CN106802350A - A kind of pathogenicity fish enteron aisle vibrios quick detection test paper - Google Patents
A kind of pathogenicity fish enteron aisle vibrios quick detection test paper Download PDFInfo
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Abstract
The present invention provides a kind of competition immune chromatography rapid detecting test paper of pathogenicity fish enteron aisle vibrios, specific fish pathogen fish enteron aisle vibrios can be used for quickly detecting, and be avoided that detection error caused by cross-immune reaction.Be provided with since near gold standard pad side four detection line T1s, T2, T3, T4 and the nature controlling line being arranged in order on the fast Test paper of fish enteron aisle vibrios its nitrocellulose filter that the present invention is provided, wherein detection line T1 be the outer membrane protein of fish enteron aisle vibrios, detection line T2 be flagellin, detection line T3 be the extracellular products of fish enteron aisle vibrios, detection line T4 be that full bacterium crushes albumen；Nature controlling line C is coated with goat anti-rabbit igg.The present invention is reacted the permutation and combination of various antigentic specificities of pathogen simultaneously with measuring samples together, and whole bacterial protein is especially set on T4 lines, cross reaction can accurately distinguish positive findings and cross reaction to the interference problem of result judgement caused by solving the common antigen of other bacterium.
Description
Technical field
The invention belongs to the pathogenic microorganism examination technical field, and in particular to a kind of pathogenicity fish enteron aisle vibrios (Vibrio
Ichthyoenteri) quick detection test paper and preparation method thereof.
Background technology
Fish enteron aisle vibrios is Gram-negative bacteria, is one of common flora in marine environment, is widely distributed in seawater and life
In thing colony, the composite factor such as its pathogenic physiological situation and water quality environment condition by host is influenceed, and is that a kind of condition is caused
Germ.Fish enteron aisle vibrios can cause various cultured fishes to be fallen ill, and serious economic loss is caused to culture fishery.It is effectively fast
The checkout and diagnosis of speed are the keys for preventing and controlling the pathogen to propagate.
The detection method of conventional fish enteron aisle vibrios has indirect immunofluorescence antibody technology, immune integrated enzyme reaction, gathers at present
Polymerase chain reaction (PCR), real-time quantitative PCR etc., but these technologies all have certain limitation in practicality, and time-consuming, behaviour
Make complexity, it is necessary to professional equipment and personnel, are not suitable for common poultry feeders at the scene to the real-time detection of fish enteron aisle vibrios.Glue
Body gold immune chromatography test paper have be easy to carry about with one, simple and efficient to handle, result naked eyes are visible, do not need professional and equipment,
Economical and practical the advantages of, be a kind of very promising field quick detection technology.Though prepared in aquatic livestock various
List (many) clonal antibodies of cultivated animals cause of disease, but field quick detection test paper technology fails to be developed well, at present still
Report without fish enteron aisle vibrios colloidal gold fast detecting test paper.
Bacterium is the complex of various antigenic components composition, its antigen include somatic antigen, flagellar antigen, capsular antigen and
Fimbrial antigen etc., can induce body and produces antibody, therefore, make a definite diagnosis disease to reach using Ag-Ab specific reaction principle
Former purpose.But, because classification position is close, between different vibrios often have common antigenic protein, cause different bacterium/
There is cross-immunoreactivity between vibrios, so as to the accurate judgement to testing result is interfered, therefore, it is accurate to judge it is certain
The key issue for solving is needed when the cross reaction of kind pathogenic bacterial infection or other bacterium is immunoassay cause of disease.Generally
Solution be by screening the distinctive antigen/antibody of pathogen to improve detection accuracy, but, aquatic livestock pathogenicity
Vibrios species is various, and the presence that various pathogens may have common antigen, common antigen with fish enteron aisle vibrios increased screening
Difficulty, workload is heavy.
The content of the invention
It is an object of the invention to provide a kind of quick detection test paper of pathogenicity fish enteron aisle vibrios, can be specific to fish
Fish enteron aisle vibrios is used for quickly detecting pathogenicity, and is avoided that detection error caused by cross-immune reaction.
The fish fast Test paper of enteron aisle vibrios that the present invention is provided, including carrier board, sample pad, gold standard pad, nitrocellulose
Film and adsorptive pads, wherein sample pad and adsorptive pads are located at the two ends of carrier board, and nitrocellulose filter is located at the middle part of carrier board；Sample
Product pad is provided with the gold standard pad for being loaded with gold mark rabbit-anti fish enteron aisle vibrios antibody, the end margin of gold standard pad one with nitrocellulose filter intersection
Overlap under sample pad, other end imbricate is on nitrocellulose filter；From near gold standard pad on nitrocellulose filter
Side starts to be provided with four detection line T1s, T2, T3, T4 and the nature controlling line being arranged in order, and wherein detection line T1 is fish enteron aisle
The outer membrane protein of vibrios, detection line T2 be flagellin, detection line T3 be the extracellular products of fish enteron aisle vibrios, detection line T4 be complete
Bacterium crushes albumen；Nature controlling line C is coated with goat anti-rabbit igg.
The above-mentioned rabbit-anti fish enteron aisle vibrios antibody for using, its preparation method is as follows：
By fish enteron aisle vibrios Amplification Culture, and bacterial concentration is adjusted to 1 × 108CFU/mL, is immunized after Formalin inactivation
Purebred new zealand white rabbit；Freund's complete adjuvant is mixed into for the first time,；Booster immunization after one week is spaced, Freund is mixed into and is not exclusively helped
Agent, interval is repeated once after two weeks；Blood is taken after one week, isolated serum, purifying obtains rabbit-anti fish enteron aisle vibrios antibody.
