Patent ID: 12259386

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, a preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings. However, the scope of the present invention is not limited thereto.

These embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the invention to those skilled in the art, and the present disclosure will only be defined by the appended claims. Well-known components, movement and techniques among the best mode for carrying out the invention will not be described in detail to prevent ambiguous interpretation.

The terminology used in the specification is intended to describe exemplary embodiments of the present invention, without departing from the spirit or scope of the present invention. In addition, the components and operations referred to as “comprises (or includes)” do not preclude the presence or addition of one or more other components and operations.

The term “R. solanacearum” pathogen used herein is a Gram-negative, rod-shaped and aerobic bacteria with several flagella generating movement, may survive for several years while being incubated in soil at 34° C. and hibernate inside the remains of sick plants. It mainly invades through wounds in the underground tissue of plants, but occasionally invades through wounds in the above-ground tissue of plants. It is known that it occurs rapidly under conditions of high temperature and high humidity.

The term “F. oxysporum” used herein is a pathogen causing severe diseases in a variety of plants globally and comprises more than 120 species according to the host specificity that causes diseases due to high host specificity. In addition, the pathogens could remain dormant for several years by formulating chlamydospore in soil without host plants, and when the environment improves, chlamydospores germinate and invade the roots of host plants, causing wilt disease orFusariumwilt disease.

According to a preferred embodiment of the present invention, the co-diagnosis for theRalstonia solanacearumandFusarium oxysporumusing semi-quantitative lateral flow immunodiagnostic technique includes the steps of: (1) providing antibodies toRalstonia solanacearum(R. solanacearum; R. S) that causes a bacterial wilt disease andFusarium oxysporum(F. oxysporum; F.O) that cause a fungal wilt disease; (2) combiningR. solanacearumandF. oxysporumwith each antibody; (3) quantifying the each amount ofR. solanacearumandF. oxysporum.

According to another preferred embodiment of the present invention, the co-diagnosis for theRalstonia solanacearumandFusarium oxysporumusing semi-quantitative lateral flow immunodiagnostic technique includes the steps of quantifying the amount ofRalstonia solanacearumandFusarium oxysporumcomprise: (1) low-titer (105cfu/ml), mid-titer (106cfu/ml) and high-titer (107cfu/ml) ofR. solanacearum; (2) low-titer (104cfu/ml), mid-titer (105cfu/ml) and high-titer (106cfu/ml) ofF. oxysporum; (3) negative (103cfu/ml) ofF. oxysporum.

According to a still another preferred embodiment of the present invention, specific antibodies toR. solanacearumorF. oxysporumcapable of detecting each specific pathogen can be selected without limits, but monoclonal antibodies that don't show cross-reactivity with bacteria and fungi that can infect plants are advised to be selected.

In addition, by using monoclonal antibodies specifically responsive tosolanacearumandF. oxysporum, the present invention provides the semi-quantitative kits that determine causative organism by detecting increased pathogens in plants with a wilt disease and analyzing the degrees of infections.

To achieve the above objectives of the present invention, the present invention usesR. solanacearum, a pathogenic bacterium, andF. oxysporum, a pathogenic fungus as immunogens for preparing antibodies. The bacteria and fungi used in the present invention was introduced and incubated by the Korean Agricultural Culture Collection (KACC) through ABC Circle, and the incubated bacteria and fungi were inactivated at high temperature and used as immunogens to produce antibodies.

To achieve the above objectives of the present invention, this invention used inactivated bacteria and inactivated fungi as immunogens to immunize mice, and conducted cell fusion for Splenocyte cells and Myeloma cells (Sp2/0 Ag-18) to produce Hybridoma. Then, each monoclonal antibody specifically responsive toR. solanacearumandF. oxysporumwere secured from the hybridoma. The sensitivity to the immunogen of the finally selected mouse's anti-R. solanacearumand anti-F. oxysporum, among the secured antibodies, is provided as the ELISA result, and the reactivity with 10 kinds of bacteria and fungi that may be present due to inflection of plants subject to the diagnosis kit, that may be present in soil, or may cause cross reactivity is provided as the ELISA result.

