CN109781980A - African swine fever virus rapid detection card and its application - Google Patents

Abstract

The invention discloses a kind of African swine fever virus rapid detection card and its applications, it is intended to solve the difficult technical problem of African swine fever virus detection.The polyclonal antibody PoAbI of anti-African swine fever virus p30, p54 and p72 albumen of colloid gold label is adsorbed in the gold-labelled pad of the detection card；The nature controlling line C that film is equipped with goat-anti or rabbit anti-mouse IgG antibody or staphylococcus aureus SPA printing is detected, the detection line T of the polyclonal antibody PoAbII printing of anti-African swine fever virus p30, p54 and p72 albumen is additionally provided with.Design a kind of application method of African swine fever virus rapid detection card: acquisition sample to be examined adds PBS or water to be suspended or grind；Instill the sample-adding end of the African swine fever virus rapid detection card；Horizontal positioned observation result.African swine fever virus rapid detection card of the invention uses double antibody sandwich method, and high specificity, sensibility are high, and detection efficiency is high, highly effective, has important practical application value.

Description

African swine fever virus rapid detection card and its application

Technical field

The present invention relates to technical field of immunoassay, and in particular to a kind of African swine fever virus rapid detection card and its answers With.

Background technique

African swine fever (African swine fever, ASF) is by African swine fever virus (African swine fever Virus, ASFV) caused by the porcines such as main infection domestic pig, wild boar, shrub pig deadly infectious disease, the death rate is high, to pig raising Industry causes huge economic loss.ASFV is the unique a kind of DNA arboviruse being currently known, and ASFV only infects domestic pig and open country Pig does not infect the mankind, is in usually subclinical infection after wild boar infection ASFV, in addition, soft ticks is also host and the communication media of ASFV. ASFV is found in nineteen twenty-one in Kenya for the first time, and then many countries in African south and east find depositing for ASFV ?.It when afternoon 17 on the 2nd of August in 2018, is diagnosed through China Animal Health and Epidemiology Center, Shenyang City, Shen North Street, Shenbeixin District Doubtful African swine fever epidemic situation occurs for five or five communities, and makes a definite diagnosis when the August morning 11 on the 3rd in road (new city).It is black by January, 2019 There is ASF epidemic situation in succession in 22 provinces such as Longjiang, Jiangsu, Anhui.Therefore, it currently needs to reinforce the quarantine and prevention and control to ASF.

The Clinical symptoms that pig infects ASFV is high fever, diarrhea, bleeding and heat flush.Some clinical symptoms and postmortem knot Fruit infects classic swine fever virus (Classical swine fever with pig such as splenomegaly, lymph node and kidney hemorrhagic disease Virus, CSFV) symptom and indistinction.The prior art be directed to not yet ASFV vaccine and effective treatment method, control mode It relies primarily on the stringent assanation of execution and destroys the mode of sick pig by fire.Therefore, the quick, reliable of ASFV detects to Guan Chong Want-the quick detection of ASFV is not only necessary to ASF prevention and control, and the pig disease similar with its clinical symptoms is identified Necessary to diagnosis.

Currently, detection African swine fever diagnostic method has hemadsorption test, direct immuno fluorescence test, animal inoculation pvaccination examination It tests, ELISA, polymerase chain reaction (Polymerase chain reaction, PCR) etc..Wherein hemadsorption test, straight The sensibility for connecing immunofluorescent test is not high and specific not strong, and animal inoculation pvaccination test is dangerous.Diagnose the conventional method of ASFV It is progress virus purification (VI) in pig bone marrow (PBM) cell culture, although this method is reliable and sensitive, which is taken Between too long (6 days).The above method is complicated for operation, time-consuming, laborious when measuring, and needs specific instrument and equipment and professional technique people Member is difficult to popularize in base.

Therefore, in view of problem above, a kind of not only high specificity, high sensitivity are developed, but quickly, it is easy, can directly detect The quick detection test paper of ASFV is of great significance.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of African swine fever virus rapid detection cards, to solve African swine fever The technical problem of viral diagnosis hardly possible.