Described fish enteron aisle vibrios outer membrane protein, its preparation method is as follows：
By fish enteron aisle vibrios Amplification Culture, phosphate buffer (PBS) washing, resuspended to original volume 1/25 after centrifugation, plus
Enter 10% sucrose, 15min is acted at -20 DEG C after mixing；Isometric 0.4%Triton-X100 is added at room temperature, when acting on one section
Between after centrifuging and taking supernatant, add 2.5 times of -20 DEG C of ice ethanol of volume overnight；Centrifuging and taking is precipitated, after washing is resuspended, the body such as addition
Product KI solution, 37 DEG C of effect 1h；Centrifuging and taking supernatant, adds 2.5 times of -20 DEG C of volume ice ethanol overnight；Centrifuging and taking precipitate, it is resuspended after
Dialysis, lyophilized obtain fish enteron aisle vibrios outer membrane protein.
Described fish enteron aisle vibrios flagellin, its preparation method is as follows：
It is PBS washings after centrifugation, resuspended to original volume 1/20 by fish enteron aisle vibrios Amplification Culture, adjusted with 1mol/L HCl
PH=2.0, low-temperature centrifugation takes supernatant after stirring 30min；Supernatant adjusts pH to neutrality, adds (NH3)2SO4, 4 DEG C were placed
Night；Low temperature ultracentrifugation, takes precipitation, it is resuspended after dialysis, lyophilized obtain fish enteron aisle vibrios flagellin.
Described fish enteron aisle vibrios extracellular products, its preparation method is as follows：
Fish enteron aisle vibrios is inoculated in after being cultivated in fluid nutrient medium, liquid culture is coated and has been pre-placed one layer
On the solid agar flat board of glassine paper, 37 DEG C of culture 36h；Hypothallus is washed with PBS, high speed centrifugation at 4 DEG C after collects thalline；Will be upper
Clearly through filtering with microporous membrane, (NH is added4)2SO4, 4 DEG C stand overnight；Then low-temperature centrifugation takes precipitation, resuspended to dialyse afterwards, freeze
Obtain fish enteron aisle vibrios extracellular products.
The broken albumen of the described full bacterium of fish enteron aisle vibrios, its preparation method is as follows：
By fish enteron aisle vibrios Amplification Culture, bacterium solution is placed in cryogenic pulverization in sonicator and obtains the broken liquid of full bacterium, freezes
It is dry to obtain the broken albumen of the full bacterium of fish enteron aisle vibrios.
The present invention is reacted the permutation and combination of various antigentic specificities of pathogen simultaneously with measuring samples together,
And whole bacterial protein is especially set on T4 lines, cross reaction is done to result judgement caused by solving the common antigen of other bacterium
Problem is disturbed, positive findings and cross reaction can be accurately distinguished.
Brief description of the drawings
The structural representation of Fig. 1, fish enteron aisle vibrios Rapid detection test strip of the present invention.
1. sample pad；2. gold standard pad；3. nitrocellulose filter；4. adsorptive pads；5. carrier base；6. detection line T1；7. examine
Survey line T2；8. detection line T3；9. detection line T4；10. nature controlling line C.T1, T2, T3, T4 line be respectively outer membrane protein, flagellin,
Extracellular products and whole bacterial protein.C lines are goat anti-rabbit igg.
The testing result schematic diagram of Fig. 2, fish enteron aisle vibrios Rapid detection test strip of the present invention.
Ⅰ：Fish enteron aisle vibrios positive findings；Ⅱ：Fish enteron aisle vibrios negative findings；Ⅲ：Fish enteron aisle vibrios negative findings, but have
The immunological cross-reaction of other bacterium；Ⅳ：Test paper fails or this operation goes wrong.
Fig. 3, fish enteron aisle vibrios quick detection test paper of the present invention detect 5 kinds of vibrios result figures.
Ⅰ：Vibrio anguillarum testing result；Ⅱ：Vibrio alginolyticus testing result；Ⅲ：Vibrio harveyi testing result；Ⅳ：Secondary haemolysis
Vibrios testing result；Ⅴ：PBS testing results；Ⅵ：Fish enteron aisle vibrios testing result.
The LDL of Fig. 4, fish enteron aisle vibrios quick detection test paper of the present invention.
Fig. 5, fish enteron aisle vibrios quick detection test paper of the present invention are applied to the detection knot of fish enteron aisle vibrios in Cynoglossus semilaevis
Really).ⅰ：The testing result of healthy Cynoglossus semilaevis tissue sample liquid；ⅱ：PBS testing results；ⅲ：Suffer from skin ulcer Cynoglossus semilaevis
The testing result of tissue sample liquid.
Specific embodiment
Applicant has found under study for action, detects fish enteron aisle vibrios using fish antiserum before, although the positive for detecting
Rate is high, and its fish antiserum and fish enteron aisle vibrios react most strong, but with Vibrio anguillarum, vibrio parahaemolytious, vibrio alginolyticus, breathe out Vickers arc
There is the cross reaction of varying degree in other vibrios such as bacterium, because other vibrios have some common anti-with fish enteron aisle vibrios
Original, common antigen can cause cross reaction in immunoassay procedures, so as to the judgement to positive findings is interfered；Cause inspection
The fish enteron aisle vibrios measured is probably false positive.
