KR101942098B1 - Rapid Kit for Diagnosing the specific antibodies against Porcine Respiratory Reproductive Syndrome Virus - Google Patents

Abstract

The present invention provides a rapid kit for the diagnosis of North American and European pig genital respiratory syndrome virus-specific antibodies and a method for diagnosing porcine reproductive and respiratory syndrome virus-specific antibodies using the same. Using the kit of the present invention, rapid immunochromatography can be used to diagnose the virus infection of North American and European pig genital respiratory syndrome in a simple and convenient manner on site without special testing equipment. The kit of the present invention shows excellent sensitivity and specificity in the measurement of porcine reproductive tract respiratory syndrome virus antibody and can be expressed more numerically using gold reader.

Description

Rapid Kit for Diagnosing the specific antibodies against Porcine Respiratory Reproductive Syndrome Virus}

The present invention relates to a rapid kit for diagnosing swine genital respiratory syndrome virus-specific antibodies and use thereof for diagnosing swine genital respiratory syndrome virus-specific antibodies.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a disease that was first reported in North America in the late 1980s and is now occurring not only in Korea but also in pig farming countries around the world.

The causative agent of swine genital respiratory syndrome is the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which is an RNA virus belonging to Nidovirales, Arterviridae, and Arterivirus. Porcine reproductive respiratory syndrome virus can be largely classified into European genotype (EU) and North American genotype (NA) according to antigenic and genetic characteristics. Symptoms of swine genital respiratory syndrome include infertility, mummified fetuses at the end of pregnancy, miscarriage, and premature birth, and it is known that frail piglets born from viral infections die due to secondary infections in the respiratory tract after birth.

It is important to discriminate between North American/European type in order to more effectively determine the infection status of swine genital respiratory syndrome in farms, and this can lead to selection of vaccines for the purpose of effective control.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have made intensive research efforts to develop a diagnostic kit capable of quickly and accurately diagnosing the infection of the swine genital respiratory syndrome virus in a biological sample. As a result, the present invention by finding out that the presence of European and North American swine genital respiratory syndrome virus antibodies can be quickly and accurately detected from samples using antigens and gold nanoparticles that specifically bind to porcine reproductive respiratory syndrome virus antibodies. Was completed.

Accordingly, an object of the present invention is to provide a rapid kit for diagnosing porcine genital respiratory syndrome virus-specific antibodies.

Another object of the present invention is to provide a method for diagnosing porcine reproductive respiratory syndrome virus-specific antibodies using the kit of the present invention.

Other objects and advantages of the present invention will become more apparent by the following detailed description and claims.

According to an aspect of the present invention, the present invention is a rapid kit for diagnosing a specific antibody of Porcine Respiratory Reproductive Syndrome Virus, comprising:

The rapid kit includes a structure in which a sample pad, a conjugate pad, a membrane, and an absorbent pad are sequentially connected,

The conjugate pad includes a first conjugate for detection of an antibody in which a porcine reproductive respiratory syndrome virus antigen and gold nanoparticles are bound, consisting of the amino acid sequence of SEQ ID NO: 1, and a porcine reproductive respiratory syndrome consisting of the amino acid sequence of SEQ ID NO: 2 It contains a second conjugate for detection of an antibody in which a viral antigen and gold nanoparticles are bound,

On the membrane, an inspection line and a control line are sequentially formed from the conjugate pad toward the absorption pad,

The test line is immobilized with a swine genital respiratory syndrome virus antigen consisting of the amino acid sequence of SEQ ID NO: 1 and a swine genital respiratory syndrome virus antigen consisting of the amino acid sequence of SEQ ID NO: 2,

In the control line, an antibody (anti-rabbit IgG) that specifically binds to the control antibody contained in the liquid sample is immobilized to confirm that the sample has been developed. to provide.

The present inventors have made intensive research efforts to develop a diagnostic kit capable of quickly and accurately diagnosing the infection of the swine genital respiratory syndrome virus in a biological sample. As a result, it was found that the presence of European and North American Swine Respiratory Syndrome virus antibodies can be quickly and accurately detected from samples using antigens and gold particles that specifically bind to Swine Respiratory Syndrome virus antibodies.

