KR101548167B1 - Method for Concentration and Detection of Norovirus or Hepatitis A Virus - Google Patents

Abstract

More particularly, the present invention relates to a method for concentrating viruses in a virus solution having a low concentration and concentrating the virus in a short period of time with high efficiency, and more particularly, Con A) to the reaction mixture; And (B) separating the virus-Concanavalin A complex from the reaction solution. The present invention also relates to a virus detection method using the same.

Description

[0001] The present invention relates to a method for concentrating and detecting norovirus or hepatitis A virus,

The present invention relates to an economical method for concentrating a virus capable of concentrating a virus in a virus solution having a low concentration at a high efficiency in a short time, and a virus detection method using the method.

"Hasho" as defined in Article 4 of the Food Hygiene Law (Prohibition of Sales of Hazardous Food, etc.) refers to biological, chemical, or physical factors or conditions that may harm the health of the human body. Foreign objects or foreign matter that does not directly affect the hygiene of the consumer are not regarded as a hazard. The hazard factors can be classified as shown in Table 1 below.

The present invention relates to the detection of biological hazards. Biological hazards include microorganisms such as fungi, bacteria, viruses, and organisms such as parasites. Biological hazards can be introduced into the workplace during the production and distribution of raw materials and may be contaminated by the workplace environment, employees, food ingredients, and the manufacturing process itself.

Accidents caused by hazards in food derived from agricultural and marine products are seriously affecting the health of the people and the domestic economic loss caused by food poisoning accidents caused by the lack of proper early detection methods and systems Reaching 300 billion won. Most of the causes of biological food poisoning are caused by microorganisms. Among them, food poisoning caused by viruses accounts for 34-40% of the total food poisoning, resulting in loss of virus food poisoning by 400 billion won.

Although the hygiene status of the modern urban environment is improving, the number of cases of norovirus infection, which is the cause of food poisoning, is increasing. In 2012, 6 people in Japan and 12 people in Europe died from the virus. Centers for Disease Control and Prevention has reported a total of 348 outbreaks from 1996 to 2000 due to Norovirus infection.

In addition, hepatitis cases are reported to increase in young adults as the rate of antibody to hepatitis A virus (HAV) decreases. Hepatitis A virus is a highly contagious waterborne disease and acute inflammatory liver disease that is transmitted through the feces and oral route and is estimated to cause 1.5 million cases of infection worldwide every year. In Korea, since the introduction of hepatitis A as a designated epidemic since 2000, the number of cases has increased from about 100 in 2001 to 7,655 in 2010.

According to this trend, in 2010, Norovirus and Hepatitis A virus were changed to Group 1 infectious diseases, and the infection control system for statutory infection was operated (Disease Control Division).

On the other hand, cases in which harmful viruses are detected in agricultural products including fruit and vegetables have been reported in domestic and foreign countries. If you eat foods that do not contain virus-infected food or drink contaminated ground water with drinking water, you can get infected by the virus. For example, Norovirus and Hepatitis A virus are susceptible to infection when they eat raw oysters. Table 2 shows the cause of infection and symptom prevention in Norovirus and hepatitis A virus.

Therefore, high-speed detection of viruses that can be utilized to prevent viral food poisoning and provide safe food to consumers from the local production of agricultural and fishery products is a way to prevent such social loss in advance.

The food poisoning virus can be diagnosed by enzyme immunoassay using monoclonal or polyclonal antibodies, and molecular biology test. Recently, a test using chip technology has also been developed.

In order to detect viruses in foods, the process of eliminating viruses from food, concentrating viruses in desalination liquids, and detecting viruses is undergoing. The virus is desorbed using a desalination liquid, and a wide variety of desalination liquids are used depending on the analysis institution. Immunomagnetic capture, organic flocculation, and PEG (polyethylene glycol) precipitation method are used as methods for concentrating the virus that has been removed. However, the immune magnetic capture method can reduce the concentration time of the virus, but it is a high-cost concentration method using an expensive antibody. Since the organic agglutination method and the PEG precipitation method take a long time to concentrate the virus and the process is complicated, The sensitivity of the detection is low.