In the result of above-mentioned analysis, applicant is using the species-specific antigen component of fish enteron aisle vibrios four as the four of test strips
Bar detection line, wherein, T4 detection lines are specifically designed to whole bacterial protein, if containing fish enteron aisle vibrios in testing sample, with inspection
Gold mark rabbit-anti fish enteron aisle vibrios antibody in four kinds of antigenic competition combination gold standard pads on survey line, analyte sample fluid is diffused into golden mark
During pad, gold mark rabbit-anti fish enteron aisle vibrios antibody is combined with fish enteron aisle vibrios in measuring samples, forms gold labeling antibody-bacterium compound,
And the binding site of fish enteron aisle vibrios specific antigen in gold labeling antibody is closed, prevent to be examined on gold labeling antibody and nitrocellulose filter
Antigen protein on survey line T1, T2, T3 and T4 is combined, then T1-T4 lines all redfree bands occur, and are as a result the positive；When compound
Thing diffuses to nature controlling line (C lines) place, is captured by the goat anti-rabbit igg at this, assembles and forms macroscopic band.Conversely,
If not containing fish enteron aisle vibrios in testing sample, analyte sample fluid does not form gold labeling antibody-bacterium compound when diffusing to gold standard pad,
Four kinds of antigen-reactives on gold labeling antibody and T1, T2, T3 and T4 line and form macroscopic band, 4 detection lines are all aobvious
Color, is as a result feminine gender.If having the pathogen of common antigen in testing sample containing other, gold mark fish enteron aisle vibrios antibody with
Its common antigen combines to form gold labeling antibody-bacterium compound, then in T1-T3 lines 1-3 bars line because the common antigen that contains not with gold
Mark fish enteron aisle vibrios antibody response and lighter or do not develop the color, due to being especially provided with the full bacterium egg of fish enteron aisle vibrios on T4 lines
In vain, wherein the common antigen for containing does not mark fish enteron aisle vibrios antibody response with gold, so that T4 line colors shoal, but on T4 lines also
Containing fish enteron aisle vibrios other antigen proteins in addition to common antigen, it is reacted so that T4 lines necessarily manifest with gold labeling antibody
Red, therefore, as long as there is C lines and the colour developing of T4 lines, 1 or several lines do not develop the color in T1-T3 lines, then testing result is other bacterium
Cross reaction.
The present invention is used as a plurality of detection line, wherein T4 by setting various antigen components of fish enteron aisle vibrios in detection layers
Whole bacterial protein is especially set in detection line, realizes to screen the peculiar antigen/antibody of fish enteron aisle vibrios, being not excluded for other diseases
It is accurate to differentiate fish enteron aisle vibrios positive findings and cross reaction in the case of the common antigen that opportunistic pathogen may contain；Especially at it
His pathogen contains in the case of various common antigens with fish enteron aisle vibrios, ensures to intersect because setting whole bacterial protein on T4 lines
Reaction is accurately differentiated with the fish enteron aisle vibrios positive.
First, the preparation method of test paper of the invention is as follows：
(1) preparation of gold standard pad
1. the preparation of collaurum and gold mark rabbit-anti
It is the collaurum of 20nm to use micro-wave oven-trisodium citrate reduction method that granular size is obtained, and uses 0.1mol/L carbonic acid
Potassium solution regulation collaurum pH value is 8.4；By rabbit-anti fish enteron aisle vibrios in suitable ratio add collaurum in, add BSA,
Liquid is preserved after centrifugal purification with gold mark to hang, as gold mark rabbit-anti liquid.
2. the preparation of gold standard pad
The spraying of gold mark rabbit-anti liquid will be made on the glass fibers, and freeze-drying is gold standard pad.
(2) the detection layers design and preparation of nitrocellulose filter
Detection layers be by various antigen components of pathogen it is arranged together with measuring samples while reacted, then the bacterium
All antigenic components all there is positive reaction with the antibody of the bacterium, and other bacterium only have common antigen to be handed over the bacteria antibody
Fork reaction, so, all antigen total overall reactions are the positive, and incomplete antigen reaction then to intersect, can exclude intersection anti-whereby
The interference that reply positive findings judges, so detection layers are designed as the various antigen components of pathogen while using.Furthermore, it is contemplated that
Detected pathogen may contain the situation of various common antigens with other bacterium, and especially setting a detection line makes it necessarily contain
Antigen component in addition to common antigen, cross reaction and positive findings are differentiated so as to accurate.Thus, respectively will be certain density
Four kinds of antigen components of fish enteron aisle vibrios, are necessarily sprayed at according to a determining deviation and sequentially on nitrocellulose membrane and are detected as four
Line T1, T2, T3 and T4, and T4 lines are specifically designed to the broken albumen of full bacterium；Goat anti-rabbit igg is sprayed on nitrocellulose membrane and is made
It is nature controlling line, dries, soaked in confining liquid, is dried after washing.
(3) making of fish enteron aisle vibrios quick detection test paper
Stick nitrocellulose filter, adsorptive pads, gold standard pad and sample pad successively on carrier board, then be cut into cutting machine
Test strips 3.75mm wide, after loading packaging gets stuck, are sealed in preservation in aluminium foil bag.
2nd, in the present invention, the research method of the LDL of fish enteron aisle vibrios quick detection test paper is as follows：
The pathogen that will have been purified PBS is adjusted to four concentration gradients, and bacterium is detected with fish enteron aisle vibrios quick detection test paper
Liquid.When certain concentration, there is all or part of colour developing in detection line T1-T4, then the first order concentration high of this concentration is tried to detect
The LDL of paper.
3rd, in the present invention, detection fish enteron aisle four kinds of spies of antigen protein of vibrios of fish enteron aisle vibrios quick detection test paper
The opposite sex simultaneously determines that the operation of antigen line concentration is as follows：
Four kinds of fish enteron aisle vibrios antigen proteins to preparing are coated with 96 hole elisa Plates, each albumen set three it is parallel,
And using aseptic PBS as negative control, 4 DEG C overnight.Coating buffer is suctioned out, is washed with the PBS containing 0.05%Tween-20, every time
5min, washes 3 times.3% bovine serum albumin(BSA) (BSA), 37 DEG C of closing 1h is added to wash per hole 3 times.Rabbit-anti fish enteron aisle is added per hole
Vibrios antibody (1:2000 dilutions), 37 DEG C of incubation 1h are washed 3 times.The goat anti-rabbit igg (1 of alkali phosphatase enzyme mark is added per hole:
8000 dilutions), 37 DEG C of incubation 1h are washed 3 times.Add substrate nitrite ion to react 10-20min in dark place per hole, add NaOH molten
Liquid color development stopping is reacted, the OD values at detection 405nm.