In order to solve the above technical problems, technical thought of the invention is as follows:

Inventor is the target in ASFV Genotyping by long-term, numerous studies and practice, the CP204L gene in conjunction with ASFV The P30 albumen of gene, coding is one of main structural proteins of ASFV, early expression and secretion in virus infection, and It plays a role during Virus entry；E183L gene is Genotyping target, and the P30 albumen of coding is ASFV main One of structural proteins, be located at virion lipoid outer membrane, play a significant role in virus infection, be related to virus and invade Enter, transmembrane domain, the formation to virus membrane antigen be it is required, there is preferable immunogenicity and than more conservative.P72 albumen It is one of main structural proteins of ASFV, is present in the surface of viral capsid, there is good immunogenicity, be expressed in virus The advanced stage of infection can occur immunological response with the serum of all infection pigs, P30, P30, P72 albumen is selected to be applied to African pig In pestivirus rapid detection card.Specific technical solution is as follows:

A kind of African swine fever virus rapid detection card, including test strips are designed, the test strips include support plate and are fixed on institute The adsorption layer in support plate is stated, the adsorption layer is followed successively by sample pad, gold-labelled pad, detection film and water absorption pad since test lead, The polyclonal antibody of anti-African swine fever virus p30, p54 and p72 albumen of colloid gold label is adsorbed in the gold-labelled pad PoAbI；The detection film is equipped with the nature controlling line C of goat-anti or rabbit anti-mouse IgG antibody or staphylococcus aureus SPA printing, It is additionally provided with the detection line T of the polyclonal antibody PoAbII containing anti-African swine fever virus p30, p54 and p72 albumen.

Wherein, the polyclonal antibody PoAbI is made by following steps:

(1) difference prokaryotic expression nucleotide sequence is as shown in SEQ ID NO.1p30Gene, nucleotide sequence such as SEQ ID Shown in NO.2p54Gene, nucleotide sequence are as shown in SEQ ID NO.3p72Gene, preparation African swine fever virus p30, P54 and p72 recombinant protein；

(2) recombination p30, p54 and p72 protein immunization rabbit is taken respectively；

(3) purifying of hyper-immune serum caprylic acid-ammonium obtains the IgG of polyclonal antibody PoAbI.

Preferably, in the step (2), the dosage of every rabbit is that 100~200 μ g/1ml recombinate p30 when animal immune Albumen, 100~200 μ g/1ml recombinate p54 albumen, and 100~200 μ g/1ml recombinate p72 albumen.

Preferably, the ELISA potency of the polyclonal antibody PoAbI is 1:2 × 10³~1:2 × 10⁷。

Wherein, the polyclonal antibody PoAbII is made through following steps:

(1) difference prokaryotic expression nucleotide sequence is as shown in SEQ ID NO.1p30Gene, nucleotide sequence such as SEQ ID Shown in NO.2p54Gene, nucleotide sequence are as shown in SEQ ID NO.3p72Gene, preparation African swine fever virus p30, P54 and p72 recombinant protein；

(2) recombination p30, p54 and p72 protein immunization mouse is taken respectively；

(3) purifying of hyper-immune serum caprylic acid-ammonium obtains the IgG of polyclonal antibody PoAbII.

Preferably, in the step (2), the dosage of every mouse is 50~100 μ g/500 μ l recombination when animal immune P30 albumen, 50~100 μ g/500 μ l recombinate p54 albumen, and 50~100 μ g/500 μ l recombinate p72 albumen.

Preferably, the ELISA potency of the polyclonal antibody PoAbII is 1:3.2 × 10⁴To 1:8.192 × 10⁶。

Preferably, the support plate is made of the toughness PVC material not absorbing water；The sample pad is by glass fibre cotton, Buddhist nun Imperial film, PVDF membrane or polyester film are made；The gold-labelled pad is made of glass fibre cotton；The detection film is by nitric acid fibre Plain film, pure cellulose film or carboxylated cellulose film is tieed up to be made；The water absorption pad is made of absorbent filter.

The application method of above-mentioned African swine fever virus rapid detection card the following steps are included:

(1) sample to be examined is acquired, adds PBS or water to be suspended or grind, obtains solution to be checked；

(2) solution to be checked is instilled to the sample-adding end of the African swine fever virus rapid detection card；Horizontal positioned 3min~6 Min observes result；

(3) it if the nature controlling line C and detection line T of detection card show two rufous bands, indicates to contain ASFV in measuring samples At least one of p30 albumen, p54 albumen, p72 proteantigen；

If the nature controlling line C for only detecting card shows a rufous band, indicate measuring samples be not detected ASFV p30 albumen and/or P54 albumen and/or p72 proteantigen；

Show to detect misoperation or detection card failure if detection card does not show any band.

Preferably, the sample is Swine serum, sick pig tissue, any one in dead porcine tissue, excrement, vaccine, body fluid.

Compared with prior art, the beneficial technical effect of the present invention lies in:

1. rapid detection card of the present invention is to rely on double antibody sandwich method, preferably rabbit-anti ASFV p30 albumen, p54 albumen and p72 Protein-specific polyclonal antibody PoAbI is as colloid gold label monoclonal antibody, preferably anti-ASFV p30 of cavy, p54 and p72 albumen Polyclonal antibody PoAbII is prepared for detection line, and high specificity, sensibility are high, and detection efficiency is high, highly effective.

