TW200848735A - Detection reagent and detection method for orchid viruses - Google Patents

Abstract

A detection reagent for orchid viruses including a solution and magnetic nanoparticles is provided. The magnetic nanoparticles are dispersed in the solution. Each of the magnetic nanoparticles includes a magnetic core, a surfactant layer and antibodies of orchid viruses. The surfactant layer is coated on the magnetic core. The antibodies of the orchid viruses are bound on the surfactant layer.

Description

200848735 24164twf.doc/〇〇6 IX. Description of the Invention: [Technical Field] The present invention relates to a detection reagent and a detection method for a living organism, and particularly relates to a detection reagent and a detection method for a orchid virus. [Prior Art] The method commonly used to detect orchid virus is light-type enzyme-linked immunosorbent assay (enZyme_linked, imrnunosorbent assay, ELISA) and biochemical polymerase-linked anti-f' (Polymerase) Two kinds of chain reaction, PCR). However, these two methods show serious shortcomings when actually applied. For example, an ELISA must have two antibodies for a single virus to be tested, and it is easy to have an incorrect test result (about 10_15% false negative) due to the color of the orchid fluid to be tested. In addition, pCR requires complicated testing procedures and related equipment. SUMMARY OF THE INVENTION In view of the above, an object of the present invention is to provide a U detection reagent for orchid virus, which can effectively detect orchid virus. Still another object of the present invention is to provide a method for detecting orchid virus, which has the advantages of simple operation and high reliability. The invention provides a detection reagent for orchid virus, which comprises a solution and a plurality of magnetic nanoparticles. The magnetic nanoparticles are distributed in a solution, and each of the magnetic nanoparticles includes a magnetic core, an intervening layer, and a plurality of blue bodies. The surfactant layer is coated on the magnetic core. The orchid virus antibody binds to the surfactant layer. ° 口 6 200848735 24164twf.doc/006 In accordance with an embodiment of the present invention, the solution is, for example, an aqueous solution in the test agent for orchid virus described above. According to an embodiment of the present invention, in the detection reagent of the orchid virus, the material of the magnetic core is, for example, Fe3〇4, Fe2〇3, _62〇4, CoFe204 or NiFe2〇4 〇 according to an implementation of the present invention. For example, in the above detection reagent for orchid virus, the diameter of the magnetic core ranges, for example, between 5 nm and 9 nm. P According to an embodiment of the present invention, in the above test agent for orchid virus, the material of the surfactant layer is, for example, a hydrophilic interface active material. According to an embodiment of the present invention, the hydrophilic interface active material is, for example, an organic acid, a protein A, a protein G, a dextran or a vesicle. According to an embodiment of the present invention, the orchid virus antibody is, for example, a monoclonal antibody or a plurality of antibodies. ◎ , ^ According to the description of the implementation of the present invention, in the detection reagent of the orchid virus mentioned above, the orchid virus is, for example, the gingival plaque virus (〇d〇nt〇gl〇ssum dngspot virus, 0RSV), East Asia A mosaic virus (Cym) or a cucumber mosaic virus (CMV). The invention provides a method for detecting orchid virus, comprising the following steps: firstly providing a detection reagent, the detection reagent includes The solution and the plurality of magnetic particles are shown in the solution. The magnetic nanoparticles are distributed in the solution towel, and each of the magnetic nanoparticles 7 200848735 24164twf. doc/006 particles include a magnetic core, an intervening agent layer and a plurality of orchid