To be coated with PBS as negative control, the absorbance value in each hole when wavelength is 405nm is determined, calculate each experimental port with the moon
Property control the ratio between absorbance value (P/N), be the positive when P/N >=2.1, calculate the P/N ratios between four kinds of antigen proteins, and root
The line concentration ratio of fish enteron aisle vibrios quick detection test paper four detection lines of detection layers is determined according to result.
4th, detect that step is as follows to testing sample using fish enteron aisle vibrios quick detection test paper：Keep flat examination of the present invention
Paper detection card, to the detection μ l of sample liquid 100 are added dropwise in sample well, waits 10~15min, testing result is visually observed, according to inspection
Survey line T1, T2, T3 and T4 develop the color situation to judge whether to infect fish enteron aisle vibrios.
Testing result decision method：
I.C lines are presented red stripes, and T1, T2, T3, T4 line neither develop the color, as a result for fish enteron aisle vibrios is positive；
All there are red stripes in II.C lines and T1, T2, T3, T4 line, as a result for fish enteron aisle vibrios is negative, or the bacteria concentration
Less than test limit；
III .C lines and the aobvious red of T4 detection lines, T1, T2, T3 line occur 1 or a plurality of line does not develop the color, and are as a result fish enteron aisle arc
Bacterium is negative, but has the cross reaction of other bacterium.
IV .C lines do not develop the color, and test paper failure or this operation go wrong, and testing result is invalid.
The present invention is further illustrated below by specific embodiment.
Instrument needed for embodiment and reagent：
Refrigerated centrifuge (Sigma companies)；High speed freezing centrifuge (Beckman companies of the U.S.)；Ultracentrifuge (day
This Hitachi companies)；Spray film instrument XYZ3060 (Biodot companies)；Semi-automatic pad pasting instrument LM5000 (Biodot companies)；Slitting
Instrument (Biodot companies)；Trisodium citrate (is purchased from the factory of Chinese Shanghai reagent one, analyze pure)；Gold chloride (Sigma companies are purchased from,
Analysis is pure), goat anti-rabbit igg (is purchased from Suo Laibao companies)；Bovine serum albumin (is purchased from Sigma companies)；Tween-20 (is purchased from Suo Lai
Precious company)；Nitrocellulose filter (is purchased from Whatman companies).
Embodiment 1：The preparation of fish enteron aisle vibrios quick detection test paper gold standard pad
1. the preparation and purifying of rabbit-anti fish enteron aisle vibrios antibody
(1) preparation of rabbit-anti fish enteron aisle vibrios antibody
1. the aseptic PBS of fish enteron aisle vibrios Amplification Culture 24h, 0.01mol/L is washed 3 times at 37 DEG C, resuspended bacterium solution, adjustment
To 1 × 108CFU/mL.To a certain amount of formalin is added in bacterium solution, 24h is inactivated.Next day takes bacterium solution plate streaking inspection and goes out
Degree living.
2. new zealand white rabbit, every minor tick one week are immunized in four times.It is immune for the first time, bacterium solution is taken according to 1:2(v:V) mix
Close Freund's complete adjuvant, the injection of 6 points of back, every 0.2mL.Second immune, takes bacterium solution according to 1:2(v:V) Freund is mixed not
Freund's complete adjuvant, the injection of 6 points of back, every 0.2mL.It is immunized twice afterwards and is not added with adjuvant, per injection 0.6mL bacterium solutions.
3. after being immunized one week for the 4th time, cardiac puncture takes whole blood, at room temperature slant setting 1h, and 4 DEG C stand overnight.Next day, 4
10 at DEG C, 000 × g centrifugation 15min abandon precipitation, obtain serum.
(2) purifying of rabbit-anti fish enteron aisle vibrios antibody
1. 4 times of volume 0.06mol/L pH=4.0 acetate buffer solutions are added to serum, blood is adjusted with 0.01mol/L NaOH
Clear dilution is to pH=4.5.
2. it is slowly added dropwise caprylic acid while stirring at room temperature, addition is every 1mL serum dilutions plus 25 μ L caprylic acids, drop
Plus after continue stir 30min.
3. 10 at 4 DEG C, 000 × g centrifugation 30min abandon precipitation, collection supernatant.Supernatant is removed with 0.45pm miillpore filters
After removing suspension, volume is measured.
4. the aseptic PBS of 0.1mol/L are added according to 10% volume, and pH=7.4 is adjusted with 5mol/L NaOH.
5. 4 DEG C of supernatant measures total liquid volume afterwards to the cold, and (NH is slowly added to according to 277g/L at being kept for 4 DEG C4)2SO4Powder
End (final concentration up to 45% saturation degree) is stirring while adding, continues to stir 30min after adding, and 4 DEG C stand overnight.
6. 10 at 4 DEG C of next day, 000 × g centrifugation 15min abandon supernatant, collect precipitation.Precipitate with a small amount of 0.01mol/L PBS
Dissolving (being the 1/10 of former serum volume), dialysed 24h, and dialyzate twice need to be changed halfway.It is lyophilized, weigh, and adjust to suitable
Concentration, is stored in -80 DEG C.