In the present invention, the immunochromatography method used to diagnose swine genital respiratory syndrome is a diagnostic method that enables the examination of various diseases such as pregnancy diagnosis or hepatitis outbreak by using an antigen-antibody reaction in general. It is a kind of immunoassay method for rapid on-site diagnosis using colored particles such as gold colloid or colored polystyrene nanoparticles as a measurement label with a solid phase.

The kit of the present invention makes it possible to specifically diagnose the presence of European and North American swine genital respiratory syndrome virus antibodies from a sample by using an antigen that specifically binds to an antibody against swine genital respiratory syndrome virus.

The kit of the present invention includes a sample pad to which a liquid sample is applied, a conjugate pad for detecting porcine genital respiratory syndrome virus antibody, a membrane in which an antigen for capturing porcine genital respiratory syndrome virus antibody is immobilized, and It includes a structure in which an absorption pad capable of absorbing a liquid sample is sequentially connected.

In the kit of the present invention, the “sample pad” refers to a pad capable of diffusing flow by receiving a sample to be analyzed, and is composed of a material having sufficient porosity to accommodate and contain a sample to be analyzed. Such porous materials include fibrous paper, microporous membranes made of cellulose materials, cellulose derivatives such as cellulose and cellulose acetate, nitrocellulose, glass fibers, naturally occurring cotton, fabrics such as nylon, or porous gels. , Is not limited thereto.

In the kit of the present invention, the "conjugate pad" is a pad that contains a sample that is diffused and moved from the sample pad, and contains a "nanoparticle-antigen conjugate" in which nanoparticles and antigens are connected. The conjugate pad, like the sample pad, is made of a material capable of diffusion flow.

In the kit of the present invention, the “membrane” may be made of any of a variety of materials through which the sample material can pass. For example, natural, synthetic, or synthetically modified naturally occurring materials, such as polysaccharides (eg, cellulosic materials, paper, cellulose derivatives such as cellulose acetate and nitrocellulose); Polyether sulfone; Polyethylene; nylon; Polyvinylidene fluoride (PVDF); Polyester; Polypropylene; Silica; Inorganic materials uniformly dispersed in a porous polymer matrix with polymers such as vinyl chloride, vinyl chloride-propylene copolymer and vinyl chloride-vinyl acetate copolymer, e.g. inactivated alumina, diatomaceous earth, MgSO4, or other inorganic finely divided materials ; Naturally occurring (eg cotton) and synthetic (eg nylon or rayon) fabrics; Porous gels such as silica gel, agarose, dextran and gelatin; Polymeric films such as polyacrylamide; It can be formed from materials such as. Preferably the membrane comprises a polymeric material such as nitrocellulose, polyethersulfone, polyethylene, nylon, polyvinylidene fluoride, polyester and polypropylene.

In the kit of the present invention, the “absorption pad” may be positioned adjacent to or near the end of the membrane. Absorbent pads generally receive a sample of fluid that travels through the entire membrane. Absorbent pads can help promote capillary action and diffuse flow of fluid through the membrane.

The kit of the present invention is configured by sequentially connecting the above-described sample pad, conjugate pad, membrane, and absorbent pad on the same support.

The support may be formed of any material as long as it can support and transport the sample pad, conjugate pad, membrane, and absorption pad, but in general, the fluid of the sample that diffuses through the membrane does not leak through the support. It is preferably liquid impermeable. For example, glass; Polymer materials include, but are not limited to, polystyrene, polypropylene, polyester, polybutadiene, polyvinyl chloride, polyamide, polycarbonate, epoxide, methacrylate, and polymelamine.

In the kit of the present invention, on the membrane, a test line and a sample in which the antigen for capturing porcine genital respiratory syndrome virus antibody binding to the porcine genital respiratory syndrome virus antibody were immobilized in the direction of the absorption pad from the conjugate pad were developed. A control line is sequentially formed to confirm that.

According to an embodiment of the present invention, the antigen for capturing porcine reproductive respiratory syndrome virus antibody immobilized on the test line is an antigen consisting of the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.

An antibody that specifically binds to the control antibody contained in the liquid sample is immobilized on the control line to confirm that the sample has been developed.