Meanwhile, tap water and ground water are mainly used for drinking water and living water. Due to the development of sanitary treatment facilities and methods, waterborne diseases such as typhoid fever, cholera, bacterial and amoebic dysplasia are greatly decreased. On the other hand, protozoa are resistant to chlorine disinfection and are difficult to remove during the water treatment process, so the onset of diseases caused by giardia and cryptosporidium is still a problem. In addition, since waterborne viruses can infect at least 1 unit, there are cases in which virus accidents are caused without detection of indicator microorganisms. Infection by viruses is caused by insufficient treatment of the contaminated raw water, or in private or simple water treatment plants. In the case of groundwater, various harmful substances are detected due to water pollution due to environmental pollution (foot-and-mouth disease, etc.). Especially, Norovirus that is transmitted through the feces of infected patients is small in size and easy to penetrate into soil. It is possible to survive, which is a problem. Therefore, there is still a need for safe drinking water provision from biological hazards such as viruses.

In order to solve the above problems, it is an object of the present invention to provide a method for efficiently concentrating and detecting viruses in a short time using low-cost equipment and reagents.

It is another object of the present invention to provide a method for producing a virus-binding crude protein which can be economically and effectively used instead of the purified virus-binding protein in the above-mentioned virus concentration.

Another object of the present invention is to produce a virus probe which can be used for concentration and detection of virus.

Yet another object of the present invention is to provide a purification method in which the removal of virus is effective.

In order to accomplish the above object, the present invention provides a method for producing a virus, comprising: (A) adding Concanavalin A (Con A) to a virus sample solution to react; And (B) separating the virus-Con A complex from the reaction solution. As a result of efforts to develop a novel method for rapidly enriching and detecting viruses, the present inventors have found that Con A specifically binds to viruses and uses them to conveniently and rapidly concentrate viruses and further to detect viruses Respectively.

The Con A may be a commercially available purified sample. However, since Con A as a reagent is expensive at a rate of several hundred thousand won per gram, when the number of samples is large and the number of repeated experiments is large, such as virus detection in a food material rather than concentration of a virus for research purpose in a laboratory, A sample that can be used as a sample is required. In the virus concentration of the present invention, a crude proteinaceous sample other than the purified Con A sample may be used. The crude proteinaceous sample has a large content of Con A, which is advantageous for effective concentration of the virus. Thus, the present inventors have developed a method for producing crude proteinaceous material having a high content of Con A. That is, Con A is a step of: (A) extracting a bean pulverized product with water; (B) obtaining a precipitate fraction precipitated by ammonium sulfate having a degree of saturation of from 0.4 to 0.8 in said water extract; And (C) removing ammonium sulfate from the precipitate fraction. The degree of saturation means the concentration ratio to the concentration in the saturated solution.

In the present invention, the reaction time of step (A) is preferably 5 minutes to 1 hour. When the reaction time is less than 5 minutes, Con A is not sufficiently bound to the virus and the concentration efficiency is lowered. The present invention is economical using low-cost samples and equipments, and is characterized in that the time required for concentration of virus is greatly shortened because the time for reaction with virus is short and the concentration efficiency is good. Therefore, if the reaction time is prolonged, the concentration efficiency is somewhat improved, but in general, it is sufficient to carry out the reaction for 10 minutes to 30 minutes. When the reaction time is more than 1 hour, other problems do not occur but the effect obtained substantially increases , The reaction time is preferably 1 hour or less.

In the following examples, only norovirus and hepatitis A virus are exemplified as the virus, but the present invention is not limited thereto.

The separation of the virus-Con A complex in step (B) may be performed by various methods, and may be appropriately selected depending on the application. For example, an organic solvent mixed with water, such as acetone, may be added to the reaction solution to precipitate a virus-CoA complex and recover it. Alternatively, a viral probe in which Con A is connected to a magnetic bead or a resin can be prepared. In Step (A), Con A may be added in the form of a virus probe, reacted, and the probe may be recovered to isolate the virus- . Con A, which is connected to magnetic beads or resin, can also be used as a purified sample, but similar results can be obtained when it is prepared in the form of crude protein.