(3) antibody titer is determined
ELISA Plate adds 100 μ L 5 × 10 per hole7CFU/mL fish enteron aisle vibrios bacterium solution is coated with overnight.Next day discards liquid in plate
Body, with the PBS (PBST containing 0.05%Tween-20：0.01mol/L PBS, pH=7.4 containing 0.05%Tween-20) washing
ELISA Plate, 200 μ L 3%BSA, 37 DEG C of closing 1h are added per hole；Washing ELISA Plate, sequentially adds the rabbit-anti of 100 μ L gradient dilutions
Fish enteron aisle vibrios antibody (table 2), setting 3 is parallel, and negative control adds 100 μ L 0.01mol/L PBS, 37 DEG C of incubation 1h；With
PBST washs ELISA Plate, and 100 μ L AP mark goat anti-rabbit igg antibodies, 37 DEG C of incubation 1h are added per hole；Washing ELISA Plate, adds per hole
Enter 200 μ L substrates nitrite ion (pNPP containing 1mg/mL, 0.5mol/L magnesium chloride and 10% diethanol amine) lucifuge color development 5min,
OD is determined with ELIASA405Value.The potency of result display rabbit-anti fish enteron aisle vibrios antibody is 1 × 105(table 1).
Table 1：The measure of rabbit-anti fish enteron aisle vibrios antibody titer
2. the preparation of colloid gold label rabbit-anti fish enteron aisle vibrios antibody
(1) preparation of collaurum
0.01% chlorauric acid solution 100mL, high-grade fire boiling 2min are taken, a small amount of 1% citric acid three is disposably added rapidly
Sodium solution, is put into micro-wave oven after continuing to heat 3min with middle-grade fire, and taking-up is cooled to room temperature and supplements loss moisture, is obtained
The collaurum of grain size 20nm or so.With 0.1mol/L solution of potassium carbonate and 0.1mol/L salt acid for adjusting pH value to 8.4, in room temperature
Carry out continuing antibody labeling step or be stored in 4 DEG C.
(2) preparation of colloid gold label rabbit-anti fish enteron aisle vibrios antibody
Take collaurum and add rabbit-anti fish enteron aisle vibrios antibody according to proper proportion, continuously stir 20min, be stored at room temperature
10min.To the final concentration of 1%BSA of its addition, 15min is continuously stirred, be stored at room temperature 10min.By above-mentioned solution at 4 DEG C
1,200 × g is centrifuged 15min, abandons precipitation and takes supernatant.In 13 at 4 DEG C, 500 × g centrifugation 30min abandon supernatant and take precipitation supernatant,
Precipitation gold labeling antibody cleaning solution (containing 1%BSA, 0.01mol/L PBS, pH=8.4) washing, then be centrifuged, carefully suck supernatant
Liquid, preserves precipitation liquid and (contains 3% sucrose, 1%BSA, 0.2%Tween-20 and 0.02%NaN with gold labeling antibody3's
0.01mol/L PBS, pH=8.4) it is resuspended to original volume 1/10, as gold is marked rabbit-anti fish enteron aisle vibrios antibody, is kept in dark place in 4
DEG C or carry out next step immediately.
3. the preparation of gold standard pad
By gold mark rabbit-anti fish enteron aisle vibrios antibody liquid even application on glass fibre, freeze-drying, lucifuge sealing is protected
It is stored in 4 DEG C.
Embodiment 2：The preparation of fish enteron aisle vibrios quick detection test paper detection layers
1st, prepared by fish enteron aisle vibrios outer membrane protein
1. Amplification Culture fish enteron aisle vibrios at 37 DEG C, 8 at 4 DEG C, 000 × g centrifugation 15min wash away culture medium, are used in combination
The resuspended washings of 0.01mol/L PBS 3 times, are eventually adding PBS and are concentrated into original volume 1/25.
2. to a small amount of 10% sucrose solution is added in the bacterium solution collected, after mixing, 15min is processed in -20 DEG C.Add etc.
Volume 0.4%Triton X-100 (PBS), mixes at room temperature, acts on 10min.12 at 4 DEG C, 000 × g centrifugation 15min take
Supernatant.2.5 times of volume ice ethanol are added, after mixing, in -20 DEG C of left overnights.Next day, 12 at 4 DEG C, 000 × g centrifugations
15min, takes precipitation.
3. precipitate after being washed through Tris-NaCl (pH=7.4), add isometric 0.6mol/L KI solution, after mixing, 37
DEG C water-bath 1h.12 at 4 DEG C, 000 × g centrifugation 15min take supernatant.2.5 times of volume ice ethanol are added, after mixing, at -20 DEG C
Stand overnight.Next day, 12 at 4 DEG C, 000 × g centrifugation 15min take precipitation.With 10mL 0.01mol/L PBS it is resuspended after, Yu Chao
Dialysed 24h in pure water, and a ultra-pure water is changed per 6h.After lyophilized, it is dissolved in PBS and is adjusted to suitable concn, and freeze in -80 DEG C.
2nd, prepared by fish enteron aisle vibrios flagellin
1. Amplification Culture fish enteron aisle vibrios at 37 DEG C, 1 at 4 DEG C, 500 × g centrifugation 30min wash away culture medium, 0.01mol/
The resuspended washings of L PBS 3 times, are eventually adding SPSS and are concentrated into original volume 1/25.
2. the bacterium solution pH of collection is adjusted to 2.0 with 1mol/L HCl, 30min is stirred continuously at room temperature, and keep pH
=2.0.4 DEG C 1,500 × g centrifugation 30min take supernatant.4 DEG C 533,000 × g centrifuged supernatant 1h abandon precipitation, take supernatant.On
Clear liquid 1mol/L NaOH adjust pH to 7.2, and are slowly added to (NH4)2SO4To final concentration of 2.67mol/L, ice bath stirring
30min.By mixed liquor, 4 DEG C stand overnight.Next day, 4 DEG C 533,000 × g centrifugation 15min abandon supernatant, take precipitation, and be dissolved in one
In quantitative 0.01mol/L PBS, dialysed 24h, and a dialyzate is changed per 6h.After lyophilized, it be dissolved in PBS and modulate suitable concn, protects
It is stored in -80 DEG C.