To achieve the objectives of the present invention, finally-selected two antibodies, mouse anti-R. solanacearumand anti-F. oxysporum, showed high sensitivity, but didn't show cross-reactivity with respect toR. solanacearumandF. oxysporum. In addition, with reference toFIG.1, two antibodies didn't show cross-reactivity to four types of bacteria (Bacillus subtilis, Lactobacillus plantarum, PeseudomonasSP.,Bacillus Amyloliquefaciens) and four types of molds (Botrysis cinerea, Colletotrichum gloeosporioides, Alternaria panax, Fusarium graminearum).

The two antibodies produced according to the present invention respond toR. solanacearumandF. oxysporumspecifically, and this invention uses the antibodies to produce the diagnostic kits according to the present invention.

To achieve the above objectives of the present invention, by using monoclonal antibody specifically responsive toR. solanacearumandF. oxysporum, this invention captures pathogens from juice of the plants and provides an analysis strip to detect eachR. solanacearumandF. oxysporumspecifically by using a rapid immunochromatography.

To achieve the above objectives of the present invention, the present invention includes the steps of inputting a fixed amount of a sample into an adjacent area of an analysis strip, combining a detecting reagent with the material for analysis of the sample to form a complex, developing the complex on a membrane, observing a change in appearance in the reaction unit having a fixed bed for each pathogen in a predetermined area of the membrane.

The above diagnostic method of the present invention includes sandwich assay or competition assay.

The present invention provides a kit, which is a kit for implementing the diagnosis method above. Referring toFIG.2, provided is a diagnosis kit for detectingRalstonia solanacearumandFusarium oxysporum, wherein the kit protects, from various contaminants, an analysis strip 2 in which a test line 221 having a fixed bed for immunoglobulin G (IgG) antibody specifically responsive toRalstonia solanacearumandFusarium oxysporum, respectively, and a control line 222 for identifying normal operation are provided in a predetermined area on a membrane 22, and a regular analysis strip, wherein at least one of a sample inlet 41 for inputting a sample and a result display window 42 for observing a response result in the test line 221 and the control line 222 on the analysis strip 2.

The above sample preferentially uses plant juice.

In order to detectR. solanacearumandF. oxysporum, which are pathogens of plant wilt disease, from plants by using the immunochromatography, the specific antibody capable of detecting pathogens in the nitrocellulose membrane is adsorbed to a predetermined position and an antibody capable of selectively binding to the pathogen is bonded to the gold particles to be dried on the pad. The dried gold conjugate pad and the pad applying the sample overlap each other to cover the nitrocellulose membrane and include a hygroscopic pad at the opposite position (Referring toFIG.3).

Membrane that can be used for producing analysis strips according to the present invention is capable of using materials generally used for diagnostic strips, and the examples include nitrocellulose, cellulose, cellulose acetate, polyethylene, and a material that is selected from various kinds of synthetic polymer.

As for the labeled reagents that can be included in the control reagents, the same can be applied as in the detection reagents above. Auxiliary specific binding materials are not specifically limited, but can be selected from, for example, avidin, biotin, FITC, anti-FITC antibody, mouse Immunoglobulin G (IgG) or anti-mouse Immunoglobulin G antibody.

The detection reagent includes a labeled reagent, an auxiliary singular coupling member, or a signal generating system that allows the presence of an analyte to be inspected from the outside through a naked eye or other mechanism. Labeled detection reagents are well known in the art to which the present invention belongs. The examples of labeled reagents include catalyst, enzyme (phosphatase, peroxidase), enzyme substrate (nitroblue tetrazolium 3,5′, 5,5′-Tetranitrobenzidine, 6-methoxy-1-naphthol, 4-Chloro-1-naphthol, 5-Bromo-4-chloro-3-indolyl phosphate), chemiluminescence enzyme substrate (dioxetane), fluorescence compounds (fluorescein, phycobiliprotein, rhodamine), Chemiluminescence compounds, metal sol, nonmetal sol, carbon sol, dye sol, particle latex, color indicator, color materials included in liposome, or the like.