Thus, the present invention also provides a virus probe in which Con A is covalently bound to a support. The support may be in any form as long as it has a functional group capable of bonding with Con A as a solid material used for fixing Con A. In the following examples, magnetic beads and resins are exemplified as supports, but species or porous carriers may be used. When the virus probe is made of magnetic beads, it can be easily recovered by magnetization or filtration, and in the case of resin or paper, it can be easily recovered by filtration. It is essential that Con A and a virus form a complex to enrich the virus, and those skilled in the art will understand that a virus probe to which Con A is attached is a conventional technique (see, for example, Shan S. Wong, and David M. Jameson protein and nucleic acid cross-linking and conjugation "CRC press, 2011). Therefore, detailed description of the production of the virus probe is omitted.

(A) preparing a sample solution; (B) concentrating the virus from the sample solution by the virus concentration method according to the present invention; And (B) detecting a virus from a concentrate of the virus.

If the sample is a solid sample such as a vegetable, the preparation of the sample solution is made through a viral desorption step. According to the present invention, the Con A solution can be used as a viral desalting solution to react with Con A simultaneously with the desorption of the virus. It is of course also possible to use crude protein as Con A in the virus detection method of the present invention.

When the above-mentioned virus probe is used as the Con A sample, the virus may be detected after the virus is desorbed from the virus probe if necessary, or the virus may be detected in a state bound to the probe. Since the cleavage of the virus from the probe is also well known in the art, a detailed description thereof will be omitted. (For example, Sousa et al. Antibody cross-linking and target elution protocols used for immunoprecipitation significantly modulate signal-to-noise ratio in downstream 2D-PAGE analysis, Proteome Science 2011, 9 (45)

Detection of viruses from the above concentrates can be carried out by those skilled in the art by using direct counting methods by electron microscopy, immunoenzymatic methods using antibodies specific for viral antigens, gene detection methods by polymerase chain reaction, Method.

The present invention also relates to a purification method characterized in that the resin through which Con A is passed is passed. As can be seen in the following examples, very small amounts of viruses contained in water can be effectively removed by passing the resin through which Concanavalin A is connected.

As described above, according to the present invention, the reaction time for concentration of the virus is only about 5 minutes to 1 hour, and the virus can be concentrated with high efficiency in a short time. In addition, since the method for concentrating viruses of the present invention can use Con A in a crude protein state, it is possible to concentrate the virus easily and economically without requiring expensive samples, expensive equipments or skilled techniques.

In the virus detection method of the present invention using the virus concentration method, further reaction time is unnecessary since binding reaction with virus occurs in the desorption process, so that it can be usefully used for virus detection in food materials requiring fast detection .

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart showing a process for producing Con A crude protein according to an embodiment of the present invention. FIG.
2A to 2C are graphs showing the binding affinities of con A and virus.
FIGS. 3A and 3B are photographs showing the concentration efficiency of hepatitis A virus. FIG.
4A and 4B are photographs showing the concentration efficiency of norovirus.
5 is a schematic diagram of a virus detection method according to an embodiment of the present invention.
6A and 6B are photographs showing the concentration efficiency of norovirus and hepatitis A virus according to the method of FIG. 5, respectively.
FIGS. 7A and 7B are diagrams showing quantitative curves of quantitation of norovirus according to another example of the present invention; and FIGS.
Fig. 8 is a comparative drawing showing the detection efficiency of hepatitis A virus in food materials. Fig.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the drawings and the embodiments are only illustrative of the contents and scope of the technical idea of the present invention, and the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

Example 1: Isolation of virus-bound crude protein

200 g of Jack-bean was ground with a blender and mixed with 2,400 mL of distilled water for 1 hour with stirring. Thereafter, the pH was adjusted to 4.6 using HCl, stirred at room temperature for 2 hours, and then immersed at 4 캜 for one day without stirring. The supernatant of the immersed reaction solution was filtered with a watman paper filter (No. 4), and the precipitate was centrifuged (6,000 rpm, 10 min). The filtered filtrate and supernatant were combined to obtain an extract of about 2 L.