3rd, prepared by fish enteron aisle vibrios extracellular products
1. fish enteron aisle vibrios is inoculated in nutrient agar fluid nutrient medium, 37 DEG C of shaking table culture 24h.Take the liquid training of 1mL
Foster thing is coated on and has been pre-placed on one layer of nutrient agar panel of glassine paper, 37 DEG C of 24~48h of culture.
2. hypothallus is washed per flat board 6mL 0.01mol/L PBS, bacteria suspension is collected 7 at 4 DEG C, 000 × g is centrifuged
30min, after supernatant takes out remaining thalline through 0.22pm filtering with microporous membrane, adds (the NH of final concentration of 2.0mol/L4)2SO4,
1h is stirred at room temperature, and 4 DEG C of sedimentations are overnight.Next day, 4 DEG C 10,000 × g centrifugations 1h takes precipitation, is dissolved in a certain amount of 0.01mol/LPBS
In, dialyse 24h in ultra-pure water, changed per 6h ultra-pure water it is lyophilized after, with suitable concn is adjusted in PBS, freeze in -80 DEG C.
4th, the preparation of the broken albumen of the full bacterium of fish enteron aisle vibrios
Fish enteron aisle vibrios is inoculated in nutrient agar fluid nutrient medium, 37 DEG C of Amplification Culture 24h.At 4 DEG C 5,000 × g from
The heart 15min, 0.01mol/L PBS washings are resuspended 3 times.Bacterium solution is adjusted to 1 × 108CFU/mL, ultrasonic disruption crusher machine
(Sonics&Materials；Amplitude 39%；Pulse on, 3sec；Pulse off, 3sec) to liquid clarification, as fish enteron aisle
The broken bacterium sample of vibrios, suitable concentration is adjusted to PBS, is frozen in -80 DEG C.
5. double crush syndrome detects the specificity of fish enteron aisle four kinds of antigen proteins of vibrios and determines antigen line concentration
1. the four kinds of fish enteron aisle vibrios antigen proteins that will be prepared are coated with (100 μ L/ holes), each albumen in 96 hole elisa Plates
If three parallel, and using aseptic PBS as negative control, 4 DEG C overnight.
2. coating buffer is suctioned out, with the PBS (PBST containing 0.05%Tween-20：0.01mol/L containing 0.05%Tween-20
PBS, pH=7.4) wash, each 5min is washed 3 times.
3. the bovine serum albumin(BSA) (PBS preparations) of 200 μ L 3%, 37 DEG C of closing 1h is added to be washed 3 times with 2. method per hole.
4. 100 μ L rabbit-anti fish enteron aisle vibrios antibody (1 are added per hole:2,000 dilutions), 37 DEG C of incubation 1h, with 2. method washing 3
It is secondary.
5. the goat anti-rabbit igg (1 of 100 μ L alkali phosphatase enzyme marks is added per hole:8,000 dilutions), 37 DEG C of incubation 1h, with 2.
Method is washed 3 times.
6. 200 μ L substrate nitrite ions, dark place reaction 10-20min are added to add 50 μ L 2mol/L NaOH molten per hole per hole
Liquid color development stopping is reacted, OD values at detection 405nm.
To be coated with PBS as negative control, each hole absorbance value when wavelength is 405nm is surveyed, calculate each experimental port right with feminine gender
The ratio between irradiation absorption value (P/N), is the positive when P/N >=2.1, calculates the P/N ratios between four kinds of antigen proteins, determines fish enteron aisle
The line concentration ratio of four detection lines of vibrios quick detection test paper detection layers.Four kinds of antigens on T1, T2, T3, T4 line of gained
The line concentration of albumen is respectively outer membrane protein 0.3mg/mL, flagellin 0.5mg/mL, extracellular products 1.0mg/mL, full bacterium and breaks
The white 0.4mg/mL of scrambled egg.
6. the preparation of fish enteron aisle vibrios quick detection test paper detection layers
Fish enteron aisle vibrios outer membrane protein, flagellin, extracellular products and full bacterium are crushed into albumen with 0.01mol/L PBS
Concentration is separately adjusted to angularly 0.3mg/mL, 0.5mg/mL, 1.0mg/mL and 0.4mg/mL, with spray film instrument respectively by outer membrane protein, whip
The broken albumen of hairless protein, extracellular products, full bacterium is sprayed on nitrocellulose filter as detection line T1, T2, T3 and T4, four inspections
At a distance of 1.5mm between survey line.It is 0.5mg/mL to adjust goat anti-rabbit igg concentration with 0.01mol/L PBS, is sprayed at nitric acid fine
As nature controlling line (C lines) on dimension film, with detection line T4 at a distance of 5mm, nature controlling line is near adsorptive pads (Fig. 1 for nature controlling line：4), detection line
T1 is near sample pad (Fig. 1：1).After the nitrocellulose filter drying at room temperature that will be coated with, at 37 DEG C confining liquid (contain 3%BSA,
0.01mol/L PBS, pH=7.4) 1h is soaked, PBST is rinsed 3 times, and each 5min is dried.
7. the making and application of fish enteron aisle vibrios quick detection test paper
In carrier base (Fig. 1：5) upper nitrocellulose filter (Fig. 1 is pasted on successively：3), gold standard pad (Fig. 1：2), sample pad
(Fig. 1：1) with adsorptive pads (Fig. 1：4), it is assembled into test strips whole plate.Wherein nitrocellulose membrane upper end-face edge is overlapped in adsorptive pads side
Under edge, and nitrocellulose filter lower edge, then under gold standard pad upper end-face edge, the upper end-face edge of sample pad overlaps gold
Mark on the lower edge of pad (Fig. 1).