The analysis strip of the present invention is produced by putting the analysis strip in a plastic single device (immunodiagnostic analyzer) with a result display 42 in a sample inlet 41 (referring toFIG.2). After grinding the soil surface of plants in the sample inlet 41 with sample diluted solution, three drops of the juice are put on the area of the sample dropping with the usage of a disposable dropping pipet.R. solanacearumandF. oxysporumin the sample respond to specific antibodies attached to gold particles and capillary action is displayed onto the nitrocellulose membrane 22. Specifically, the specific antibody capable of detecting each of thesolanacearumorF. oxysporumis adsorbed at a predetermined position on the test line 221 of the membrane. The pathogen present in the sample is combined with the antibody conjugated to the gold particles to form a composite, and the composite forms a violet (red) band by gold particle color at the corresponding position by combining with an antibody specific to thesolanacearumorF. oxysporumlocated in the test line while passing through the test line. In the control line 222, rabbit anti-chicken IgY is adsorbed to constantly react with the presence of pathogens in the sample regardless of whether pathogen is present in the sample, thereby showing a violet (red) band.

The non-responsive contents are absorbed in wicks, and it is easy to identify the formulated bands since membrane looks pure white. In addition, whenR. solanacearumandF. oxysporumdon't exist in the sample, purple or red bands are formulated in the control lines of the strip (referring toFIG.5).

With respect to a method of diagnosing the status and degrees of infections ofR. solanacearumandF. oxysporumin the plants, the interpretation of each result will be described with reference toFIG.6in detail.

Hereinafter, a raw material, an analysis strip, and a manufacturing method thereof according to an embodiment of the present invention will be described in detail with reference to the following examples. Although these embodiments are presented in order to understand the contents of the present invention, the scope of the present invention should not be construed as limited to the embodiment.

Exemplary Embodiment 1. Production of Antibodies to Mouse Anti-R. solanacearumand Anti-F. oxysporum

A. Preparation of Immunogens,R. solanacearumandF. oxysporum

The bacteria and fungi used as immunogens in mice were introduced and incubated in Korean Agricultural Culture Collection (KACC) through ABC Circle, and the incubated bacteria and fungi were inactivated at high temperature and used as immunogens to produce antibodies.

B. Production of Hybridoma

Two hybridomas were produced to create antibodies specifically responsive to pathogens of wilt diseases. An emulsion mixed at a ratio of 1:1 with complete adjuvant or incomplete adjuvant (Sigma), which is an immunity enhancer, was injected into a belly of a mouse (Balb/cA mouse, 8 week, and female). For two months, six times of immunity were carried out, and mice with high antibody production rate were selected and spleen was extracted. Only red blood cells were selectively removed using RBC hemolytic buffer (Sigma) in splenocytes obtained by crushing the spleen. The spleen cells which were washed three times were mixed with myeloma cells (Sp2/0: Ag-18) at a ratio of 5:1, and a cell fusion was induced by mixing PEG1500 (polyethylene glycol 1500, Sigma) by 1 ml. Hybridoma produced by fusing two kinds of cells was dispensed in the 96 well culture plate, and incubated for 1 week in the selective medium with DMEM (Hyclone) including 10% FBS (Fetal bovine serum, Hyclone) and 1×HAT media supplement (Sigma), and unfused cells were removed. After being incubated for 1 week in the culture media with DMEM (Hyclone) including 10% FBS (Fetal bovine serum, Hyclone) and 1×HAT media supplement (Sigma), cell culture media was collected, and clones that could generate specific antibodies to antigens were selected via the 1st screening. The selected clones went through the isolation process and 2nd screening to secure final monoclonal hybridoma.

C. Selection of Specific Antibody

When it comes to the selection of antibodies to anti-R. solanacearumand anti-F. oxysporum, the antibodies that shows high sensitivity to antibodies were selected, and the antibodies that do not show response to the cross-reacting materials were selected. The sensitivity and specificity of antibodies were identified via the ELISA test.

When selecting antibodies that meet the above conditions, five types of bacteria (Ralstonia solanacearum, Bacillus subtilis, Lactobacillus plantarum, PeseudomonasS.P,Bacillus amyloliquefaciens), five types of fungi (Fusarium oxysporum, Botrysis cinerea, Colletotrichum gloeosporioides, Alternaria ponax, Fusarium graminearum) were identified to determine reactivity through the ELISA test. The above 5 types of bacteria and 5 types of fungi were introduced and incubated through ABC Circle.

According to the result of the test, the anti-R. solanacearumantibody is selected as a final antibody which exhibits high sensitivity toR. solanacearum, and does not exhibit cross reactivity with respect to nine bacteria and fungi (refer toFIG.1).