virus antibodies. The agent layer is coated on the magnetic core. The orchid virus antibody is bound to the surfactant layer. Next, the first magnetic susceptibility of the detection reagent is measured (哗10). Then, the orchid liquid to be tested is mixed with the detection reagent. Next, the second magnetic susceptibility of the test reagent and the orchid liquid to be tested is measured. Thereafter, the difference between the magnetic susceptibility and the first magnetic susceptibility when the second magnetic susceptibility is different from the first magnetic susceptibility indicates that the orchid liquid to be tested contains orchid virus. In the embodiment described above, in the above-mentioned orchid virus test: = 1 / the magnetic susceptibility and the second magnetic susceptibility measuring instrument are, for example, AC^(V^H) (AC magnetosusceptometer). : 方方:J Ben Haoyue - described in the embodiment 'In the above-mentioned orchid virus detection, the second round of the Wei-Wing, for example, is the AC magnetic susceptibility. ΐ=;!=:: In the above-mentioned orchid virus, the amount of orchid disease ================================================================================= The material of the above-mentioned orchid virus for detecting the magnetic core includes Fe 〇F 〇 or NiFe 2 〇4.峨 MnFe2〇4, the test method described in the examples, between the above-mentioned orchid virus tests. The diameter of the 'magnetic core' is, for example, in the range of 5 nm to _ nanometer according to the invention, in the above-mentioned orchid virus test 200848735 24164 twf.doc/006 = method, the surfactant layer The material is, for example, a hydrophilic interface active material. According to an embodiment of the present invention, in the above test method, the hydrophilic interface active material is, for example, an organic acid, a protein: a protein G, a dextran or a vesicle. In accordance with an embodiment of the present invention, in the above-described orchid disease detection method, the orchid virus antibody is, for example, a monoclonal antibody or a plurality of strains, which are described above according to an embodiment of the present invention. In the main method of orchid disease, orchid virus such as spoon detection or courgette virus. In the fruit 阑肷 阑肷 , 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈λ有, 夕卜 The orchid virus of the present invention is mixed with the flower and liquid, and then compared; the orchid virus is to be tested, so it is quite simple in operation (4) The method of the above-mentioned results obtained by the detection method does not have the effect of the color of the orchid liquid, and the detection method of the orchid virus proposed by ί Lanming can be determined in addition to the qualitative analysis of the singularity of the heart. The above and other objects, features and advantages of the present invention will become more apparent from the following description. [Embodiment] Fig. 1 is a schematic view showing a detection reagent for orchid virus according to an embodiment of the present invention. Fig. 2 is a schematic view showing the combination of magnetic nanoparticles and a sputum virus to be tested according to an embodiment of the present invention. Please refer to Figure 1. The detection reagents for orchid virus include solution 1〇〇 and magnetic 丨 奈 nanoparticle 1〇2. The solution 1 is, for example, an aqueous solution. The orchid virus to be tested may be any pre-detected orchid virus, such as the mushroom leaf spot virus, the East Asian orchid mosaic virus, or the orchid disease or the courgette virus. The magnetic nanoparticles 102 are distributed in the solution 1 , and each of the magnetic nanoparticles 102 includes a magnetic core 104, an intervening agent layer, and an orchid virus antibody 108. Magnetic core 104 is the source that causes the detection reagent to possess magnetic properties. The material of the magnetic core 104 is, for example, Fe3〇4, Fe2〇3, 〇4, c〇FQ〇4 or