The test strips justifying cutting machine that will be assembled is cut into 3.75mm test strips wide, is fitted into during packaging gets stuck, and close
Envelope is stored in the aluminium foil bag of drier.
Embodiment 3：The detection method of fish enteron aisle vibrios quick detection test paper
(1) preparation of measuring samples
Take the doubtful fish lesion tissue for suffering from fish enteron aisle vibrios, kidney or blood, per 1g tissue samples with 5mL 0.01mol/L without
Bacterium PBS or 0.9% SPSS, 3~5min is fully ground under condition of ice bath, the lapping liquid conduct obtained with silk cover filtering
Detection sample liquid.
(2) fish enteron aisle vibrios quick detection test paper detection measuring samples
Detection paper card of the present invention is kept flat, to the detection μ l of sample liquid 100 are added dropwise in sample well, 10~15min, naked eyes is waited
Observation testing result, judges whether aquatic livestock infects fish enteron aisle vibrios according to detection line T1, T2, T3 and T4 colour developing situation.
(3) judgement (Fig. 2) of result
Ⅰ：C lines are presented red stripes, and T1, T2, T3, T4 line neither develop the color, and show that the individuality has infected fish enteron aisle vibrios.
Ⅱ：All there are red stripes in C lines and detection line T1, T2, T3, T4 line, as a result for fish enteron aisle vibrios is negative, or should
Bacteria concentration is less than test limit.
Ⅲ：C lines and the aobvious red of T4 detection lines, T1, T2, T3 line occur 1 or a plurality of line does not develop the color, and show that the individuality is not felt
Dye fish enteron aisle vibrios, but other pathogens that there is common antigen with fish enteron aisle vibrios have been infected, as a result it is judged to cross reaction.
Ⅳ：C lines do not develop the color, and test paper failure or this operation go wrong, and testing result is invalid.
Embodiment 4：Specific, the repeated and stability test of fish enteron aisle vibrios quick detection test paper
(1) specificity
The Vibrio anguillarum of purifying, vibrio alginolyticus, Vibrio harveyi, vibrio parahaemolytious and fish enteron aisle vibrios are adjusted to appropriate dense
Degree, using PBS as negative control, using more than the invention detection paper 6 groups of samples, as a result show, Vibrio anguillarum, vibrio alginolyticus,
Vibrio harveyi, vibrio parahaemolytious all make detection line T1, T2, T3, T4 and nature controlling line develop the color, wherein T3 line colors it is shallow compared with PBS (I,
IIth, III, IV), show that there is common antigen with fish enteron aisle vibrios in this 4 kinds of extracellular productses of bacterium；PBS make detection line T1, T2,
T3, T4 and nature controlling line develop the color (V)；Fish enteron aisle vibrios makes detection line T1, T2, T3 and T4 not develop the color, nature controlling line colour developing
(Ⅵ).These results show that quick detection test paper of the invention has good specificity, can accurately distinguish fish enteron aisle vibrios
Infection and the cross reaction of other bacterium.(Fig. 3)
(2) repeatability
Using different batches detection paper fish enteron aisle vibrios sample and PBS, every group is repeated 5 times, as a result without significant difference.
(3) stability
Test paper of the present invention with 4 DEG C and at room temperature lucifuge sealed storage respectively, detection paper fish enteron aisle arc was used every 1 month
Bacterium sample and PBS.Result is 12 months to store the term of validity at 4 DEG C, and the term of validity is 6 months at room temperature.
(4) test strips LDL
The fish enteron aisle vibrios that will have been purified is adjusted to 5 × 10 with PBS7、5×106、5×105、5×104CFU/mL.Use fish intestines
Road vibrios quick detection test paper detects this five kinds bacterium solution from concentration is high to low, as a result shows, 5 × 107、5×106、5×
105During CFU/mL, detection line T1-T4 does not develop the color all, and 5 × 104CFU/mL makes T1-T4 lines develop the color, then test paper of the present invention is to fish
The lowest detection lower limit of enteron aisle vibrios is 5 × 105CFU/mL.(Fig. 4)
Embodiment 5：Suffer from the detection of fish enteron aisle vibrios in canker Cynoglossus semilaevis
Morbidity Cynoglossus semilaevis skin surface has different size of ulcer, a face of festering, and the bleeding of fin base portion, festers, the sick fish having
Belly has ascites in expanding, and enteron aisle bulging is almost deviate from outside anus, and intestinal wall is thin, and enteral is hydraulically full, dissects and finds sick fish liver hair
White or congested rubescent, enteron aisle is without food, gollbladder dilation.Sterile working takes the tissue such as disease fish liver,spleen,kidney, per 1g tissue samples 5mL
The aseptic PBS of 0.01mol/L, 3~5min is fully ground under condition of ice bath, and the lapping liquid obtained with silk cover filtering is used as detection sample
Liquid.Healthy Cynoglossus semilaevis tissue and PBS are detected as negative control simultaneously.
Detection paper card of the present invention is kept flat, to the detection μ l of sample liquid 100 are added dropwise in sample well, 10~15min, naked eyes is waited
Observation testing result, it is found that ill Cynoglossus semilaevis sample liquid makes C lines line red, and T1-T4 detection lines neither develop the color, and show sample liquid
In have fish enteron aisle vibrios (Fig. 5：ⅲ).Healthy Cynoglossus semilaevis tissue sample liquid and PBS all make C lines and 4 aobvious red of detection line
(Fig. 5：ⅰ、ⅱ).
The tissue grinder liquid such as the liver,spleen,kidney of above-mentioned ill Cynoglossus semilaevis, streak inoculation is in 2216E flat boards, 25 DEG C of cultures
24h, the consistent dominant colony of picking form carries out purifying culture, until pure culture bacterial strain is obtained, using Physiology and biochemistry and molecule
Biological method identified, the amplification of 16S rRNA gene orders is carried out using 27F and 1492R, and carry out hsp60 gene sequences
The amplification of row, amplified production gives certain Gene Tech. Company Limited to be sequenced, and analyzes and identifies result and is shown as fish enteron aisle vibrios sense
Dye, shows that fish enteron aisle vibrios quick detection test paper of the invention has detection specificity well.