In addition, antibodies to anti-F. oxysporumare selected as a final antibody which showed high sensitivity toF. oxysporum, and does not show cross-reactivity to 9 types of bacteria and fungi (refer toFIG.1).

D. Bulk Security of Antibody

In order to mass-produce an antibody in a secured hybridoma, a method of mouse ascites generation is used. The hybridoma was cultured and 5×10-6 cells per mouse (Balb/cA mouse, 8 week old, female) were injected into the abdominal cavity, and a plurality of ascites generated in mouse abdominal cavity was recovered after 2-3 weeks. The antibody purification was carried out using a protein G resin (HiTrap protection G HP column, GE). The purified monoclonal antibody was obtained, and a sandwich pair test was carried out to specifically react to each ofR. solanacearumandF. oxysporum, and an antibody that does not show cross reactivity with other bacteria and fungi was finally selected. Finally, an antibody showing high sensitivity and specificity toR. solanacearum, and an antibody showing high sensitivity and specificity toF. oxysporumwere obtained and used in the preparation of a kit according to the present invention.

Exemplary Embodiment 2. Production of Detection Strips and Diagnostic Kits forR. solanacearumandF. oxysporum

A. Preparation of membrane coated with antibodies toR. solanacearumandF. oxysporum

Two specific antibodies to each pathogen (Vetall Lab.) of which the final concentration was the 1 mg/ml concentration is used for test line, the control line is rabbit anti-chicken IgY (Rabbit anti-Chicken IgY) with the 1 mg/ml concentration. The solution of the antibody and control line of the test line was coated in a nitrocellulose membrane using a dispensing mechanism (KINAMETICS, USA). It was dried overnight in a laboratory of low humidity or dried in a pan for at least 5 hours. The plate of the manufactured membrane was kept in a sealed container or in a laboratory with low humidity with a drying agent.

B. Production of Antibody Gold Conjugates

Mouse Anti-R. solanacearumantibody (Vetall Lab.) was agitated so that the final concentration reaches 18 ug/ml and was added dropwise to the gold solution. In addition, the mouse anti-F. oxysporumantibody (Vetall Lab) was agitated so that the final concentration reaches 16 ug/ml and added dropwise to the gold solution. Each two solution was again agitated for 15 minutes. Then, a 10% BSA solution was added to each gold particle suspension. After agitating the solution again for 15 minutes, combined gold solution was isolated through the process of centrifugation, and the supernatant was deserted to eliminate uncombined antibodies. By adding 5 mM Sodium Tetraborate (pH 7.2) with 1% BSA which is three times of the capacity of pellet to combined gold solution (pellet), and then the above pellet was again suspended. After suspensions were again isolated through the process of centrifugation, final pellet with 5 mM Sodium Tetraborate (pH 7.2) added with 1% of BSA was produced by adjusting the absorbance to 10±1 O.D. in the spectrophotometer (530 nm).

C. Production of Chicken IgY Antibody Combined with Gold Conjugates

While agitating the chicken IgY (Fitzgerald, 70-B9093RA00-A0) so that the final concentration reaches 20 ug/ml, and was added dropwise to the gold solution, and the solution were again agitated for 15 minutes. After that, 10% BSA solution was added to the suspensions of gold particles. After agitating the solution again for 15 minutes, combined gold solution was isolated through the process of centrifugation, and the supernatant was deserted to eliminate uncombined antibodies. By adding 5 mM Sodium Tetraborate (pH 7.2) with 1% BSA with 3× capacity of pellet to combined gold solution (pellet), the above pellet was again suspended. After the suspensions were again isolated through the process of centrifugation, final pellet with 5 mM Sodium Tetraborate (pH 7.2) added with 1% of BSA was produced by adjusting the absorbance to 10±1 O.D. in the spectrophotometer (530 nm).

D. Preparation of Pads for Gold Conjugate

The above gold conjugate which was produced in B and C was manufactured by adding 5% Trehalose.

For diagnosis strips forR. solanacearum, the 0.5×20 cm glass fiber was prepared such that a mouse anti-Rsolanacearumantibody-gold conjugate has a final concentration of 3.0 optical density (O.D) and the chicken IgY-gold conjugate at a final concentration of 0.5 O.D.