According to an embodiment of the present invention, rabbit IgG, mouse IgG, etc. may be used as a control antibody in the present invention, but the present invention is not limited thereto. Preferably, the control antibody contained in the liquid sample is rabbit IgG, and the antibody immobilized on the control line is anti-rabbit IgG. When the liquid sample is normally developed, the conjugate of the control antibody and gold nanoparticles contained in the liquid sample reacts with the antibody immobilized on the control line to develop color.

According to one embodiment of the present invention, a biological sample is appropriately diluted in a sample dilution solution and then added to a sample dropping window of a sample pad, and applied to the kit of the present invention.

As biological samples applied to the kit of the present invention, pig-derived whole blood, serum, plasma, saliva, and other body fluids (e.g. urine) may be exemplified, but are not limited thereto.

According to one embodiment of the present invention, the biological sample applied to the kit of the present invention is whole blood, plasma or serum of pigs.

More specifically, after appropriately diluting a biological sample in a sample dilution solution, it is placed in a sample dropping window located on a sample pad of a rapid kit for measuring swine genital respiratory syndrome virus antibody and deployed. If there is an antibody against the swine genital respiratory syndrome virus in the sample, the antibody is combined with an antigen-gold nanoparticle binding agent for detecting porcine genital respiratory syndrome virus antibody, and an antigen for capturing porcine genital respiratory syndrome virus antibody fixed to the test line. A gold color will appear on the test line. Therefore, when there is an antibody against swine genital respiratory syndrome virus in a biological sample, it develops at both the control line and the test line of the membrane, and when there is no antibody against the porcine reproductive syndrome virus in the biological sample, it develops only at the control line (Fig. 2). Reference).

In the case of color development in both the control line and the test line in the kit of the present invention, the biological sample is determined to have been produced by infection with the swine genital respiratory syndrome virus or by administration of a vaccine against the swine genital respiratory syndrome virus, and the color is developed only in the control line, The biological sample was determined not to be infected with the swine genital respiratory syndrome virus or to produce no antibodies after administration of the vaccine against the swine genital respiratory syndrome virus.

The kit of the present invention can be used to determine whether a pig-derived biological sample has an antibody specific for the swine genital respiratory syndrome virus. By judging whether the pig-derived biological sample from the pig-derived respiratory syndrome virus-specific antibody is present, whether the pig is infected with the pig-genital respiratory syndrome virus or whether the pig-genital respiratory syndrome virus-specific antibody was produced by vaccination. I can confirm.

According to one embodiment of the present invention, the kit of the present invention does not show cross-reaction with respect to CSFV, BVDV, FMDV, PCV, APP5, HP, PCV2, APP2 and MH.

According to another aspect of the present invention, the present invention provides a method for diagnosing a porcine genital respiratory syndrome specific antibody comprising the following steps:

(a) diluting the biological sample in a sample diluent and contacting the sample pad of the kit of the present invention; And

(b) analyzing the signal generated by the kit to determine the presence or absence of a porcine genital respiratory syndrome virus-specific antibody in the biological sample.

Using the present invention, it is possible to diagnose both North American or European type porcine genital respiratory syndrome specific antibodies.

Since the present invention is a method of diagnosing specific antibodies for porcine reproductive respiratory syndrome using the kit, descriptions of the contents in common between the two are omitted in order to avoid undue complexity in the present specification.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a rapid kit for diagnosing a virus-specific antibody of North American and European type swine genital respiratory syndrome, and a method for diagnosing a virus-specific antibody of swine genital respiratory syndrome using the same.

(b) Using the kit of the present invention, it is possible to diagnose whether North American-type and European-type swine reproductive respiratory syndrome virus infection is performed simply and quickly in the field without special testing equipment using rapid immunochromatography.

(c) The kit of the present invention exhibits excellent sensitivity and specificity in the measurement of porcine genital respiratory syndrome virus antibody, and because the result can be expressed numerically using a gold reader, it is more convenient and more accurate than the existing kits to be checked with the naked eye. It is possible.