Claims (8)
1. a kind of fast Test paper of fish enteron aisle vibrios, described Test paper includes that carrier board, sample pad, gold standard pad, nitric acid are fine
The plain film of dimension and adsorptive pads, wherein sample pad and adsorptive pads are located at the two ends of carrier board, and nitrocellulose filter is located in carrier board
Portion；Sample pad is provided with the gold standard pad for being loaded with gold mark rabbit-anti fish enteron aisle vibrios antibody, gold standard pad one with nitrocellulose filter intersection
End margin is overlapped under sample pad, and other end imbricate is on nitrocellulose filter；Characterized in that, described nitric acid
Since being provided with four detection line T1s, T2, T3, T4 and the nature controlling line that are arranged in order near the gold standard pad side on cellulose membrane
C, wherein detection line T1 are the outer membrane protein of fish enteron aisle vibrios, detection line T2 is that flagellin, detection line T3 are fish enteron aisle vibrios
Extracellular products, detection line T4 be full bacterium crush albumen；Nature controlling line C is coated with goat anti-rabbit igg.
2. the fast Test paper of fish enteron aisle vibrios as claimed in claim 1, the preparation side of described rabbit-anti fish enteron aisle vibrios antibody
Method is as follows：
By fish enteron aisle vibrios Amplification Culture, and bacterial concentration is adjusted to 1 × 108CFU/mL, is immunized purebred new after Formalin inactivation
Western orchid White Rabbit；Freund's complete adjuvant is mixed into for the first time,；Booster immunization after one week is spaced, incomplete Freund's adjuvant is mixed into, is spaced
It is repeated once after two weeks；Blood is taken after one week, isolated serum, purifying obtains rabbit-anti fish enteron aisle vibrios antibody.
3. the fast Test paper of fish enteron aisle vibrios as claimed in claim 1, described fish enteron aisle vibrios outer membrane protein, its preparation side
Method is as follows：
By fish enteron aisle vibrios Amplification Culture, phosphate buffer washing after centrifugation is resuspended to original volume 1/25, adds 10% sugarcane
Sugar, 15min is acted on after mixing at -20 DEG C；Isometric 0.4%Triton-X100 is added at room temperature, and effect is centrifuged after a period of time
Supernatant is taken, 2.5 times of -20 DEG C of ice ethanol of volume is added overnight；Centrifuging and taking is precipitated, and after washing is resuspended, adds isometric KI solution,
37 DEG C of effect 1h；Centrifuging and taking supernatant, adds 2.5 times of -20 DEG C of volume ice ethanol overnight；Centrifuging and taking is precipitated, resuspended to dialyse afterwards, freeze
Obtain fish enteron aisle vibrios outer membrane protein.
4. the fast Test paper of fish enteron aisle vibrios as claimed in claim 1, described fish enteron aisle vibrios flagellin, its preparation side
Method is as follows：
It is PBS washings after centrifugation, resuspended to original volume 1/20 by fish enteron aisle vibrios Amplification Culture, adjust pH=with 1mol/L HCl
2.0, low-temperature centrifugation takes supernatant after stirring 30min；Supernatant adjusts pH to neutrality, adds (NH3)2SO4, 4 DEG C stand overnight；It is low
Warm ultracentrifugation, takes precipitation, it is resuspended after dialysis, lyophilized obtain fish enteron aisle vibrios flagellin.
5. the fast Test paper of fish enteron aisle vibrios as claimed in claim 1, described fish enteron aisle vibrios extracellular products, its preparation side
Method is as follows：Fish enteron aisle vibrios is inoculated in after being cultivated in fluid nutrient medium, liquid culture is coated and has been pre-placed one layer
On the solid agar flat board of glassine paper, 37 DEG C of culture 36h；Hypothallus is washed with PBS, high speed centrifugation at 4 DEG C after collects thalline；Will be upper
Clearly through filtering with microporous membrane, (NH is added4)2SO4, 4 DEG C stand overnight；Then low-temperature centrifugation takes precipitation, resuspended to dialyse afterwards, freeze
Obtain fish enteron aisle vibrios extracellular products.
6. the fast Test paper of fish enteron aisle vibrios as claimed in claim 1, the broken albumen of the described full bacterium of fish enteron aisle vibrios, its system
Preparation Method is as follows：
By fish enteron aisle vibrios Amplification Culture, bacterium solution is placed in cryogenic pulverization in sonicator and obtains the broken liquid of full bacterium, freezes
Albumen is crushed to the full bacterium of fish enteron aisle vibrios.
7. the fast Test paper of fish enteron aisle vibrios described in claim 1 is in non-diseases diagnoses and treatment purpose detection fish enteron aisle vibrios
Application.
8. a kind of method that non-diseases diagnoses and treatment purpose detects fish enteron aisle vibrios, it is characterised in that described method is to use
The fast Test paper of fish enteron aisle vibrios described in claim 1 detects testing sample, and the criterion of described method is as follows：
If nature controlling line C line is presented red stripes, and detection line T1, T2, T3, T4 line neither develop the color, and are as a result fish enteron aisle vibrios
It is positive；
If red stripes all occur in nature controlling line C line and detection line T1, T2, T3, T4 line, as a result for fish enteron aisle vibrios is negative；
If nature controlling line C line and detection line T4 detection lines develop the color, and detection line T1, T2, T3 line occur 1 or a plurality of line does not show
Color, then testing result is negative fish enteron aisle vibrios, but has the cross reaction of other bacterium.
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