The extract was stirred at 4 ° C while adding 560 g of ammonium sulfate to saturation degree of 0.4, stirred at 4 ° C for 4 hours, and then immersed for one day without stirring. Then, the immersion liquid was centrifuged (6,000 rpm, 10 min) to recover about 2,560 mL of the supernatant. The supernatant recovered at 4 ° C was stirred while adding an additional 717 g of ammonium sulfate so that the degree of saturation was 0.8, followed by agitation for 2 hours and then immersion for one day. The next day, the supernatant obtained by centrifugation (10,000 rpm, 15 min) was discarded and the precipitate was recovered using 60 mL of distilled water.

A solution of the precipitate (soy protein fraction) obtained by ammonium sulfate saturation of about 0.4 to 0.8 by the above method was dialyzed using distilled water (Spectra / Por 6, Spectrum Laboratories, USA) to remove ammonium sulfate. For further purification, the dialysate was centrifuged (10,000 rpm, 15 min) to obtain about 132 mL of supernatant.

74 g of ammonium sulfate was added to the supernatant, and the soybean extract having a saturation degree of 0.8 dissolved therein was stirred at 4 ° C. for 1 hour and centrifuged (6,000 rpm, 10 min). The precipitate obtained by centrifugation was dissolved in 20 mL of 0.05 M phosphate solution (pH 6.1) and dialyzed against 60% ethanol at -8 ° C for 48 hours. Thereafter, 60% ethanol was removed and dialyzed with tertiary distilled water for 24 hours. The dialyzed dialysis solution was centrifuged (15,000 rpm, 20 min), and the obtained supernatant was lyophilized to isolate 0.43 g of crude protein containing concanavalin A (Con A) from the soybean.

FIG. 1 is a flow chart showing the separation process of crude protein including Con A.

Example 2: Confirmation of binding between Con A and virus

ELISA and Sulface Plasmon Resonance (SPR) analysis showed that con A and the virus were able to bind to each other. As a virus, GI.4, GII.3 or GII.4 type norovirus, which is a type frequently appearing in Korea, was received from Gwangju Health and Environment Research Institute.

In the ELISA, 50 μl of Con A was dispensed into 96-well plates (Nunc) at 0, 0.01, 0.05, 0.1, 1, 50 and 100 μg / ml for 12 hours at 4 ° C. Sigma, USA), and blocked with PBS solution containing 5% skim milk (SKI400, Bioshop) for 1 hour at room temperature. After this, the norovirus was reacted at room temperature for 2 hours and then washed three times with PBS containing 0.02% Tween 20. After incubation for 1 hour with 5% skim milk and 1000 times diluted primary antibody (Anti-Norovirus Capsid protein VP1 antibody, ab92976, Abcam), the cells were washed and incubated with secondary antibody diluted 2000 times with 5% skim milk Goat Anti-Rabbit Immunoglobulins / HRP, P0448, Dako) for one hour and washed. The o-phenylenediamine (OPD) base (P8287, Sigma) was added, incubated at room temperature for 20 minutes, stopped with 0.5 MH ₂ SO ₄ , and absorbance was measured at 495 nm. The left-hand side of FIGS. 2A-2C are ELISA results for GI.4, GII.3 or GII.4 type Norovirus, respectively. The concentration of Con A was found to increase with increasing concentration of Norovirus, and it was found to be significant at a concentration of at least 50 ㎍ / ㎖.

