KR101512484B1 - Rapid Methods for Detection of Norovirus in Food Using a Magnetic Bead and Quantum Dot - Google Patents

Abstract

The present invention relates to a method for detecting fluorescence of norovirus, and more particularly, to a method for detecting norovirus fluorescence comprising the steps of: (a) detecting a magenetic bead-binding G protein Preparing a virus probe by binding with an anti-norovirus antibody; (b) contacting the virus probe with an assay sample to form a virus probe-virus complex; (c) binding quantum dots, which are fluorescent materials, to the viral probe-virus complex. According to the present invention, the present invention can purify Norovirus from a sample mixed with virus and can detect it faster than RT-PCR.

Description

TECHNICAL FIELD The present invention relates to a method for rapidly detecting norovirus in foods using magnetic beads and quantum dots,

The present invention relates to a method for pure separation and rapid detection of a single species of Norovirus in various viruses by utilizing a magnetic bead and a quantum dot to which a mixed antibody is attached.

Accidents caused by hazardous substances in agricultural and marine products are seriously affecting people's health and the economic loss caused by domestic food poisoning cases caused by lack of proper early detection methods and systems reaches 1.3 trillion won a year . Even though virus food poisoning accounts for 34-40% of the total food poisoning, the loss due to virus food poisoning reaches 4000 billion won, so it is used for prevention of viral food poisoning from the production of agricultural and fishery products and safe food for consumers and producers. Possible research on the development of virus diagnosis methods is a way to prevent such social loss in advance.

Although the hygiene status of the modern urban environment has been greatly improved, the incidence of norovirus infection has been increasing recently, and the detection of norovirus in agricultural products including fruits and vegetables has also been reported at home and abroad have. Norovirus is most likely to occur when fresh oysters are eaten raw, and can also be transmitted from vegetables. It is susceptible to infection when ingesting Norovirus-infected food, and it is also infected by drinking water from groundwater contamination. This method of diagnosing norovirus is limited to enzymatic immunoassay using molecular cloning or polyclonal antibody and molecular biology test, which is not suitable for the end user to use in the field. It mainly involves groundwater, fish and shellfish products and some vegetables It is a reality that testing methods are being performed.

Therefore, it is necessary to solve the limitations of currently used virus pure separation method and molecular biological diagnosis method as follows for the rapid diagnosis method for various enriched aquatic products.

First, in general, pure isolation of virus is carried out by inoculating host cells, so that other species are diluted and a large number of target species are cultured and separated. However, this method was time-consuming and costly. Therefore, in this study, we aimed to shorten the time and isolate only the target virus accurately.

Second, there are various substances in food, and some of them act as a substance that interferes with the reaction of molecular biologically specific gene amplification method. Therefore, if the virus is mistakenly determined to be not contaminated by disturbing gene amplification despite the existence of virus Therefore, in this study, it is effective to concentrate the virus efficiently by using a magnetic immune probe to detect the virus in the sample, and also to efficiently remove the interfering substance. This technique enhances the detection power, and the immunological method and the microfluidic method It can be a way to overcome the disadvantages of sensitivity of conventional molecular biology alone experiments.

Third, polymerase chain reaction (PCR), a method of amplifying specific genes for molecular biology to detect viruses, has been used rapidly and accurately to date. However, in order to perform such a reaction, it requires specialized techniques and time such as separation of DNA or RNA, cDNA synthesis, and PCR reaction. Therefore, in this study, it is easier to apply in the field than in the field, We tried to detect a virus quickly by using a quantum dot.

The present inventors have sought to develop a novel method for rapidly detecting norovirus. As a result, the present inventors confirmed that it is possible to rapidly detect norovirus using a magnetic bead and a target-binding moiety associated therewith, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for detecting norovirus.

Another object of the present invention is to provide a method for detecting norovirus in a sample.

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

According to one aspect of the present invention, there is provided a composition for detecting norovirus comprising a magnetic particle and a target-binding moiety bonded to the magnetic particle.

The present inventors have sought to develop a novel method for rapidly detecting norovirus. As a result, the present inventors confirmed that the magnetic beads and the target-binding moiety associated therewith can be used to rapidly detect norovirus.