1 is a schematic diagram of an immunochromatography method.
2 is a schematic diagram showing the principle of positive/negative discrimination for the diagnostic kit of the present invention.
3 is a schematic diagram of a strip.
4 is a result of measuring the detection limit for the diagnostic kit.
5 is a result of measuring the cross-reaction to antibodies to other diseases.
6 is a result of evaluating reproducibility.
7 is a result of comparing the reactivity of the commercially available ELISA (IDEXX PRRS X3 Ab) and the PRRS antibody rapid kit of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Development content and method

The “Swine Genital Respiratory Syndrome Virus Antibody Diagnosis Rapid Kit” is a kit that specifically detects antibodies against the swine genital respiratory syndrome virus (PRRSV) contained in pig serum, and is detected using immunochromatography (Fig. 1). .

IFA and ELISA methods are widely used in the PRRSV antibody diagnosis method, but this diagnosis method requires several processes, takes a lot of time, and requires expensive equipment, which makes it difficult to diagnose simple and fast, and it is more expensive than the rapid kit. have.

Advantages and disadvantages of the inspection method method of inspection Advantages Disadvantages IFA -Low price
PRRSV antibody diagnosis standard test method -Large reading error depending on the experimenter
-Requires expensive equipment ELISA -Simultaneous measurement of multiple samples
-Relatively inexpensive -Inspection time 2~3 hours
-Complex inspection process
Requires expensive equipment RAPID -Quick and easy inspection
(Within 10-20 minutes)
-Low cost Indirect rapid-quantitative analysis difficulty

This diagnostic kit has the advantage of being able to detect PRRSV antibodies 10 minutes after inserting a sample and being inexpensive. In addition, both antibodies caused by North American PRRSV and European PRRSV infection can be detected and diagnosed in the early stages of infection. When used in parallel with a commercially available ELISA, the time of infection (initial infection) can be determined, so it can be usefully applied to initial countermeasures to prevent the spread of infection after screening. Compared to IFA ELISA, it has a more sensitive detection limit, making it suitable for early disease screening in the field, and it is easy to diagnose and identify the presence or absence of antibodies produced by the vaccine after vaccination.

The method of using this kit is to mix the serum sample with the dilution buffer and put it in the inlet after 5 minutes. If PRRSV antibody is present in the serum, it binds to the PRRSV recombinant antigen-gold conjugate and is dispensed on the membrane while moving the membrane by capillary force. It is a sandwich type diagnostic kit in which a red line appears on the test line in response to the PRRSV recombinant antigen (FIG. 2).

Example 1: Preparation of Rapid Diagnostic Kit

1-1: Preparation of gold conjugate

Gold conjugate refers to a conjugate between an antigen and gold particles (40 nm), and was prepared as follows using an adsorption method to prepare a conjugate.

Manufacturing method

1) Antigen preparation: North American PRRSV recombinant nucleocapsid antigen and European PRRSV recombinant nucleocapsid antigen are prepared for test line reaction, and rabbit antibodies are prepared for control line reaction (10 μg each for 1 ml gold solution coupling). need).

2) Gold solution preparation: Prepare 40 nm colloidal gold using a spectrophotometer so that the OD value becomes 1 at a wavelength of 525 nm, and the pH is 10.

3) Preparation of conjugate: Mix the antigen and gold prepared above at 10 μg/ml and react at room temperature for 1 hour.

4) Blocking and washing: In order to block the portion of the gold solution that did not react with the antigen, 0.3% casein was added and reacted at 37°C for 20 minutes. To remove the unreacted antigen and blocking solution other than the prepared conjugate, the supernatant was discarded by centrifugation, and then centrifuged again by adding 1% BSA solution, and then the supernatant was removed, and 1% BSA solution was added again. After centrifugation, the supernatant is removed and added to about 1/10 of the volume initially started with a 1% BSA solution, and then the OD value is measured at a wavelength of 525 nm using a spectrophotometer.

5) Drying to maintain gold conjugate stability

Gold conjugates have a disadvantage that their activity decreases over time. In order to overcome this shortcoming and ensure stability, a method of drying by adding three chemical substances to the glass fiber was used. Add 5% sucrose, 0.1% casein, 0.1% NaN ₃ , respectively, rabbit IgG gold conjugate was added to a spectrophotometer OD value of 0.7, and PRRSV recombinant antigen-gold conjugate OD 5 was added using a dryer. Dry for a minute.