NiFe^4. The diameter of the magnetic core 1 〇 4 is, for example, between 5 nm and 9 〇〇 〇 。. The non-activator layer 106 is coated on the magnetic core 104, and the magnetic core 1〇4 can be stably dispersed in the aqueous solution by the interface active agent layer 106 and Brownian motion. Therefore, the magnetic core 104 can be uniformly and stably dispersed in the solution without being aggregated or precipitated. The material of the surfactant layer 106 is, for example, a hydrophilic interface active material such as an organic acid, protein A, protein G, dextran or vesicles. The Lancai virus antibody 108 is bound to the surfactant layer 1Q6, so that the 200848735 24164twf.doc/006 magnetic nanoparticle 102 can be combined with the corresponding orchid virus, causing the magnetic nanoparticle 102 to become larger or It is to integrate a plurality of magnetic nanoparticles 102 into a cluster of magnetic particles. The orchid virus antibody 1〇8 is, for example, a monoclonal antibody or a polyclonal antibody (p〇lyCi〇nai). Hereinafter, the detection principle of the present invention will be described using FIG. 1 and FIG.

Ο Refer to Figure 1, each individual magnetic core; the magnetic dipole direction of 1〇4 is indicated by the shovel head. In the absence of an external magnetic field, due to thermal motion, the magnetic dipole direction of each magnetic core is directed to the random (rand〇m) direction, that is, to the 夂 direction, and the probability is isotropic. Therefore, the detection reagent does not exhibit magnetic properties without a magnetic field. When the applied magnetic field is a change in alternating current (about tens to tens of thousands of Hz), the single magnetic nanoparticle 102 is magnetically excited by the applied alternating current, because it detects the alternating magnetic field (Xac). Please refer to Figure 2. When the detection reagent is mixed with the orchid liquid to be tested, some of the magnetic nanoparticles in the reagent will be combined with the orchid virus to be tested by the orchid virus antibody 1〇8 to cause these magnetic nanoparticles. Particle ι〇2 = the actual volume becomes larger or the multiple magnetic nanoparticles are depolymerized into a cluster of magnetic r1〇fr^2. In this way, the magnetic nanoparticles (10) which are caused by the orchid virus can no longer be reduced with the addition of an alternating magnetic field. If there are more blues to be tested, the more magnetic nanoparticles will be withdrawn and the blue tH11G combined with the denier to be tested. The current AC magnetization is the intersection of the test reagent and the _ orchid liquid before and after mixing. The rate change 'can detect whether the orchid liquid to be tested contains the orchid to be tested 200848735 24164twf.doc/006 virus 110. The flow of the orchid virus is shown in the embodiment of the present invention. The second step S100 'provides a detection reagent, and the detection reagent is as described in the embodiment of FIG. , rate = #WS102' measurement detection reagent: the first - magnetic susceptibility H-type brother - measurement method of magnetic susceptibility, for example, profit & Ο u

The magnetic permeability meter measures the AC susceptibility of the detection reagent. /; IL person then 'please refer to the step, mix the orchid liquid to be tested with the detection reagent. Please refer to step S106. After the magnetic liquid in the reagent to be detected is fully reacted, measure the detection reagent and the reagent: blue solution The second magnetic susceptibility after the combination. The heart of the heart 'Please refer to the step, the difference between the magnetic susceptibility and the second magnetic susceptibility /, 'when the second magnetic susceptibility is different from the first magnetic susceptibility, for example, the second, the magnetic susceptibility of the younger brother' represents The orchid liquid contains orchid disease. : When the test sputum liquid does not contain the orchid virus to be tested, the second magnetic susceptibility is almost in agreement with the second magnetic susceptibility. The value of the county means that the case where the second rate is different from the first susceptibility does not include the difference caused by the error of the quantity. In the above, the detection method of the orchid virus of the present embodiment can be carried out only by measuring the antibody of the spleen disease surface to perform any separation process (4) = the test procedure is very simple, as long as the test of the orchid virus is tested. And measuring the blue liquid mixture 're-measuring the detection reagent before and after mixing the magnetic susceptibility Θ additionally 'using the orchid virus detection method of the present embodiment, the result, will not be affected by the color of the orchid liquid to be tested and the wrong The measurement results are obtained, so that the obtained measurement results have high reliability.曰D, Ο

(J 200848735 24164twf.doc/006 changes can be found whether the orchid liquid to be tested contains the orchid to be tested, and the calculation of the amount of change in magnetic susceptibility can be carried out and quantitative analysis. In the following 'in order to prove the effectiveness of the present invention, two experimental examples are specifically described. The two experimental examples use an AC magnetic permeability meter to measure the parental permeability of the detection reagent, and the orchid viruses to be tested are Odontoglossmn ringSpot virus (〇RSV) and Cymbidium mosaic virus (CyMV) 〇 Figure 4 shows the percentage change of AC susceptibility of 〇RSV test results in an experimental example of the present invention. The bar chart. (1) Design of the orchid liquid to be tested In this experimental example, the design of the orchid liquid to be tested is shown in the table. The number of the orchid liquid to be tested is SI, S2, S3, and the orchid liquid to be tested si Contains 〇RSV but no CyMV 'The orchid liquid S2 to be tested contains CyMV but no 〇 rsV, and there is neither ORSV nor CyMV in the orchid liquid S3 to be tested. H The orchid liquid to be tested is blue: the composition of the poison -, one, ·, . 1yi ~来口,J Group is the orchid liquid number to be tested OR SV " ---------------------- CyMV S1 has ----- .. no S2 benefits S3 benefits --------- —— No (b) test procedure 13 200848735 24164twf.doc/006 Early too οι精的检测试剂. The test reagent contains magnetic nanoparticles, and the tree U particles are made of nucleus, coated with 介:=吉合合面 surfactant The anti-ORS V layer on the layer is composed of the AC-magnetization rate Xac of the measuring AC-measuring instrument before the mixing with the test liquid to be tested. 3. Take another 4G microliter of the orchid liquid to be tested and mix it thoroughly with the detection reagent. ο ο After the magnetic nevus is fully reacted with the orchid liquid to be tested, put it into the AC magnetic permeability meter. The AC susceptibility Zac after mixing with the orchid liquid to be tested.