Currently, as the number of cases of norovirus infection and cases of detection increases, the present inventors have found that by using an immunoprecipitated antibody-bound quantum dot to rapidly and accurately detect the virus from a virus-mixed sample, By developing a rapid and accurate detection method, a method of isolating and rapidly detecting Norovirus is provided.

In the composition of the present invention, the magnetic particles of the present invention and the target-binding moiety bonded thereto are bonded through an affinity substance, and the target-binding moiety binds specifically to the norovirus So that the Norovirus can be selectively detected in the sample.

According to embodiments of the present invention, the magnetic particles used in the composition of the present invention include, but are not limited to, magnetic beads.

As used herein, the term " quantum dots " (QDs) generally refers to semiconductor or metal nanoparticles that absorb light at different colors and re-emit quickly, depending on the size of the dots. Nanocrystals, which are nano-sized crystal grains and nano-sized single crystals themselves or aggregates of nano-sized single crystals, are largely classified according to their dimensions. They are a 0-dimensional point, a single sphere, Dimensional nanowire, nanorod, nano-needle, and two-dimensional nanodisk. The form of zero-dimensional nanocrystals is often referred to as a quantum dot (dots), in which droplet-shaped hemispheres of several to several tens of nanometers in diameter are scattered on a substrate. The energy level can be increased by limiting the size of the quantum dot to a region that is shorter than the normal state of the material wave. Depending on the energy state, the color of the emitted light changes when each material is excited, so the wavelength of the emitted light can be controlled by adjusting the size of the quantum dot. Thus, if the very small quantum dots themselves are the source of light, they can be used in a variety of applications, including cell-based therapies, in vivo imaging and tracking, and in biological studies to understand intracellular functions.

According to some embodiments of the present invention, the target-binding moieties of the present invention include, but are not limited to, monoclonal antibodies or polyclonal antibodies capable of binding Norovirus.

According to some embodiments of the present invention, the target-binding moiety of the present invention is a Norovirus antibody conjugated to the magnetic particle through association with the affinity substance.

According to some embodiments of the present invention, the composition of the present invention forms a magnetic particle-target-binding moiety-norovirus complex through binding with norovirus in a sample.

According to some embodiments of the present invention, the detection of norovirus in the composition of the present invention is carried out using reverse transcription-polymerase chain reactions (RT-PCR) or antigen-antibody reaction.

According to another aspect of the present invention, the present invention provides a method for detecting norovirus in a sample comprising the steps of:

(a) preparing a virus probe by binding a magnetic particle-binding protein G and a Norovirus antibody;

(b) adding the virus probe to a sample; And

(c) analyzing the presence of the antigen by separating the target antigen from the virus probe, wherein the analysis is performed by quantitatively confirming the norovirus through the intensity of the fluorescence of the secondary antibody to which the quantum dot is attached ≪ / RTI >

Since the method of the present invention comprises the composition of the present invention as an active ingredient, its description is omitted in order to avoid excessive complexity between the two.

The term "sample" in the present invention means all samples obtained from human or mammals. According to some embodiments of the present invention, the sample to which the method of the present invention can be applied is a blood sample, plasma, serum, virus, bacteria, tissue, cell, lymph, bone marrow, saliva, milk, urine, feces, , Brain extracts, spinal fluid, joint fluids, thymus fluids, ascites, amniotic fluid, cell tissue fluids, and cell culture fluids.

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

(a) The present invention relates to a composition for detecting norovirus comprising a magnetic particle and a target-binding moiety bonded to the magnetic particle bound thereto and a method for detecting the same.

(b) The compositions and methods of the present invention can quickly and easily detect Norovirus in a sample using a target-binding moiety.

(c) Thus, the method of the present invention can be applied to accurately and easily diagnose damage caused by a norovirus infection.