2. rapid detection card of the present invention is adaptable, do not limited by the source of host sample, sample can be Swine serum, disease Porcine tissue, dead porcine tissue, excrement, vaccine, the body fluid being even secreted from animal, including blood, serum, blood plasma, urine, eye Tear, saliva etc..

3. the testing result intuitive display of rapid detection card of the present invention, easily judgement, result judgement is intuitive, accurate, simple bright , for example, using be displayed in red or brownish red detection trace and compare trace as detect the positive and negative marker, i.e., in fibre It ties up and only shows that a brownish red compares trace C in plain film layer, indicate to infect in test sample without ASFV；If in cellulose film layer Two brownish reds of upper display, a control trace C and a brownish red detect trace T, then it represents that tested animal has ASFV infection.

4. rapid detection card of the present invention is at low cost, do not needed separately using colloidal gold immuno-chromatography test paper strip of the present invention with other Instrument, equipment and reagent save big measuring appratus, equipment and additive reagent expense；Profession and layman can be whenever and wherever possible Real-time online detection is carried out, without paying expert diagnosis expense and correlative charges, testing cost can be greatlyd save, reduce check fee With.

5. rapid detection card of the present invention is easy to operate, quickly and is convenient for carrying and saves, as long as being waited in detection process It is added dropwise to test lead after the dilution of sample product, can determine that testing result within 5 minutes or so, is suitble to scene ASFV diagnosis, can satisfy The demand that grassroots organization, slaughterhouse, raiser diagnose ASFV, has a vast market application scenarios.

Detailed description of the invention

Fig. 1 is the SDS-PAGE electrophoresis of p30, p54 and p72 albumen of recombinant bacterium expression；

In figure, M Marker；1 is p30 ultrasound supernatant；2 be p30 ultrasound precipitation；3 compare for pET28a zero load；4 be p54 ultrasound Supernatant；5 be p54 ultrasound precipitation；6 be p72 ultrasound supernatant；7 be p72 ultrasound precipitation；

Fig. 2 is the SDS-PAGE electrophoresis of recombinant bacterium expression p30, p54 and p72 albumen of purifying；

In figure, M Marker；1 is p30 albumen；2 be p54 albumen；3 be p72 albumen；

Fig. 3 is the structural schematic diagram of test strips inside rapid detection card；

In figure, 1 is support plate, and 2 be detection film, and 3 be sample pad, and 4 be gold-labelled pad, and 5 be nature controlling line C, and 6 be detection line T, and 7 be to inhale Water cushion；

Fig. 4 is rapid detection card complete structure schematic diagram；

Fig. 5 is that rapid detection card testing result judges schematic diagram；

In figure, A indicates ASFV infection；B indicates negative, i.e., is uninfected by ASFV in sample；Nature controlling line C line does not develop the color in figure C, indicates Test result is invalid.

Specific embodiment

Illustrate a specific embodiment of the invention with reference to the accompanying drawings and examples, but following embodiment is used only in detail It describes the bright present invention in detail, does not limit the scope of the invention in any way.

Related instrument and equipment is routine instrument device unless otherwise instructed in the examples below；It is related Reagent is commercially available conventional reagent unless otherwise instructed；Related test method is unless otherwise instructed conventional method.

Embodiment one: preparation African swine fever virus rapid detection card

1. codon is transformed

According to the 2008 strain sequence (GenBank:MH910495.1) of Georgia that GenBank is announced, studied in conjunction with inventor Practical experience simultaneously considers large intestine bar expression system codon preference, avoids hairpin structure in combination with mRNA secondary structure, especially It is the RNA structure near ribosome bind site, carries out codon transformation, optimization, improved nucleotide sequence such as SEQ The p54 albumen base of the p30 protein gene of ASFV shown in ID NO.1, nucleotide sequence ASFV as shown in SEQ ID NO.2 Cause, the p72 protein gene of nucleotide sequence ASFV as shown in SEQ ID NO.3；MRNA can be effectively improved by genetic modification Stability and protein translation efficiency, finally improve expressing quantity, provide material base for the great expression of protein.

