containing 1% cow's serum Albumin, 0.02% Sodium azide, the 0.01M PBS of 0.5% Tween-20 are resuspended, and 4 DEG C save backup.

2.3 gold labeling antibodies spray film

By above-mentioned gold labeling antibody liquid storage with film instrument is quantitatively sprayed with 3 μ L/cm flow velocity even application on pad, 37 DEG C are dried It is dry, 4 DEG C of lucifuge kept dries.

3 chromatography film preparations

The anti-WSSV monoclonal antibodies and sheep anti-mouse igg of debita spissitudo, nitric acid fibre is sprayed on quantitative spray film instrument by reference picture 1 On the plain film of dimension, respectively as detection line and control line, 37 DEG C of oven drying 8h.

The assembling of 4 WSSV immune colloid gold quick detection kits

Reference picture 1, using PVC board as backing, be stained with order with adhesive sticker thereon sample pad, gold labeling antibody pad, Nitrocellulose filter and blotting paper.The wide test strips of 3.00mm are cut into cutting machine, is fitted into plastic casing and detection examination is made Agent box, places into sealed storage in the aluminium foil bag with drier.

The operating method of the WSSV immune colloid gold quick detection kit of example two

1 sample treatment

Weigh shrimp gill 2g to be detected, add 0.01M PBS and mixed by 1: 10 (W/V) ratio, plus on a small quantity quartz sand with grinding Alms bowl grinding, homogenate 1min, 5~10min of reprecipitation, take supernatant as detection sample, to be checked.

2 kit application methods

Kit is taken out from packaging bag, the μ L of measuring samples solution 100 is drawn and is added drop-wise in well, start meter after sample-adding When；As a result it should be read in 3-5min, other times interpretation is invalid.During observation, kit level is positioned over observer front.

3 experimental results judge

T lines and C lines develop the color for the positive in observation window, represent that the content of WSSV in detection sample is higher than the μ g/ of detection limit 1 mL.As shown in Fig. 3-a；T lines colour developing in observation window and C lines do not develop the color for feminine gender, represent in detection sample without WSSV or WSSV contents are extremely low.As shown in Fig. 3-b；Do not occur T lines and C lines in observation window, it may be possible to which misoperation or kit have failed. Specification should be read again, and is retested with new kit.As shown in Fig. 3-c.

The detection limit of example three is determined

1 operating procedure

Weigh the shrimp gill 2g of healthy prawn (PCR detection without WSSV infect), add WSSV solution to final concentration of 0.25, 0.5th, 0.75,1.0,2.0,4.0mg/kg, adds 0.01M PBS and is mixed in 1: 10 (W/V) ratio, plus a small amount of quartz sand is used Mortar grinder, homogenate 1min, 5~10min of reprecipitation, take supernatant as mark-on and detect sample, the reagent prepared with example one Box detects that negative control is 0.01mol/L PBS solutions to it respectively.Every group sets 6 Duplicate Samples.

2 experimental results

As shown in table 1, detection line color is incremented by experimental result in gradient.Negative control and WSSV final concentration of 0.25, In the range of 0.5 mg/kg, C lines develop the color and T lines do not develop the color, and the stability of experimental result is good；Under 0.75mg/kg concentration C lines with T lines band develops the color, but the stability of experimental result is bad；C lines develop the color with T lines and with stable in the range of 1.0~4.0mg/kg The characteristics of property is good.Test result indicates that, the WSSV detections of this kit are limited to 1.0mg/kg.

The WSSV immune colloid gold quick detection kit detection limit of table 1

^aConcentration unit：mg/kg；^bColour developing degree：- represent and do not develop the color ,+represent colour developing；^cStability：- represent it is unstable ,+ Represent stable.

Claims (6)

1. a kind of WSSV immune colloid gold quick detection kit, including：In plastic casing (1) and shell Test strips, it is characterised in that the plastic casing upper shell is provided with adding mouth (2) and observation window (3), and the test strips include Backing (4) and the end to end sample pad (5) sticked on backing, gold labeling antibody pad (6), chromatographic film (7) and adsorptive pads (10), joint has 0.2~0.3cm's overlapping, and the sample pad (5) is located at the lower section of adding mouth (2), the chromatographic film (7) The detection line (8) being parallel to each other and control line (9) are coated with lower section positioned at observation window (3), the chromatographic film (7).

2. WSSV immune colloid gold quick detection kit as claimed in claim 1, it is characterised in that The backing (4) is made up of PVC board, and sample pad (5) is made up of glass fibre, and gold labeling antibody pad (6) can be by glass fibre Or polyester film is made, chromatographic film (7) can be made up of nitrocellulose filter, cellulose acetate film or poly tetrafluoroethylene, adsorptive pads (10) it is made up of filter paper.

3. WSSV immune colloid gold quick detection kit as claimed in claim 1, it is characterised in that The colloid gold label albumen of white spot syndrome virus resisting monoclonal antibody is sprayed with the gold labeling antibody pad (6).

4. WSSV immune colloid gold quick detection kit as claimed in claim 1, it is characterised in that The detection line (8) is coated with white spot syndrome virus resisting monoclonal antibody, control line (9) coating secondary antibody.

5. the WSSV immune colloid gold quick detection kit as described in claim 3 or 4, its feature exists It is comprehensive in, white spot syndrome virus resisting monoclonal antibody on the gold labeling antibody pad (6) and anti-hickie in detection line (8) Close syndrome virus monoclonal antibody has different binding sites on white spot syndrome virus.

6. WSSV immune colloid gold quick detection kit as claimed in claim 4, it is characterised in that The secondary antibody can be sheep anti-mouse igg, goat anti-rabbit igg.