Plasmon resonance analysis was performed using a Biacore 2000 SPR biosensor (GE, USA). First, a CM5 chip (carboxymethylated dextran surface chip, Biacore, Inc.) was mixed with N-ethyl-N '- (3- dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccine (NHS). ≪ / RTI > Thereafter, GI.4, GII.3 or GII.4 type Norovirus was injected to inject about 350 reaction units (RU) to be immobilized, and 1M ethanolamine was injected to block unreacted groups. Con A (Sigma-aldrich, USA) diluted to various concentrations of 0.25-100 nm was injected into the chip for 2 minutes at a rate of 20 μl / min. 2A to 2C are plasmon resonance spectra for GI.4, GII.3 or GII.4 type Norovirus, respectively. Give BIA evaluation. 3.2 The coupling speed (Kon) and the dissociation speed (Koff) were calculated using software, and the equilibrium dissociation constant (Kd) Koff / Kon was obtained therefrom. The equilibrium dissociation constants of GI.4, GII.3 or GII.4 type Norovirus were 4.47 × 10 ^-9 , 4.62 × 10 ^-9 and 2.19 × 10 ^-9 , respectively, and the binding affinities of all three types of Norovirus to Con A It was confirmed that it was high.

Example 3: Concentration of norovirus and hepatitis A virus using crude protein containing Con A or Con A

Norovirus and hepatitis A virus were enriched by three methods using crude protein containing Con A or Con A prepared in Example 1. In the following experiment, Norovirus was used in GII.4 type.

1) Concentration of virus by precipitation

(1) Concentration of virus

Con A (Sigma-aldrich, USA) or the crude protein prepared in Example 1 in 10 ⁴ TCID ₅₀ / mL The reaction was carried out in a concentration of 0, 0.5, 1 or 5 mg / mL in a hepatitis A virus sample (VR-1402, ATCC, USA) for 10 minutes. After that, -20 ° C acetone was added at 6 times of the amount of the sample and stirred, followed by centrifugation at 2,000 rpm for 3 minutes. The supernatant was discarded and the concentrated protein was recovered. The recovered protein was washed with cold 90% acetone, centrifuged under the same conditions, and the supernatant was removed and dried for 5 minutes to obtain a concentrated hepatitis A virus precipitate. A concentrated hepatitis A virus precipitate was obtained by the same method as described above, except that Bovine Serum albumin (BSA) (BioShop, Canada) was added at a concentration of 5 mg / mL instead of Con A as a control and reacted for 10 minutes .

A virus concentrate was prepared by PEG precipitation for comparison with the prior art. More specifically, a 2-fold PEG solution [16% PEG 6000 (Sigma chemical Co., USA), 0.525 M NaCl (BioShop, Canada)] was added to a hepatitis A virus sample and reacted at 4 ° C for 10 minutes. The reaction solution was centrifuged at 2,000 rpm for 3 minutes to remove the supernatant, and a hepatitis A virus concentrate was obtained in the form of a precipitate. In the PEG precipitation method, the reaction is usually carried out for 4 to 6 hours. However, for comparison with the method of the present invention in which viruses can be concentrated in a short time, the reaction time is limited to the same.

Norovirus samples (Gwangju Institute of Health and Environment, 100 RT-PCR units / mL) were obtained by the same method as the hepatitis A virus except for the reaction with Con A, BSA or PEG for 30 minutes .

(2) Identification of virus in concentrated sample

The precipitates prepared in (1) were each suspended in 200 μl of TE solution (10 mM Tris-Cl, 1 mM EDTA, pH 8.0), diluted 10-fold each, and subjected to RT-PCR. Or norovirus were detected, and the results are shown in Fig. 3 and Fig. Fig. 3A shows Con A, Fig. 3B shows the results of detection of hepatitis A virus using crude protein produced in Example 1, Fig. 4A shows Con A, Fig. 4B shows the results of detection of Norovirus using crude protein prepared in Example 1 Detection result.

HAV_VP1_F (SEQ ID NO: 1: 5'-TGG TTT GCC ATC AAC ACT GAG G-3 ') and HAV_VP1_R (SEQ ID NO: 2: 5'-ACC CAA GGA GTA TCA ACG GGA AG 3 ') and Noro_Mon 431_F (SEQ ID NO: 3: 5'-TGG ACI AGR GGI CCY AAY CA-3') and Noro_Mon 433_R (SEQ ID NO: 4) were used as primers for detection of norovirus '-GGA YCT CAT CCA YCT GAA CAT-3').