2. the preparation of prokaryotic expression carrier

(1) using pUC57 plasmid as frame, by Shanghai, Sheng Gong bioengineering Co., Ltd is respectively synthesized containing ASFVp30、p54、p72 The recombinant vector of gene, and it is respectively designated as pUC57-p30, pUC57-p54, pUC57-p72；

(2) using above-mentioned synthesis recombinant vector pUC57-p30, pUC57-p54, pUC57-p72 as template, respectively according to templet gene Sequence design clone genep30、p54、p72Specific primer to as follows:

p30:

P30-F:5 '-AGGATCCATGGACTTCATCCTGAACAT-3 ' (SEQ ID NO.4)；

P30-R:5 '-GCCTCGAGTTATTTTTTTTTCAGCAG -3 ' (SEQ ID NO.5)；

p54:

P54-F:5 '-AGGATCCATGGACTCTGAATTCTTCCA-3 ' (SEQ ID NO.6)；

P54-R:5 '-GCCTCGAGTTACAGAGAGTTTTCCAG-3 ' (SEQ ID NO.7)；

P72:

P72-F:5 '-AGGATCCATGGCTTCTGGTGGTGCTTTCTG-3 ' (SEQ ID NO.8)；

P54-R:5 '-ACTCGAGTTAGGTAGAGTAACGCAGAAC-3 ' (SEQ ID NO.9)；

(3) PCR amplification is carried out, obtains ASFV respectivelyp30、p54、p72Gene is simultaneously cloned and is connected to pET28a carrier, structure Build prokaryotic expression carrier pET28a-p30, pET28a-p54 and pET28a-p72；

(4) prokaryotic expression carrier pET28a-p30, pET28a-p54 and pET28a-p72 are converted into e. coli bl21 respectively DE3 competent cell obtains expression bacterial strain pET28a-p30-BL21, pET28a-p72-BL21 and pET28a-54-BL21；

(5) ASFV p30, p54, p72 albumen are expressed respectively and is purified using escherichia expression system, the specific method is as follows:

Recombinant bacterium is cultivated in LB culture medium to OD₆₀₀When=0.6~0.8, the isopropyl-of final concentration of 0.3 mmol/L is added β-D- thiogalactoside (IPTG) is used as inducer, and 30 DEG C, 12 h of 220r/min inducing expression, harvest expresses bacterium；It will obtain Thallus be resuspended in 0.01mol/L PBS buffer solution according to 1g/ml, be collected by centrifugation after ultrasonication supernatant precipitating, SDS- PAGE electrophoretogram is as shown in Figure 1；Through Ni column purification albumen；Albumen dialysis after purification is removed into imidazoles, is collected in bag filter Solution discards precipitating through 8000r/min, 4 DEG C of 10 min of centrifugation, collects supernatant and obtains p30, p54 and p72 albumen as immune Antigen and envelope antigen, SDS-PAGE electrophoresis is as shown in Fig. 2, -20 DEG C save backup.

3. animal immune

4 groups of tests: 2 groups of immune new zealand white rabbits, 2 groups of immune guinea pigs are set；

New zealand white rabbit is immune by the method for dorsal sc multi-point injection, immune with purifying respectively with Freund's complete adjuvant The emulsification of antigen p30, p54 and p72 protein mixture, immunizing dose are respectively (+100 μ g/ of+100 μ g/p54 of 100 μ g/p30 P72) and (200 μ g/p30+200 μ g/p54+200 μ g/p72) every rabbit be immunized 1ml, every group each 4；After first immunisation 21 days It is carried out in the same way with dosage booster immunization 3 times with incomplete Freund's adjuvant immunizing antigen respectively, after the 3rd booster immunization Two weeks, ear edge vein exploitating blood, indirect ELISA surveys serum titer the results show that rabbit polyvalent antibody potency is in 1:2 × 10³To 1:2 × 10⁷Between.

Cavy is immune by the method for dorsal sc multi-point injection, with Freund's complete adjuvant respectively with immunizing antigen p30, The emulsification of p54 and p72 protein mixture, difference immune guinea pig, every group each 4, immunizing dose is respectively (50 μ g/p30+50 μ g/ P54+50 μ g/p72) and (100 μ g/p30+100 μ g/p54+100 μ g/p72) every 500 μ l of immunized guinea pigs；In first immunisation 21 It is carried out in the same way with dosage booster immunization 3 times with incomplete Freund's adjuvant immunizing antigen respectively after it, the 3rd reinforcement is exempted from Two weeks after epidemic disease, carry on the back mesopodium venous blood sampling, indirect ELISA survey serum titer, as the result is shown cavy polyvalent antibody potency 1:3.2 × 10⁴To 1:8.192 × 10⁶Between.

4. the preparation of polyclonal antibody

New zealand white rabbit is lain supine upon on station, pareordia hair is cut off, with the tincture of iodine, alcohol disinfecting skin.It is touched with left hand The 3rd~4 intercostal space of left border of sternum selects the most obvious place of heartbeat to make point of puncture, and right hand syringes insert the needle into chest Syringe needle when feeling heartbeat by syringe needle, then is lunged heart, then extracts blood out by chamber.Hyper-immune serum caprylic acid-ammonium Purifying obtains the IgG of polyclonal antibody PoAbI, and indirect ELISA surveys potency, and -80 DEG C save backup.