For diagnosis strips forF. oxysporum, the 0.5×20 cm glass fiber was prepared such that a mouse anti-F. oxysporumantibody-gold conjugate has a final concentration of 2.0 optical density (O.D) and the chicken IgY-gold conjugate at a final concentration of 0.5 O.D.

E. Production of Wicks and Sample Pads

The wicks and sample pads were produced after dehydration to absorb reacting solution.

F. Assembling of the Device

Each of the membranes and the pads prepared above was cut into a strip size suitable for the size of the immunoassay device by overlapping each of the membranes and the pads, respectively, with a sample pad, a gold pad (gold conjugate treatment pad), a nitrocellulose membrane, and finally a moisture absorption pad. The cut strip was finally placed on the lower plate of the immunoassay device for diagnosis, and a kit for co-diagnosis was prepared.

G. Product Configuration

As described above, the immunoassay device and the sample diluent, the sample dilution liquid bottle, the homogenizer, and the dropper are configured as a final product.

Exemplary Embodiment 3. Selection of Cut-Off Level of Pathogen According to the Present Invention

A. To select the cut-off levels of pathogens for co-diagnosis kits for theR. solanacearumandF. oxysporumproduced in the above exemplary embodiment 3, the expression concentration life test in the plant ofR. solanacearumandF. oxysporumcausing wilt disease was carried out by the ABC Circle. The three types of seeding in host plants (tomato, cucumber, chili) were sown for cultivation. CultivatedR. solanacearumandF. oxysporumwere injected into each plant. By identifying symptoms of wilt diseases that appear in plants at 25° C. or higher, and movement and density of microorganism inside the sap were measured in the interval of five days.

As a result, when the early symptoms of the wilt disease in theR. solanacearumplant were confirmed with the naked eye, the pathogen density of 1×105cfu/ml or higher was confirmed, and at least 1×106cfu/ml or more, the wilt disease was rapidly progressed to kill plants. Density measurements were not accurately performed with microorganism separation and identification method in 1×104cfu/m ofR. solanacearum. When the early symptoms of the wilt disease in theF. oxysporumplant were confirmed with the naked eye, the pathogen density of 1×104cfu/ml or higher was confirmed, and at least 1×105cfu/ml or more, the wilt disease was rapidly progressed to kill plants. Density measurements were not accurately performed with microorganism separation and identification method in 1×103cfu/m ofF. oxysporum.

B. Based on the symptoms of wilt diseases and pathogen density, the cut-off level of pathogens in theR. solanacearumandF. oxysporumdiagnosis kit was selected, and for semi-quantitative analysis, the level was determined into three tiers (low, mid, and high) as in <Table 1>.

Exemplary Embodiment 4. Effect Test of Diagnostic Strips forR. solanacearum

A. By using the diagnostic kits forR. solanacearumproduced according to the above exemplary embodiment 2, the plant samples of domestic farmers were provided from ABC Circle for diagnosis. The inflection was identified by using 30 positive samples where bacterial wilt disease occurs and 20 negative samples. By using the microorganism separation and identification method, the pathogen density was measured to detect the signs of infections as in <Table 2>.

With the diagnostic kits of the present invention and Agdia Rs Immuno Strip Test, examination was conducted, and the relative sensitivity and relative specificity of the diagnostic kits were identified by measuring the number of detected samples as below <Table 3A>. The relative sensitivity shows the ratio of positive samples in the Agdia Rs Immuno Strip Test that was found positive in the diagnostic kits of the present invention, and the relative specificity shows the ratio of negative samples in the Agdia Rs Immuno Strip Test that was found negative in the diagnostic kits of the present invention.

As a result, all of the 50 samples showed the same result compared to the Agdia Rs Immuno Strip Test, and the relative sensitivity and relative specificity were 100%.

B. The examination was conducted based on the diagnostic kits of the present invention and the microorganism separation and identification method, and the titer-specific relative sensitivity and relative specificity of the diagnostic kits were identified by measuring the number of detected samples as below <Table 3B>.

The titer-specific relative sensitivity shows the ratio in the density of pathogen from microorganism separation and identification method that also showed the same titer in the diagnostic kits forR. solanacearumaccording to the present invention.

As a result, the titer-specific relative sensitivity was 100% (high titer), 90% (medium titer) and 92% (low titer).

Therefore, the performance of the diagnostic kits according to the present invention was outstanding.