士 匕 乂 ac and 乂 this,. The difference. When the orchid fluid to be tested contains ORSV, 'Xac and Xac,. The values will not be the same and will be less than Lc. . When the blue sputum to be tested does not contain 0RSV, Xac and Xac,. Almost the same, the difference between them comes from the error of the measurement system. (-) Detection result Refer to the absolute value of the representative (Xadac, .) in the vertical axis title in Fig. 4. Through the above detection procedure, the detection reagents were mixed with the three orchid liquids si, S2, and S3, respectively, and the results showed that the orchid liquid S1 containing only the ORSV had a large change in the alternating magnetic susceptibility, and the other two did not. There is almost no change in the 0RSV orchid liquids S2 and S3 to be tested. Therefore, the method proposed by the present invention can correctly and conveniently detect the blue liquid to be tested containing 〇 R S V . FIG. 5 is a bar graph showing the percentage change of the magnetic susceptibility of the CyMV detection result of an experimental example of the present invention. 14 200848735 24164twf.doc/006 () Design of the test orchid liquid

"In this experimental example, the design of the orchid liquid to be tested is shown in Table 2. The numbers of the ^ are each w, S2, S3, and the orchid liquid S1 to be tested contains 0RSV.

Si Wait: · Flower liquid S2 contains CyMV but no. RSV, and there is neither 0RSV nor CyMv in the sputum S3 to be tested.

-~ The composition of the virus The number of orchids to be tested

(2) Testing procedures; 2 slanting agent. The inspector's money contains magnetic nanoparticles. The nanoparticles are composed of a magnetic core, an interface coated on the surface of the heteronuclear, and an anti-CyMV of the East Asian blue-stained virus combined with the surfactant layer. Ο 2 The above-mentioned detection of the sensitive magnetic flux of Lai Min people, the detection reagent in the AC magnetic susceptibility before the mixing with the flower liquid. . 3. Take another 4 liters of the orchid liquid to be tested and mix well with the test reagent. After a full reaction of the magnetic green chlorophyll and the cypress, the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _士 5·Compared with the difference between Xac and 0. When the orchid liquid to be tested contains CyMV 4 'Xac and Xac,. The value will not be the same, L will be less than ^. . And when the blue sputum to be tested does not contain cyMV, n. Almost _, 'the difference between them 15 200848735 24164twf.doc/006 error from the measurement system. (III) Test results Refer to Figure 5 for the absolute value of the representative (χ^χ^.) in the vertical axis title. Through the above detection procedure, the detection reagents were mixed with the three orchid liquids SI, S2, and S3, respectively, and the results showed that the orchid liquid S2 containing only CyMV had a large change in the alternating magnetic susceptibility, and the other two were not included.