The PCR conditions for detection of each virus were as follows;

Hepatitis A virus PCR conditions: 30 cycles of denaturation at 94 ° C for 30 seconds, annealing at 57 ° C for 30 seconds, extension at 72 ° C for 30 seconds, and final extension at 72 ° C for 10 minutes.

Norovirus PCR conditions: Denaturation was carried out at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 30 seconds, and final extension at 72 ° C for 10 minutes.

As shown in FIG. 3A, when Con A was added at a concentration of 0.5 mg / mL, the concentration efficiency of hepatitis A virus was increased by about 10 times as compared with the case without addition of Con A, . The concentration of BSA, a negative control protein of Con A, was 0.4 times lower than that of Con A at 0.5 mg / mL. The concentration of PEG in the conventional concentration method was 0.5 times lower than that of 0.5 mg / mL of Con A, indicating that the concentration of Con A was excellent even at a low concentration of 0.5 mg / mL.

3B showing the concentration efficiency of the crude protein produced in Example 1, rather than the purified Con A, also showed a somewhat lower concentration efficiency than that of the purified Con A, but when the crude protein was used at 5 mg / mL, And the concentration efficiency was remarkable as compared with the conventional PEG precipitation.

FIG. 4A showing the concentration efficiency for Norovirus also shows that Norovirus is effectively concentrated by Con A than PEG in the prior art. Concentration of BSA, a negative control protein, was lower than that of actone alone without protein addition. 4b for the crude protein produced in Example 1 also had a lower concentration efficiency than the purified Con A, similar to the results of the hepatitis A virus, but showed similar concentration efficiency as the PEG precipitation method according to the prior art.

2) Concentration of virus by magnetic beads

(1) Production of magnetic beads connected with Con A

A magnetic bead to which Con A was linked was prepared by reacting magnetic beads with streptavidin linked thereto and biotinylated Con A (concanavalin A, con A) (FIG. 5).

More specifically, Con A (Sigma-aldrich, USA) was biotinylated according to the method of preparing a Biotin-XX microscale protein labeling kit (B30010; Molecular Probes). Briefly, 100 μl of 1 mg / ml Con A solution and 100 μl of 1 M sodium bicarbonate were added to the reaction tube (Component C) and mixed. Subsequently, 1 μl of a solution prepared by adding 10 μl of ionized water to biotin-XX and SSE (component A) was added and reacted at room temperature for 15 minutes. The biotinylated Con A was then purified using a spin filter (component D) and a tabletting resin (component E) included in the kit.

Magnetic beads (10 mg / mL, M-280 streptavidin, 11206D; Invitrogen) conjugated with 1 mg of streptavidin were added to 10 μg of biotinylated con A prepared by the above method and reacted at room temperature for 1 hour The magnetic beads were recovered and washed with PBS containing 0.02% Tween 20 (Sigma, USA) to produce magnetic beads connected with Con A.

(2) Concentration of virus

Is a Con A prepared in (1) to the sample tube containing the linked magnetic beads 10 ⁴ TCID ₅₀ / mL of hepatitis A virus sample (VR-1402, ATCC, USA) or Norovirus sample (Gwangju Institute of Health and Environment, 100 RT-PCR units / mL) was diluted to a concentration of 10 ^-1 to 10 ^-4 , and the mixture was reacted at room temperature for 10 minutes. After the reaction, the magnetic beads were collected using a magnet, and washed three times with 200 μl of PBS (Phosphate Buffered Saline; BioWorld, Korea). The washed magnetic beads and 100 μl of PBS were placed in a 1.5 ml tube and suspended to remove supernatant.

To separate the viral conjugate bound to the magnetic beads, 50 μl of the eluant (50 mM Glycine, pH 2.8) was placed in a tube containing magnetic beads and neutralized to pH 7.5 with 100 mM Tris solution.

(3) Detection of virus

(2), the target virus was subjected to RT-PCR by the method described in 1) (2) and electrophoretically detected for each of the solution diluted with magnetic beads and the diluted virus solution. 6A and 6B are electrophoretic images showing the results of detection of norovirus and hepatitis A virus.