Cavy is lain supine upon on station, pareordia hair is cut off, with the tincture of iodine, alcohol disinfecting skin.Breastbone is touched with left hand The 3rd~4 intercostal space of left border selects the most obvious place of heartbeat to make point of puncture, and right hand syringes insert the needle into thoracic cavity, leads to When crossing syringe needle and feeling heartbeat, then syringe needle is lunged into heart, then extracts blood out.The purifying of hyper-immune serum caprylic acid-ammonium The IgG of polyclonal antibody PoAbII is obtained, indirect ELISA surveys potency, and -80 DEG C save backup.

Indirect ELISA is surveyed polyclonal antibody bioactivity result and is taken shown in table 1:

1 indirect ELISA of table surveys polyclonal antibody potency

。

5. the preparation of gold labeling antibody

(1) preparation of colloidal gold:

Taking 100 mL ultrapure waters to be placed in the conical flask of 500 mL cleaning, 1 mL 1%(w/v be added) chlorauric acid solution boils；It is stirring Mix the 1 mL 1%(w/v that Fresh is rapidly added under state) sodium citrate solution, boils about 3 min to solution colour by Huang Discoloration is aubergine, continues to boil 2 min；It is cooled to room temperature to solution, mends ultrapure water to 100 mL, with 0.2 mol/L K₂CO₃ Adjust pH to 9.0,4 DEG C be kept in dark place it is spare.

(2) most suitable labelled protein concentration mensuration

The IgG for taking polyclonal antibody PoAbI to be marked is dialyzed overnight with 20 4 DEG C of mmol/L dobell's solutions (pH 9.0). With 25 μ L ultrapure water 1:2,1:4,1:8 ... difference doubling dilution viral monoclonal antibody to be marked in microwell plate；Each hole is added 125 μ L colloidal gold solution, is stored at room temperature 5 min；125 μ L, 1 mol/L NaCl solution is added；Each hole color with protein concentration drop It is low and from red become blue.Using the protein concentration of the IgG highest dilution of the unchanged indigo plant of color as the most suitable label concentration of colloidal gold, When colloid gold label, protein concentration increases by 20%；

The optimum mark concentration of interpretation of result PoAbI is respectively as follows: 10 μ g/mL after measured.

(3) colloid gold label of polyclonal antibody PoAbI IgG

The IgG of the polyclonal antibody PoAbI to be marked of the most suitable protein concentration of 2 mL is taken, 10 mL colloidal gold solution (pH are added 9.0) it, mixes rapidly, room temperature acts on 30 min；Mixeding liquid volume 10% is added contains 10%(w/v) bovine serum albumin(BSA) (BSA) 20 mmol/L dobell's solutions mix rapidly, and room temperature acts on 10 min-15 min；4 DEG C, 12000 r/min centrifugation 30min, Carefully remove supernatant；Precipitating is resuspended with the 20 mmol/L dobell's solutions containing 1%(w/v) BSA, is ibid centrifuged, abandons supernatant；Weight After backwashing is washed 1 time, precipitating 1 mL TB buffer (20 mmol/L Na2B4O7,1%(w/v) BSA, 0.1%(w/v) NaN₃) adjustment Gold labeling antibody is to 0.2 mg/mL, and sufficiently after piping and druming, 4 DEG C are saved backup.

6. detecting the preparation of film

Can will be identified simultaneously respectively with physiological saline the polyclonal antibody PoAbII of ASFV p30, p54 and p72 albumen IgG and SPA is diluted to 2.0mg/mL and 1.0mg/mL, spare with 0.22 μm of filtering with microporous membrane.NC film is placed in 3000 specking of XYZ It on instrument platform, and is fixed with press strip, then with 50 dots/ μ L/cm respectively by IgG the or SPA solution of polyclonal antibody PoAbII Specking forms detection line (T line) and nature controlling line (C line) trace in NC film center；Detection line and nature controlling line are at a distance of 5mm.It will test After 72 h of film natural drying at room temperature, desiccant sealing is added, room temperature preservation is spare.

7. the preparation of gold-labelled pad

With containing 1%(w/v) BSA and 0.1%(w/v) NaN₃20 mmol/L Na₂B₄O₇Buffer is uniformly sprayed on glass fibre cotton On, gold labeling antibody bonding pad is made；Then 10cm is spread according to every milliliter of solution²Amount the gold labeling antibody mixed liquor of acquisition is sprayed In on gold labeling antibody bonding pad.

3 h are dried in vacuo, sets in the hermetic bag equipped with desiccant and saves backup.

8. the preparation of sample pad

Sample pad select glass fibre cotton, with contain 0.1 mol/L NaCl, 0.2% Tween 20(v/v) and 0.1%(w/v) fold The PBS(pH 7.2 of nitrogen sodium) solution immersion glass sliver；It is dry to set 50 DEG C of 30 min of drying box, adds the closed preservation of desiccant room temperature It is spare.