CyMV's orchid liquids to be tested showed little change in si and S3. Therefore, the method proposed by the present invention can correctly and conveniently detect the orchid liquid to be tested containing CyMV. According to the above, 1, the rush has at least the following advantages. The detection reagent of the orchid virus proposed by the present invention can effectively measure whether the orchid liquid to be tested contains the orchid virus to be tested. Double single 2. · The invention is based on the fact that the remedy of the remedy of the remedy of the remedy of the disease is quite quantitative. The radix of the orchid orchid obtained by the method of detecting the orchid virus can be carried out. Although the present invention has been disclosed in the preferred embodiment as above The iron is limited to the present invention, and any person skilled in the art can not make a departure from the scope of the patent application. Protection of X Ming [Simplified Description of the Drawings] Fig. 1 is a schematic view showing the detection of orchid virus according to an embodiment of the invention _ 16 200848735 24164twf.doc/006. 2 is a schematic view showing the combination of magnetic nanoparticles and the orchid virus to be tested according to an embodiment of the present invention. FIG. 3 is a flow chart showing a method for detecting orchid virus according to an embodiment of the present invention. Fig. 4 is a bar graph showing the percentage change of the AC susceptibility of the 〇 r s V detection result of an experimental example of the present invention. Fig. 5 is a bar graph showing the percentage change of the AC susceptibility of the CyMV detection result of an experimental example of the present invention. [Explanation of main components] 100 Solution 102 Magnetic nanoparticles 104 Magnetic core 106 Interfacial layer 108 Orchid virus antibody 110 · Orchid virus to be tested SI, S2, S3 · Number of orchid liquid to be tested

Sioo, S102, S104, S106, S108: Step number 17

Claims (1)

200848735 24164twf.doc/006 X. Patent application scope ··· A detection reagent for orchid virus, including: a solution; and particles = miscellaneous recordings, distributed in a bribe liquid towel, each of the magnetic nano-magnetic core; ο ο ; 活性, an active agent layer, coated on the magnetic core; and an M-flower virus antibody, bound to the surfactant layer. The detection of the orchid virus according to the invention is as follows: The reagent of the orchid virus according to the first item of the patent scope, wherein the magnetic core material comprises F / CoFe2〇4 or NiFe2〇4 . 3 must be 4, the agent please check the orchid virus test mentioned in the first paragraph of the patent scope _ "the diameter of the magnetic core in the range of 5 nanometers to _ nanometers ^ 2 patents _ 1 The detection of the orchid virus·^/, the medium; the material of the I-side activity layer includes a hydrophilic interface active material, such as the detection agent of the orchid virus according to the fifth aspect of the patent scope, wherein the hydrophilic interface active material Including organic acid, egg protein G, dextran or vesicles., 7. The agent of the H virus as described in the application of the ' 其中 病毒 virus antibody including the monoclonal antibody π 8. The test of the orchid virus described in item i 18 200848735 24164twf.doc/006 == The virus includes a method of detection of the tooth flanking virus, the East Asian blue virus 9 · a orchid virus, comprising: providing a detection reagent, including: a solution; and a nano magnetic core; a magnetic active layer, coated on the magnetic core; and a scorpion virus antibody, combined with the interface activity measuring the detection reagent a first magnetic susceptibility; Mixing the detection reagent; and measuring the second magnetic susceptibility of the system and the orchid liquid; comparing the difference between the first magnetic susceptibility and the second magnetic susceptibility, and each of the = ratios is different from the first susceptibility , indicating the orchid flower to be tested for the scorpion virus. 啕, 10. The method for detecting orchid virus according to item 9 of the patent scope, the measurement of the first-wei rate and the second magnetic susceptibility includes The method of detecting an orchid virus according to the invention of claim 9, wherein the first magnetic susceptibility and the second magnetic susceptibility comprise an alternating magnetic susceptibility. 12. The method for detecting orchid virus according to the nine item, wherein the method further comprises: changing the first magnetic susceptibility and the second magnetic susceptibility to change 19 200848735 24164 twf.doc/006 to reduce the weight of the π ... 13· For example, the method described in the ninth item of the patent application, the new method includes the water seam. The method of the orchid virus described in the patent of the patent of the nuclear compound (4), wherein the magnetic core Material package #, C〇Fe2〇4 or NiFe2〇4. 1 〇 、, ^, (4) 〇 4, The method for the detection of the orchid virus described in item 9 is from the middle of the 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈 奈. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ = Λ 或 or multiple antibodies. The method of 'the orchid virus includes 4 detection ΐ orchid virus test side or cucurbit vine virus. Mother-in-law rotatory ring spot virus, East Asian blue mosaic virus 20