1 is a schematic diagram showing a method for detecting norovirus of the present invention.
Figure 2 shows RT-PCR results showing norovirus specifically detected by the particles of the present invention.
Fig. 3 shows the detection results of the concentration of norovirus isolated by the magnetic beads of the present invention.
Fig. 4 shows the detection results of concentration of norovirus isolated by quantum dots of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Immunoprecipitation using magnetic beads

In the present invention, virus probes were prepared by linking magnetic bead-binding protein G, which is a magnetic substance, with norovirus antibody. The procedure for preparing the viral probes in which the magnetic bead-binding protein G is linked to the norovirus antibody is as follows: 50 μl of magnetic bead-binding protein G (Invitrogen, USA) is placed in a 1.5 ml tube, Respectively. Thereafter, 200 μl of a PBS (Phosphate Buffered Saline; Bio-World, Korea) containing 0.02% Tween 20 (Sigma, USA) was added with Norovirus monoclonal antibody (0.1 mg / ml; mouse monoclonal anti- UK) was added to the tube containing magnetic bead-binding protein G in which the supernatant was removed, and the mixture was allowed to react for 10 minutes. After the reaction is completed, the magnet is attached to the tube and the supernatant is removed, leaving only a virus probe with magnetic bead-binding protein G and antibody bound thereto. To remove impurities other than the virus probes, the virus was washed twice with PBS containing 0.02% tween 20, and then the supernatant was removed to prepare a virus antibody probe comprising a magnetic bead-binding protein G for virus detection.

The reaction conditions of Norovirus, HAV (Hepatitis A Vrirus) and HEV (Hepatitis E Virus) mixed with virus probes are as follows: 1 ml of mixed virus (Norovirus, HAV, HEV) And the mixture was reacted at room temperature for 10 minutes. After the immunological reaction, the magnetic bead portion of the complex in which the antigen is bound to the virus antibody attached to the magnetic bead-binding protein G by attaching a magnet is attached to the magnet. The supernatant containing other viruses that failed to bind was transferred to a new 1.5 ml tube for PCR analysis. The antigen-antibody conjugate attached to the protein G was washed three times with 200 μl of PBS to remove impurities, and then suspended in 100 μl of PBS and transferred to a new 1.5 ml tube to remove the supernatant. The antigen-antibody conjugate attached to protein G in the new tube was separated by elution with an eluent (50 mM glycine, pH 2.8) and neutralized to pH 7.5 with 100 mM Tris solution. To confirm the isolation of Norovirus, RT-PCR was performed to confirm the virus.

The reaction conditions for the detection of fluorescence on the antigen-antibody conjugate attached to protein G are as follows: 200 μl of PBS containing 0.02% tween 20 in the antigen-antibody conjugate attached to protein G was coated with primary polyclonal Norovirus antibody 10 Were dissolved and allowed to react for 10 minutes. After the reaction is completed, the magnet is attached to the tube to remove the supernatant, and then the supernatant is washed three times with 200 μl of PBS to remove impurities. Thus, only the primary antibody bound to the antigen-antibody complex attached to the protein G is left. The secondary antibody with a quantum dot attached specifically to the primary antibody was dissolved in 200 μl of PBS containing 0.02% tween 20 and allowed to react for 10 minutes. Then, a magnet was attached to the tube and the supernatant . After washing with 200 μl of PBS three times to remove impurities, only the primary antibody-secondary antibody bound to the antigen-antibody conjugate attached to the protein G is left. To detect the fluorescence, the excitation wavelength is changed from 360 nm to Spectra Max GEMINI -XS spectrofluorometer (Molecular Devices Corp., Sunnyvale, Calif., USA).

Example 2: Confirmation of pure isolation of norovirus

The virus was detected by immunoprecipitation using magnetic beads by performing RT-PCR on the eluted product to confirm isolation of the desired virus from the virus mixture. In order to identify the virus, polymerase chain reaction (PCR) was carried out using a primer specific for norovirus, HAV and HEV. The primer sequences were as follows: MON_431_F (5'-TGG ACI AGR GGI CCY AAY CA-3 ') and MON_433_R (5'-GGA YCT CAT CCA YCT GAA CAT-3) VP1_F (5'-TGG TTT GCC ATC AAC ACT GAG G-3 ') and VP1_R (5'-ACC CAA GGA GTA TCA ACG GCA AG-3') were used for identification of HEV, HEV_IN_F (5'-GTA TTT CGG CCT GGA GTA AGA C-3 ') and HEV_IN_R (5'-TCA CCG GAG TGY TTC TTC CAG AA-3').