1-2: Preparation of strip

The strip has a shape in which a sample pad (Ahlstrom), a membrane (Millipore), and an absorbent pad (Ahlstrom) are overlapped so as to react with capture while flowing in a certain direction (FIG. 3).

Manufacturing method

_{1) Antigen preparation: 0.5% sucrose (Sigma) and 0.1% NaN 3} (Sigma) in 0.6 mg/ml of anti-rabbit IgG (Arista) 1 mg/ml and North American and European PRRSV recombinant antigen (nucleocapsid) mixture solution Prepare by adding.

2) Antigen dispensing and fixation: Attach a membrane to a backing card (PJ Corporation), anti-rabbit IgG to the control line, and a mixture of North American and European PRRSV recombinant antigens (nucleocapsid) to the test line at bed speed 10 Dispense at cm/sec, dispensing volume 0.9 μl/cm. After that, it is dried in a dehumidifying cabinet for at least 24 hours to fix the antigen on the membrane.

3) Attaching and cutting the pad: When the antigen is fixed, attach the sample pad and the absorption pad to the designated positions, and cut them at 4 mm intervals using a rotary slitter.

4) Assembly and packaging: Assemble the finished strip into the housing and seal it with silica gel in an aluminum pouch and package.

1-3: Preparation of detection solution

Add 0.4% Tween20 (Sigma), 0.5 N NaCl, and 0.01% NaN ₃ to 50 mM Borax buffer.

1-4: How to use and judge the result

1) Take the serum sample to be tested with a sample dropper, put 2 drops into the dilution tube, add 8 drops of the dilution buffer using the dropper, mix well, and react for 5 minutes.

2) Take out the device, place it on a flat surface, and add 3 drops of the solution mixed in step 1 to the sample inlet.

3) Load at room temperature for 10 minutes and measure the numerical value using a gold reader.

4) If the numerical value is less than 40, it is judged as negative, and if it is above 40, it is judged as positive.

1-5: Analysis of results

Detection limit comparison

This kit was found to detect a concentration that is about two times lower than the detection limit of the existing ELISA kit (IDEXX indirect ELISA) (FIG. 4).

Sample IFA IDEXX PRRS X3 Ab ELISA (S/P) VDRG ^® PRRSV AB RAPID KIT Cal.1 1/80 (+) 2.579 (+) + Cal.2 1/40 (+) 1.634 (+) + Cal.3 1/20 (+) 1.013 (+) + Cal.4 - 0.568 (+) + Cal.5 - 0.268 (-) + Cal.6 - 0.171 (-) - Cal.7 - 0.074 (-) - Cal.8 - 0.047 (-) -

IDEXX PRRS X3 Ab ELISA (S/P) cut-off value:

negative result: S/P <0.4, positive result: S/P ≥ 0.4

Sensitivity specificity measurement

The sensitivity and specificity were measured compared to the ELISA method, which is the top method for measuring PRRSV antibodies.

Median Dinostic Co., Ltd. test results IDEXX PRRS X3 Ab ELISA Rapid Measurement sensitivity
(Rapid positive/ELISA positive) 100% positivity voice synthesis ELISA positivity 100 0 100 Measurement specificity
(Rapid voice/ELISA voice) 97.1% voice 3 102 105

Results of evaluation request by the Ministry of Agriculture, Forestry and Livestock Quarantine IDEXX PRRS X3 Ab ELISA Rapid Measurement sensitivity
(Rapid positive/ELISA positive) 93.2% positivity voice synthesis ELISA positivity 218 16 234 Measurement specificity
(Rapid voice/ELISA voice) 95.7% voice 10 221 231

As a result of the Mediandinostic test, 100 out of 100 samples positive in IDEXX PRRS X3 Ab ELISA were detected as positive in Rapid, and the sensitivity was 100%, and 102 out of 105 samples negative in IDEXX PRRS X3 Ab ELISA were detected as negative. Therefore, the specificity is 97.1%.