9. the preparation of water absorption pad

Water absorption pad selects the preferable absorbent filter of water imbibition, and room temperature is closed to be saved backup.

10. the preparation of support plate

Support plate is the PVC plastic flitch of single side adhesive sticker.

11. detecting the assembling of card

It first will test film and be pasted on support plate center, then gold-labelled pad and sample pad are successively pasted on to the sample end of detection film, Each interlayer is overlapped the mm of 1 mm~2, then water absorption pad is pasted on to the other end of detection film, 1 mm~2 mm Chong Die with detection film.

The mechanism of test strips is as shown in Figure 3:

Test strips include support plate 1 and fixed adsorption layer on the supporting plate, and adsorption layer is followed successively by sample pad since test lead 3, gold-labelled pad 4, detection film 2 and water absorption pad 7, are adsorbed with rabbit-anti ASFV p30, p54, p72 of colloid gold label in gold-labelled pad 4 The mixture of protein-specific polyclonal antibody PoAbI, detection film are equipped with the nature controlling line C of staphylococcus aureus SPA printing 5, the detection line T 6 of anti-ASFV p30 of cavy, p54 and p72 protein polyclone antibody PoAbII printing.

Wherein, supporting plate material is the toughness PVC material not absorbed water.Sample cushion material is glass fibre cotton, nylon membrane, gathers Vinylidene fluoride film or polyester film.Gold-labelled pad material is glass fibre cotton.Detection membrane material is nitrocellulose filter, pure cellulose Film or carboxylated cellulose film.Water suction cushion material is absorbent filter.

After the completion of test strips preparation, test strips are fitted into during plastics get stuck, covers tightly and gets stuck, rapid detection card, and general is made It is fitted into the aluminium foil bag being protected from light, and room temperature kept dry is spare after adding desiccant to seal.

The structure of card is detected as shown in figure 4, including getting stuck and test strips.

The present invention detects the detection reaction principle of card are as follows:

When will contain the measuring samples of ASFV p30 albumen or p54 albumen or p72 proteantigen be added colloid gold test paper sample application zone (S), solution to be checked drives antigen to be checked and gold labeling antibody mixture together to nitrocellulose membrane diffusion by siphon, and final It penetrating into filter paper layer, gold labeling antibody is combined with antigen p30 albumen to be checked or p54 albumen or p72 albumen in diffusion process, Gold labeling antibody is combined with antigen p30 albumen to be checked or p54 albumen or p72 albumen, forms gold labeling antibody-antigenic compound PoAbI-p30 or PoAbI-p54 or PoAbI-p72.Gold mark compound can be detected with the polyclonal antibody PoAbII on detection film Trace, which combines, generates rufous " | " label, and part cannot not continue with the gold labeling antibody of antigen binding in conjunction with detection trace Diffusion, generates reddish brown color marker " | " on detection film in conjunction with the SPA in Quality Control trace, and two kinds of label combination superpositions form two Rufous positive mark " || " indicates to contain ASFV(Fig. 5 A in sample)；When in sample be free of ASFV p30 albumen or p54 egg When white or p72 proteantigen, no gold labeling antibody-antigenic compound is formed, and cannot then generate feminine gender in conjunction with T detection trace It marks " | " (Fig. 5 B).Show that detection card has failed if there is no reddish brown color marker to show (Fig. 5 C) on detection film.

Wherein, detection line and nature controlling line are Chu " ∣ ∣ " in addition to linear trace, trace, " ┬ can also be arranged for " 10 " font ┬ " font arranges trace, " ┴ ┴ " font arrangement trace, " ├ ├ " font arrangement trace or " ┤ ┤ " font and arranges trace.

Embodiment two: African swine fever virus is detected using African swine fever virus rapid detection card

1. the preparation of test sample solution

The different detection samples such as Swine serum, disease (dead) porcine tissue, excrement and vaccine to be checked are acquired, appropriate PBS or water is added to carry out letter It is single to be suspended or grind.

2. detection card detection

Solution to be checked is instilled into colloidal gold immuno-chromatography test paper strip and is loaded end；The horizontal positioned min of 3min~6 observes result.

3. testing result determines

Detection card shows two rufous bands (detection line and nature controlling line develop the color) " || ", indicates to contain ASFV in measuring samples Antigen；Only show a rufous band (nature controlling line) " | " for feminine gender, indicate measuring samples be not detected ASFV p30 albumen or P54 albumen or p72 proteantigen；Detection card does not show any band and shows to detect misoperation or detection card failure, need to be with another Detection detection card is taken to detect again.