PCR conditions for virus detection were as follows: PCR conditions for norovirus were denaturation (95 ° C / 30 sec), annealing (60 ° C / 30 sec) and extension (72占 폚 / 30 seconds) was carried out 40 times and the final elongation was carried out at 72 占 폚 for 10 minutes. The PCR conditions of HAV were performed 40 times for denaturation (95 ° C / 30 sec), annealing (57 ° C / 30 sec) and extension (72 ° C / 30 sec) Gt; 72 C < / RTI > for 10 minutes. HEV was subjected to denaturation (94 ° C / 30 sec), annealing (61 ° C / 30 sec) and extension (72 ° C / 30 sec) for 40 times and final extension at 72 ° C for 10 min .

As a result, Norovirus, HAV and HEV were detected in the washed fraction (FIG. 2B) obtained by washing the magnetic beads bound with the Norovirus antibody with the PBS solution, and the magnetic bead antigen-antibody conjugate washed with the PBS solution was detected in the glycine solution (Fig. 2), only the norovirus was detected in the eluted fraction (Fig. 2-A) neutralized with a Tris solution (pH 7.6) after eluting the virus with a buffer solution (pH 2.8).

Example 3: Detection of the ability of norovirus to be detected by dilution through immunomagnetic separation

In order to confirm the detection ability of the desired virus by the virus dilution with the magnetic bead, 30 μl of Norovirus diluted in 15 g of lettuce was inoculated 10 times. Subsequently, the lettuce was dried in a clean bench for 30 minutes and homogenized in 60 ml of Tris solution (100 mM Tris-HCl, 50 mM glycine, 1% beef extract, pH 9.5). The virus supernatant was taken by centrifugation at 15,000 x g, 4 < 0 > C. Then, the virus prepared by reacting the immune probe prepared in Example 1 with the virus supernatant was detected by RT-PCR.

Norovirus was detected on a dilution basis. As a result, norovirus was detected from the undiluted sample up to 100-fold diluted samples (Fig. 3A) Detection was carried out in diluted samples (Fig. 3B).

Example 4: Confirmation of fluorescence detection ability of Norovirus isolated by IMS using quantum dots

After isolating the virus by immunoprecipitation using magnetic beads, a virus-specific primary antibody and a secondary antibody specific for the primary antibody were reacted to detect the fluorescence. Quantum dots are attached to the secondary antibody to quantitatively confirm norovirus through the intensity of fluorescence. Fluorescent detection of Norovirus by dilution resulted in a decrease in fluorescence intensity as the virus was diluted. The fluorescence intensity was the same as that of the negative control PBS and magnetic beads in the 1000-fold diluted samples (FIG. 4). The above results are consistent with the results of FIG. 3, which detects up to 100-fold diluted Norovirus in RT-PCR.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is obvious that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A composition for the detection of norovirus comprising magnetic particles and a target-binding moiety coupled to the magnetic particles, wherein the magnetic particles and the target-binding moiety are capable of interacting with the norovirus in the sample Binding agent to form a magnetic particle-target-binding moiety-norovirus complex through the binding, and the detection of norovirus in the composition is carried out by measuring the intensity of the secondary antibody-derived fluorescence with the quantum dot attached thereto Lt; RTI ID = 0.0 > of Norovirus. ≪ / RTI >
The composition of claim 1, wherein the magnetic particles are magnetic beads.
2. The composition of claim 1, wherein the magnetic particles are bound to an affinity material for the target-binding moiety.
4. The composition of claim 3, wherein the affinity material is Protein G.
2. The composition of claim 1, wherein the target-binding moiety is an antibody capable of binding to norovirus.
4. The composition of claim 3, wherein the target-binding moiety is a Norovirus antibody conjugated to the magnetic particle through association with the affinity material.
delete delete A method for detecting norovirus in a sample comprising the steps of:
(a) preparing a virus probe by binding a magnetic particle-binding protein G and a Norovirus antibody;
(b) adding the virus probe to a sample; And
(c) analyzing the presence of the antigen by separating the target antigen from the virus probe, wherein the analysis is performed by quantitatively confirming the norovirus through the intensity of the fluorescence of the secondary antibody to which the quantum dot is attached ≪ / RTI >

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