As a result of commissioning the assessment by the Ministry of Agriculture, Forestry and Livestock Quarantine, 218 out of 234 samples that were positive in IDEXX PRRS X3 Ab ELISA were detected as positive in Rapid, and the sensitivity was 93.2%, and out of 231 samples that were negative in IDEXX PRRS X3 Ab ELISA. 221 cases were detected as negative and showed a specificity of 95.7%.

Example 2: Cross-reactivity evaluation

In order to confirm the cross-reaction with other antisera, antibodies to other diseases in the pig serum were checked through ELISA, and a sample that was negative for PRRSV and positive for antibodies to other disease agents was tested as a prototype to confirm cross-reaction to other antibodies. I did. As a result of the test, it was confirmed that there was no cross-reaction with other disease antisera (FIG. 5 ).

number One 2 3 4 5 6 7 8 9 10 PRRSV S/P 0.06 0.02 0.01 0.03 0.01 0.01 0.02 0.01 0.05 0.16 CSFV PC% 99.7 104.8 102.3 95.1 33.1 95.8 88.1 84.6 103.3 101.0 BVDV S/N 0.80 0.98 0.57 1.05 0.98 0.77 1.02 1.02 0.63 1.03 FMDV S/N 0.67 0.41 0.82 0.80 0.76 0.81 0.17 0.83 0.36 0.68 PCV S/P 2.08 2.28 0.28 2.10 0.54 2.04 0.72 1.68 1.65 2.18 APP5 S/P 0.40 0.90 0.90 1.77 0.68 1.39 1.06 1.52 0.53 0.17 HP S/P 0.28 0.35 0.29 0.79 0.46 0.45 0.33 0.40 0.34 0.36 PCV2 S/P 1.44 1.57 0.51 1.65 0.47 1.40 0.78 1.21 0.82 1.18 APP2 S/P 0.58 0.78 0.78 1.47 0.66 1.15 1.09 1.86 0.56 0.13 MH S/P 0.61 0.60 0.26 0.36 0.41 0.09 0.18 0.10 0.37 0.97 PMA S/P 1.50 1.00 0.56 1.60 0.93 0.71 0.57 1.01 0.74 0.53

Example 3: Reproducibility test

In order to confirm the reproducibility of the rapid test, a reproducibility test was performed with three strips with two positive and one negative specimen. In terms of the numerical values of the gold reader, it was found that CV was excellent in reproducibility within 10% (FIG. 6).

Sample No. 1 strip 2 strips 3 strips Average STDEV CV% Voice 110866 16 18 15 16 2 9 Low-positive 110752 242 239 251 244 6 3 Medium-positive 110816 384 403 423 403 20 5