Exemplary Embodiment 5. Effect Test of Diagnostic Strips forF. oxysporum

A. By using the diagnostic kits forF. oxysporumproduced according to the above exemplary embodiment 2, the plant samples of domestic farmers were provided from ABC Circle for diagnosis. Here, 19 positive samples where fungal wilt disease occurred and 31 negative samples were used to detect the signs of infections. By using the microorganism separation and identification method, the pathogen density was measured to detect the signs of infections as in <Table 2>.

With the diagnostic kits of the present invention and the microorganism separation and identification method, the examination was conducted, and the relative sensitivity and relative specificity of the diagnostic kits were identified by measuring the number of detected samples as below <Table 4>.

The titer-specific relative sensitivity shows the ratio in the density of pathogen from the microorganism separation and identification method that also showed the same titer in the diagnostic kits forF. oxysporumaccording to the present invention.

As a result, the titer-specific relative sensitivity was 100% (high-titer), 90% (mid-titer) and 100% (low-titer). Therefore, the performance of the diagnostic kits according to the present invention was outstanding.

TABLE 1Selection of cut-of level of diagnostic kitsforR. SolanacearumandF. OxysporumSymptoms ofR. SolanacearumF. OxysporumWilt DiseaseHigh-Titer107cfu/ml or106cfu/ml orLate stageaboveaboveMid-Titer106cfu/ml105cfu/mlMid stageLow-Titer105cfu/ml or104cfu/ml orEarly stagebelowbelowNegative104cfu/ml or103cfu/ml orNo Symptomsbelowbelow

TABLE 2Comparison of Performance of Diagnostic Kits forR. SolanacearumandF. OxysporumMicro-Micro-organismorganismsepara-separa-tion andtion andidenti-identi-ficationficationmethodAgida RsThe kit ofmethodThe kit offor R.SImmuno-the presentfor F.Othe presentNo.Sample(cfu/ml)Stripinvention(cfu/ml)invention1Tomato4.8 × 107PositiveHigh-Titer≤103Negative2Tomato1.2 × 105PositiveLow-Titer≤103Negative3Tomato≤103NegativeNegative5.4 × 106High-Titer4Tomato2.6 × 105PositiveLow-Titer≤103Negative5Cucumber1.0 × 105PositiveLow-Titer≤103Negative6Cucumber4.0 × 106PositiveMid-Titer≤103Negative7Chili5.4 × 105PositiveMid-Titer≤103Negative8Chili1.8 × 108PositiveHigh-Titer≤103Negative9Chili1.1 × 105PositiveLow-Titer2.6 × 105Mid-Titer10Tomato1.4 × 105PositiveLow-Titer7.0 × 106High-Titer11Chili2.6 × 105PositiveLow-Titer1.6 × 105Low-Titer12Cucumber≤104NegativeNegative≤103Low-Titer13Chili≤103NegativeNegative≤103Low-Titer14Chili≤103NegativeNegative3.8 × 104Low-Titer15Tomato≤103NegativeNegative2.2 × 104Low-Titer16Tomato4.2 × 107PositiveHigh-Titer≤103Negative17Tomato5.2 × 104NegativeNegative≤103Negative18Chili≤104NegativeNegative8.6 × 105Mid-Titer19Cucumber5.1 × 106PositiveMid-Titer≤103Negative20Cucumber1.2 × 106PositiveMid-Titer≤103Negative21Cucumber4.2 × 104NegativeNegative≤103Negative22Tomato3.2 × 106PositiveMid-Titer≤103Negative23Tomato≤103NegativeNegative≤103Negative24Tomato≤103NegativeNegative≤103Negative25Cucumber≤103NegativeNegative≤103Negative26Chili8.4 × 104NegativeNegative≤103Negative27Cucumber≤103NegativeNegative1.0 × 107High-Titer28Tomato≤103NegativeNegative9.2 × 106High-Titer29Cucumber≤103PositiveLow-Titer≤103Negative30Chili7.2 × 105PositiveLow-Titer≤103Negative31Chili2.6 × 104NegativeNegative≤103Negative32Cucumber≤103NegativeNegative5.5 × 105Mid-Titer33Cucumber≤104NegativeNegative1.2 × 105Mid-Titer34Chili4.6 × 104NegativeNegative3.2 × 105Mid-Titer35Tomato≤103PositiveLow-Titer≤103Negative36Tomato≤103NegativeNegative≤103Negative37Tomato≤103NegativeNegative6.8 × 105Mid-Titer38Tomato6.2 × 107PositiveHigh-Titer1.2 × 104Low-Titer39Tomato1.2 × 108PositiveHigh-Titer5.0 × 104Low-Titer40Tomato5.4 × 106PositiveMid-Titer3.4 × 104Low-Titer41Tomato4.0 × 105PositiveLow-Titer≤103Negative42Tomato6.0 × 105PositiveLow-Titer2.8 × 105Mid-titer43Chili6.0 × 106PositiveMid-Titer≤103Negative44Chili2.4 × 106PositiveMid-Titer≤103Negative45Chili2.6 × 105PositiveLow-Titer6.0 × 105Mid-titer46Cucumber5.2 × 105PositiveLow-Titer7.6 × 105Mid-titer47Cucumber3.0 × 105PositiveLow-Titer≤103Negative48Cucumber3.8 × 106PositiveMid-Titer≤103Negative49Tomato4.4 × 106PositiveMid-Titer≤103Negative50Tomato5.8 × 106PositiveMid-Titer≤103Negative* When the microorganism density ofR. Solanacearumwas at 104cfu/m or below, the measurement was not accurately conducted.* When the microorganism density ofF. Oxysporumwas at 103cfu/m or below, the measurement was not accurately conducted.