The present invention is described in detail above in conjunction with drawings and examples, still, those of skill in the art Member is it is understood that without departing from the purpose of the present invention, can also carry out each design parameter in above-described embodiment Change, forms multiple specific embodiments, is common variation range of the invention, is no longer described in detail one by one herein.

SEQUENCE LISTING

<110>Henan Zhong Ze bioengineering Co., Ltd, Zhengzhou University

<120>African swine fever virus rapid detection card and its application

<130> 2019

<160> 9

<170> PatentIn version 3.2

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ctggctcagg acctgctgaa ctctcgtatc tctaacatca aaaacgttaa caaatcttac 120

ggtaaaccgg acccggaacc gaccctgtct cagatcgaag aaacccacct ggttcacttc 180

aacgctcact tcaaaccgta cgttccagtt ggcttcgaat acaacaaggt tcgtccgcac 240

accggtaccc cgaccctggg taacaaactg accttcggta tcccgcagta cggtgacttc 300

ttccacgaca tggttggtca ccacatcctg ggtgcttgcc actcttcttg gcaggacgct 360

ccgatccagg gtacctctca gatgggtgct cacggtcagc tgcagacctt cccgcgtaac 420

ggttacgact gggacaacca gaccccgctg gaaggtgctg tttacaccct ggttgacccg 480

ttcggtcgtc cgatcgttcc gggtaccaaa aacgcttacc gtaacctggt ttactactgc 540

gaatacccgg gtgaacgtct gtacgaaaat gttaggttcg acgttaacgg caactctctg 600

gacgaatact cttctgacgt taccaccctg gttcgtaaat tctgcatccc gggtgacaaa 660

atgaccggtt acaaacacct cgttggtcag gaagtaagcg ttgaaggtac ctctggtccg 720

ctgctgtgca acatccacga cctgcacaaa ccgcaccagt ctaaaccgat cctgaccgac 780

gaaaacgaca cccagcgtac ctgctctcac accaacccga aattcctgtc tcagcacttc 840

ccggaaaact ctcacaacat ccagaccgct ggtaaacagg acatcacccc gatcaccgac 900

gctacctacc tggacatccg tcgtaacgtt cactactctt gcaacggtcc gcagaccccg 960

aaatactacc agccgccgct ggctctgtgg atcaaactgc gtttctggtt caacgaaaac 1020

gttaacctgg ctatcccgtc tgtttctatc ccgttcggcg agaggttcat caccatcaag 1080

ctggcttctc agaaagacct ggttaacgaa ttcccgggtc tgttcgttcg tcagtctcgt 1140

ttcatcgctg gtcgtccgtc tcgtcgtaac atccgtttca aaccgtggtt catcccgggt 1200

gttatcaacg aaatctctct gaccaacaac gaactgtaca tcaacaacct gttcgttacc 1260

ccggaaatcc acaacctgtt cgttaaacgt gttcgtttct ctctgatccg tgttcacaaa 1320

acccaggtta cccacaccaa caacaaccac cacgacgaaa aactgatgtc tgctctgaaa 1380

tggccgatcg aatacatgtt catcggtctg aaaccgacct ggaacatctc tgaccagaac 1440

ccgcaccagc accgtgactg gcacaaattc ggtcacgttg ttaacgctat catgcagccg 1500

acccaccacg ctgaaatctc tttccaggac cgtgacaccg ctctgccgga cgcttgctct 1560

tctatctctg acatctctcc ggttacctac ccgatcaccc tgccgatcat caaaaacatc 1620

tctgttaccg ctcacggtat caacctgatc gacaaattcc cgtctaaatt ctgctcttct 1680

tacatcccgt tccactacgg tggtaacgct atcaaaaccc cggacgaccc gggtgctatg 1740

atgatcacct tcgctctgaa accgcgtgaa gaataccagc cgtctggtca catcaacgtt 1800

tctcgtgctc gtgaattcta catctcttgg gacaccgact acgttggttc tatcaccacc 1860

gctgacctgg ttgtttctgc ttctgctatc aacttcctgc tgctgcagaa cggttctgct 1920

gttctgcgtt actctacc 1938

<210> 4

<211> 27

<212> DNA

<213>artificial synthesized

<400> 4

aggatccatg gacttcatcc tgaacat 27

<210> 5

<211> 26

<212> DNA

<213>artificial synthesized

<400> 5

gcctcgagtt attttttttt cagcag 26

<210> 6

<211> 27

<212> DNA

<213>artificial synthesized

<400> 6

aggatccatg gactctgaat tcttcca 27

<210> 7

<211> 26

<212> DNA

<213>artificial synthesized

<400> 7

gcctcgagtt acagagagtt ttccag 26

<210> 8

<211> 30

<212> DNA

<213>artificial synthesized

<400> 8

aggatccatg gcttctggtg gtgctttctg 30

<210> 9

<211> 28

<212> DNA

<213>artificial synthesized

<400> 9

actcgagtta ggtagagtaa cgcagaac 28

Claims (10)