Example 4: Reactivity comparison

The reactivity of the commercially available ELISA (IDEXX PRRS X3 Ab) and the rapid kit for diagnosing porcine reproductive respiratory syndrome virus antibodies was compared with the commercially available ELISA (IDEXX PRRS X3 Ab) after attacking SPF pigs with three North American PRRSV and three European PRRSV inoculated with SPF pigs (Fig. 7 ). In the rapid kit for diagnosing pig reproductive respiratory syndrome virus antibody, the result of detecting the antibody that is believed to be induced at the early stage of infection compared to ELISA was confirmed, and it is believed that it is possible to determine whether it is an early infection when used in combination with a commercially available ELISA, thus preventing the spread of infection. It is expected that it will be easy to cope with the initial action.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is clear that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> MEDIAN Diagnostics Inc. <120> Rapid Kit for Diagnosing the specific antibodies against Porcine Respiratory Reproductive Syndrome Virus <130> PN150433 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 123 <212> PRT <213> North American genotype PRRSV(VR2332) Nucleocapsid protein <400> 1 Met Pro Asn Asn Asn Gly Lys Gln Gln Asn Arg Lys Lys Gly Asp Gly 1 5 10 15 Gln Pro Val Asn Gln Leu Cys Gln Met Leu Gly Lys Ile Ile Ala Gln 20 25 30 Gln Asn Gln Ser Arg Gly Lys Gly Pro Gly Lys Lys Asn Lys Lys Lys 35 40 45 Asn Pro Glu Lys Pro His Phe Pro Leu Ala Thr Glu Asp Asp Val Arg 50 55 60 His His Phe Thr Pro Ser Glu Arg Gln Leu Cys Leu Ser Ser Ile Gln 65 70 75 80 Thr Ala Phe Asn Gln Gly Ala Gly Thr Cys Thr Leu Ser Asp Ser Gly 85 90 95 Arg Ile Ser Tyr Thr Val Glu Phe Ser Leu Pro Thr His His Thr Val 100 105 110 Arg Leu Ile Arg Val Thr Ala Ser Pro Ser Ala 115 120 <210> 2 <211> 128 <212> PRT <213> European genotype PRRSV (Lelystad) Nucleocapsid protein <400> 2 Met Ala Gly Lys Asn Gln Ser Gln Lys Lys Lys Lys Ser Thr Ala Pro 1 5 10 15 Met Gly Asn Gly Gln Pro Val Asn Gln Leu Cys Gln Leu Leu Gly Ala 20 25 30 Met Ile Lys Ser Gln Arg Gln Gln Pro Arg Gly Gly Gln Ala Lys Lys 35 40 45 Lys Lys Pro Glu Lys Pro His Phe Pro Leu Ala Ala Glu Asp Asp Ile 50 55 60 Arg His His Leu Thr Gln Thr Glu Arg Ser Leu Cys Leu Gln Ser Ile 65 70 75 80 Gln Thr Ala Phe Asn Gln Gly Ala Gly Thr Ala Ser Leu Ser Ser Ser 85 90 95 Gly Lys Val Ser Phe Gln Val Glu Phe Met Leu Pro Val Ala His Thr 100 105 110 Val Arg Leu Ile Arg Val Thr Ser Thr Ser Ala Ser Gln Gly Ala Ser 115 120 125

Claims (5)

Sample pad;
A first conjugate for detecting an antibody bound to a porcine reproductive / respiratory syndrome virus antigen and gold nanoparticles comprising the amino acid sequence of SEQ ID NO: 1, and a porcine reproductive / respiratory syndrome virus antigen comprising the amino acid sequence of SEQ ID NO: 2 and gold nanoparticles A conjugate pad comprising a second conjugate for antibody detection;
A membrane comprising a porcine reproductive / respiratory syndrome virus antigen consisting of the amino acid sequence of SEQ ID NO: 1 and a test line to which a porcine reproductive / respiratory syndrome virus antigen consisting of the amino acid sequence of SEQ ID NO: 2 is immobilized and a tank antibody is immobilized; And
Absorption pad;
/ RTI &gt;
The sample pad, the conjugate pad, the membrane, and the absorbent pad are sequentially connected,
Wherein the inspection line and the reference line are sequentially formed in the direction from the conjugate pad to the absorption pad.
Rapid kit for simultaneous diagnosis of European and North American porcine respiratory reproductive syndrome virus (PRRSV) specific antibodies. The rapid kit for simultaneous diagnosis of European and North American pig genital respiratory syndrome virus-specific antibodies according to claim 1, wherein said sample pad, conjugate pad, membrane and absorbent pad are sequentially connected on a support. The rapid kit for simultaneous diagnosis of European and North American pig genital respiratory syndrome virus specific antibodies according to claim 1, wherein said anti-rabbit antibody is anti-rabbit IgG or anti-mouse IgG. . Methods for simultaneous diagnosis of European and North American Porcine Respiratory Reproductive Syndrome Virus (PRRSV) -specific antibodies including the following steps:
Contacting the biological sample with a sample pad of a rapid kit for simultaneous diagnosis of European and North American pig genital respiratory syndrome virus-specific antibodies of claim 1 by diluting the sample with a sample dilution; And
Analyzing signals generated in the rapid kit for simultaneous diagnosis of European and North American pig genital respiratory syndrome virus specific antibodies and simultaneously measuring the presence of European and North American pig genital respiratory syndrome virus specific antibodies in the biological sample. The method for simultaneous diagnosis of European and North American pig genital respiratory syndrome virus specific antibodies according to claim 4, wherein said biological sample is whole blood, plasma or serum of a pig.

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