6a and 6b, the detection of hepatitis A virus and norovirus was confirmed up to a 1,000-fold dilution range in the concentrate, and even when using magnetic beads with Con A attached thereto, The samples were also confirmed by RT-PCR.

3) Enrichment of virus by resin

(1) Production of resin with Con A attached

To activate the carboxyl group in the resin, 0.5 g of ion-exchange resin (WK60L, Yukrung Chemical Co.) was added to 30% EtOH solution in which 10 mg of EDC (N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride) Followed by reaction for 10 minutes. After removing the supernatant, 30% EtOH solution in which 10 mg of con A was dissolved was added and further reaction was performed for 12 hours. The resin was recovered from the reaction solution and washed with 30% EtOH.

(2) Concentration of virus

(1) was used to concentrate norovirus. First, 0.34 g of resin was filled in a column (80 mm * 200 mm), 0.5 ml of a dilution of Norovirus (GII.4, 4.07 × 10 ² / mL) was poured, and the mixture was diluted with PBS containing 0.02% Tween 20 Phosphate Buffered Saline (BioWorld, Korea) solution (0.5 ml). The filtrate and the resin were respectively collected and the virus contained was qualitatively and quantitatively analyzed below.

(3) Detection of virus

Qualitative and quantitative analysis of the virus diluent and resin contained in the resin were conducted. For the purpose of comparison, a virus diluted solution was passed through the same method as (2) using a resin not bonded with Con A, and the amount of virus contained in the resin and the filtrate passed through the resin was analyzed together.

First, the virus diluent stock solution, the filtrate passing through the resin to which Con A is connected, and the resin collected after washing the filtrate were collected and subjected to RT-PCR according to the method described in 1) (2) The result of reaction was confirmed after EtBr staining and the results are shown in FIG. 8B. Resin was extracted with 0.5 ml of Trizol (Invitrogen, Carlsbad, CA, USA). As can be seen from FIG. 8B, no virus was detected in the virus dilution solution that passed through the resin column connected with con A, and the virus was detected in the recovered resin. On the other hand, no virus was detected in the recovered resin, while Norovirus was detected in the virus diluent that passed through the resin column not connected to con A.

For more detailed analysis, quantitative analysis was performed.

To obtain a standard curve for quantification, Norovirus RNA was extracted and cDNA was synthesized. Norovirus RNA was cloned using Noro_Mon431_F and Noro_Mon 433_R as primers. The cloned RNA was inserted into pTOP TA V2 (Enjinomics, EZ001S) to prepare a plasmid and used as a standard sample of real-time RT-PCR using a 10-fold dilution method.

For the standard reaction solution, 2 μl of the cloned plasmid per reaction, 10 μl of SYBR Premix Ex Taq (Takara, RR420A) and 0.5 μl of Forward and Reverse primer (10 pmol) were used, and the final volume was adjusted to 20 μl with PCR grade water Followed by RT RT PCR machine (Takara, TP850). The PCR reaction was repeated 45 times at 95 ° C for 10 seconds, at 55 ° C for 20 seconds, and at 72 ° C for 30 seconds. The amplified fluorescence values were continuously measured once for 10 seconds at 72 ° C as each cycle of the polymerase chain reaction proceeded.

Figure 7a is y = -3.729x + 42.55 a standard curve obtained by the above method, was calculated as R ² = 0.991. From the y-intercept value of 42.55, it can be seen that all values amplified within 42 cycles between real-time RT-PCR runs can be relied on.

Through the equations obtained from the standard samples, the stock solution used in the experiment was 3.39 copies / μL. Con A was 2.16 copies / μL when the concentrated virus was quantified through a coupled resin column. On the other hand, ct value was not confirmed in the resin not connected Con A, and 2.15 copies / μL was detected in filtrate passing through the column.

Example 4: Comparison of detection efficiency of hepatitis A virus in fresh vegetables

According to the present invention and the prior art, the concentration efficiency of hepatitis A virus from actual fresh vegetable samples was compared.