TABLE 3Relative Sensitivity and Relative Specificity of DiagnosticKits forR. Solanacearumand Those of Other CompaniesAgida Rs ImmunoStrip TestKits of the present inventionPositiveNegativeTotal40 SamplesPositive30030Negative02020Total302050Relative sensitivity100%Relative specificity100%

TABLE 4Relative Sensitivity and Relative Specificity ofDiagnostic Kits forR. SolanacearumandMicroorganism separation and identification methodMicroorganism separation andidentification method(R. Solanacearum)NegativeKits of the≥107=106≤105≤104present inventioncfu/mlcfu/mlcfu/mlcfu/mlTotal50 SamplesHigh-Titer51006≥107cfu/mlMid-Titer091010=106cfu/mlLow-Titer0012214≤105cfu/mlNegative0002020Total510132250High-Titer Relative100%SensitivityMid-Titer Relative90%SensitivityLow-Titer Relative92%SensitivityRelative Specificity90%

TABLE 5Relative Sensitivity and Relative Specificity ofDiagnostic Kits forF. OxysporumandMicroorganism separation and identification methodMicroorganism separation andidentification method(F. Oxysporum)NegativeKits of the≥106=105≤104≤103present inventioncfu/mlcfu/mlcfu/mlcfu/mlTotal50 SamplesHigh-Titer40004≥106cfu/mlMid-Titer09009=105cfu/mlLow-Titer01528≤104cfu/mlNegative0002929Total41053150High-Titer Relative100%SensitivityMid-Titer Relative90%SensitivityLow-Titer Relative100%SensitivityRelative Specificity93%

As described above, it should be noted that the method for co-diagnosis ofRalstonia solanacearumandFusarium oxysporumby using semi-quantitative lateral flow immunodiagnostic technique and kit for use therein according to the present invention has been specifically described in the preferred embodiment, but the above-described embodiments are for purposes of illustration and are not intended to limit the scope thereof. It will be apparent to those skilled in the art that various modifications and variations are possible within the spirit and scope of the present invention, and thus these modifications and variations will be apparent to those skilled in the art.

REFERENCE NUMERALS

1IMMUNOASSAY DEVICE2ANALYSIS STRIP3LOWER CASE3A,3B FIRST STRIP SUPPORT UNIT, SECOND STRIP SUPPORT UNIT4UPPER CASE4A,4B FIRST STRIP RESPONSE UNIT, SECOND STRIP RESPONSE UNIT21SAMPLE PAD221TEST LINE222CONTROL LINE22MEMBRANE23WICK24SHORT SIDE25LONG SIDE31SHORT-SIDE FIXING UNIT32LONG-SIDE FIXING UNIT33LOWER PORTION BLOCKING UNIT41SAMPLE INLET42CHARACTER DISPLAY42RESULT DISPLAY WINDOW