1. a kind of African swine fever virus rapid detection card, including test strips, the test strips include support plate and are fixed on described Adsorption layer in support plate, the adsorption layer are followed successively by sample pad, gold-labelled pad, detection film and water absorption pad since test lead, It is characterized in that, the polyclonal of anti-African swine fever virus p30, p54 and p72 albumen of colloid gold label is adsorbed in the gold-labelled pad Antibody PoAbI；The detection film is equipped with the Quality Control of goat-anti or rabbit anti-mouse IgG antibody or staphylococcus aureus SPA printing Line C is additionally provided with the detection line T of the polyclonal antibody PoAbII containing anti-African swine fever virus p30, p54 and p72 albumen.

2. African swine fever virus rapid detection card according to claim 1, which is characterized in that the polyclonal antibody PoAbI is made by following steps:

(1) difference prokaryotic expression nucleotide sequence is as shown in SEQ ID NO.1p30Gene, nucleotide sequence such as SEQ ID Shown in NO.2p54Gene, nucleotide sequence are as shown in SEQ ID NO.3p72Gene, preparation African swine fever virus p30, P54 and p72 recombinant protein；

(2) recombination p30, p54 and p72 protein immunization rabbit is taken respectively；

(3) purifying of hyper-immune serum caprylic acid-ammonium obtains the IgG of polyclonal antibody PoAbI.

3. African swine fever virus rapid detection card according to claim 2, which is characterized in that in the step (2), move The dosage of every rabbit is that 100~200 μ g/1ml recombinate p30 albumen when object is immune, and 100~200 μ g/1ml recombinate p54 albumen, 100~200 μ g/1ml recombinate p72 albumen.

4. African swine fever virus rapid detection card according to claim 1, which is characterized in that the polyclonal antibody The ELISA potency of PoAbI is 1:2 × 10³~1:2 × 10⁷。

5. African swine fever virus rapid detection card according to claim 1, which is characterized in that the polyclonal antibody PoAbII is made through following steps:

(1) difference prokaryotic expression nucleotide sequence is as shown in SEQ ID NO.1p30Gene, nucleotide sequence such as SEQ ID Shown in NO.2p54Gene, nucleotide sequence are as shown in SEQ ID NO.3p72Gene, preparation African swine fever virus p30, P54 and p72 recombinant protein；

(2) recombination p30, p54 and p72 protein immunization mouse is taken respectively；

(3) purifying of hyper-immune serum caprylic acid-ammonium obtains the IgG of polyclonal antibody PoAbII.

6. African swine fever virus rapid detection card according to claim 5, which is characterized in that in the step (2), move The dosage of every mouse is that 50~100 μ g/500ml recombinate p30 albumen when object is immune, and 50~100 μ g/500ml recombinate p54 egg White, 50~100 μ g/500ml recombinate p72 albumen.

7. African swine fever virus rapid detection card according to claim 1, which is characterized in that the polyclonal antibody The ELISA potency of PoAbII is 1:3.2 × 10⁴To 1:8.192 × 10⁶。

8. African swine fever virus rapid detection card according to claim 1, which is characterized in that the support plate is not by absorbing water Toughness PVC material be made；The sample pad is made of glass fibre cotton, nylon membrane, PVDF membrane or polyester film；Institute Gold-labelled pad is stated to be made of glass fibre cotton；The detection film is by nitrocellulose filter, pure cellulose film or carboxylated cellulose film system At；The water absorption pad is made of absorbent filter.

9. the application method of African swine fever virus rapid detection card described in claim 1, which comprises the following steps:

(1) sample to be examined is acquired, adds PBS or water to be suspended or grind, obtains solution to be checked；

(2) solution to be checked is instilled to the sample-adding end of African swine fever virus rapid detection card described in claim 1；It is horizontal positioned The min of 3min~6 observes result；

(3) it if the nature controlling line C and detection line T of detection card show two rufous bands, indicates to contain ASFV in measuring samples At least one of p30 albumen, p54 albumen, p72 proteantigen；

If the nature controlling line C for only detecting card shows a rufous band, indicate measuring samples be not detected ASFV p30 albumen and/or P54 albumen and/or p72 proteantigen；

Show to detect misoperation or detection card failure if detection card does not show any band.

10. the application method of African swine fever virus rapid detection card according to claim 9, which is characterized in that the sample This is Swine serum, sick pig tissue, any one in dead porcine tissue, excrement, vaccine, body fluid.