In the prior art, the virus was desorbed by a desolvation solution containing beef extract and then concentrated by PEG. For concentration using Con A, the precipitation method according to 1) of Example 3 was used. More concrete experimental methods are as follows.

10 μl of Hepatitis A virus diluted to 10 ³ TCID ₅₀ in 10 g of lettuce was inoculated 10 times with 20 μl each, and the sample was prepared by drying in a clean bench for 30 minutes.

40 mL of the Tris solution and 5 mL of the aqueous solution prepared by dissolving the crude protein prepared in Example 1 were added to the sample and the mixture was subjected to physical impact for 5 minutes using a stomacher (SIBATA Science Technology), followed by stirring for 10 minutes . After 10 minutes, the solution was centrifuged at 15,000 x g at 4 ° C to take the virus supernatant. To the supernatant, -20 ° C acetone was added at 6 times the volume of the sample, and the mixture was stirred. Then, the concentrated protein was recovered by centrifugation at 2,000 rpm for 3 minutes. In the subsequent step, hepatitis A virus was detected by the same method as described in (2) of Example 3 1).

For comparison, 80 mL of beef extract desalting solution (100 mM Tris-HCl, 50 mM glycine, 1% beef extract, pH 9.5) was added to the same type A virus infected lettuce samples and the stomaker (SIBATA Science Technology) After physical impact for 5 minutes, the mixture was stirred for 10 minutes. After 10 minutes, the solution was centrifuged at 15,000 x g, 4 to take the virus supernatant. PEG solution [16% PEG 6000 (Sigma chemical Co., St. Louis, Mo., USA), 0.525 M NaCl] was added to the supernatant for 10 minutes. Thereafter, the concentrated protein was recovered by centrifugation at 2,000 rpm for 3 minutes, and hepatitis A virus was detected by the same method as in (2) of Example 3-1.

8 is a view showing the main characteristics and results of the Con A precipitation method and the PEG precipitation method. In FIG. 8, although the concentration of the virus according to the method of the present invention is based on the crude protein, it was confirmed that the virus was detected about twice as effectively as the method using the PEG solution.

<110> KOREA BASIC SCIENCE INSTITUTE <120> Method for Concentration and Detection of Virus <130> P0414-113 <150> KR 10-2013-44609 <151> 2013-04-23 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer for Hepatitis A Virus <400> 1 tggtttgcca tcaacactga gg 22 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer for Hepatitis A Virus <400> 2 acccaaggag tatcaacggc aag 23 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for Norovirus <220> <221> modified_base <222> (6) <223> i <220> <221> modified_base <12> <223> i <400> 3 tggacgagrg ggccyaayca 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer for Norovirus <400> 4 ggayctcatc cayctgaaca t 21

Claims (15)

(A) adding Concanavalin A to a sample solution suspected of the presence of norovirus or hepatitis A virus; And
(B) separating Norovirus-Concanavalin A complex or Hepatitis A virus-Concanavalin A complex from the reaction solution;
Wherein the method comprises the steps of:
delete The method according to claim 1,
Wherein the reaction time of step (A) is from 5 minutes to 1 hour.
delete The method according to claim 1 or 3,
Wherein the step (B) comprises adding acetone to the reaction solution of step (A) to precipitate and recover the virus-concanavalin A complex.
delete The method according to claim 1 or 3,
The method of concentrating norovirus or hepatitis A virus according to any one of the preceding claims, wherein the Concanavalin A in step (A) is added in a state of being connected to the resin.
delete delete delete (A) preparing a sample solution suspected of the presence of norovirus or hepatitis A virus;
(B) concentrating the virus from the sample solution by the method of claim 1 or 3; And
(C) detecting norovirus or hepatitis A virus from the concentrate of the virus;
And detecting the virus.
12. The method of claim 11,
Wherein when the sample of step (A) is a solid, the sample solution is prepared through the step of removing the virus, and the method for detecting norovirus or hepatitis A virus.
13. The method of claim 12,
Wherein the step of removing the virus and the step of reacting with Concanavalin A in the virus concentration step (B) are simultaneously carried out. delete delete

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