KR101621190B1 - Kit and method for detecting negatively charged virus using charge switchable micro-bead beating - Google Patents

Abstract

The present invention relates to a kit for the detection of negative charged viruses using a transformable microbead and a detection method thereof. The present invention is a simple but effective method for detecting norovirus, which according to the present invention minimizes the loss of norovirus from 1) sample, 2) the destruction of effective Norovirus, and 3) simple microfluidic systems . In the present invention, microvalves capable of 1) concentration of norovirus and bead beads, 2) micro valves capable of vibrating the micro chamber space change control and bead beating were used. This simple platform technology is applicable to a variety of viruses as well as other pathogenic bacteria such as E. coli and salmonella through optimization of microbeads and simple control of driving conditions.

Description

TECHNICAL FIELD [0001] The present invention relates to a kit for detecting negative charge using micro-beads and a method for detecting charge-

The present invention relates to the development of microfluidic chip modules for detecting negative charge viruses, especially Norovirus, present in food samples. The present invention also relates to a kit for concentrating, destroying and detecting Norovirus, which is carried out in a module in a yearly manner, and includes a technique for concentrating and destroying virus using substratum microbeads.

Food poisoning is one of the most important diseases in the world and is a leading cause of toxins and pathogens. In addition, over 80% are caused by pathogens. Therefore, it is very important to quickly identify the cause of food poisoning and the surrounding environment when it occurs. Such food poisoning is divided into viral food poisoning and bacterial food poisoning. Viral food poisoning is not possible to propagate in vitro, so the propagation rate is relatively slow, and it can occur with traces of individuals (10 ~ 100), and there is no general treatment or vaccine. In addition, continuous monitoring or sequestration is required for at least three to two weeks, even if the patient is cured to prevent secondary infection.

Norovirus is the most common cause of viral food poisoning. The number of viruses causing food poisoning is smaller than that of bacteria. Among them, norovirus is considered to be the main cause of food poisoning in winter and it also causes group infection.

Norovirus has a single positive strand RNA of about 7.6 kb in size and is a non-enveloped virus consisting of phospholipid and glycoprotein similar to the cell membrane on the outside. It has no outer membrane and only the protein encapsulates the gene in the form of a true twenty- It is very small, 27 ~ 40nm. Thus, about 40% of all viruses are genes and the remaining 60% are in the simplest form of protein.

The capsid protein of norovirus forms a rigid structure by cross-linking between protein and protein to retain the nucleic acid inside. The structure of the outer membrane of Norovirus differs from that of a general protein by a structure in which the protein bond and the dissociation function are exerted by immobilization or covalent bonding between protein and protein.

An important issue in detecting pathogens from actual specimens is 1) how much total process time can be reduced (including all sample preprocessing), 2) limit of detection (LOD) to detect very small samples in large samples How high can it be. To date, various methods have been developed to detect sensitive and accurate pathogens. Selective media methods, specific reaction methods using specific antibodies or antigens, and nucleic acid amplification methods. The detection limit of these methods has been drastically improved by the introduction of detection signal amplification using several biological or chemical materials such as nanoparticles, enzymes, chemiluminescent reagents, and liposomes. Several of these methods have already been commercialized. Nonetheless, signal amplification and detection times remain a significant development challenge in methods for detecting pathogens from food. This is because the sample preprocessing process (concentration, purification) requires a long time.

Biological detection methods such as antibody-based assays or enzymatic reactions are difficult to achieve without pretreatment because of the large amounts of salt, oil, and complex components contained in the food. In addition, this enrichment process is difficult to apply to targets that can not be cultured, such as Norovirus.

The physical, chemical and biological methods of enrichment of pathogens in drinking water, fruits and vegetables can be carried out by centrifugation, sedimentation using PEG, concentration using a charged filter, (Ca ^{2 +} , Mg ^{2 +} ), and separation using antibodies.

Although a variety of enrichment methods have been developed, there are two important issues to quickly and productively detect viruses contained in complex samples such as salt-containing foods: shortened long pre-treatment times and increased detection efficiency at low concentrations. Therefore, there is a need for a method for concentrating viruses both scientifically and commercially.

Korean Patent Publication No. 10-2013-0128348 (published on November 26, 2013)

It is an object of the present invention to develop a detection kit which is a microfluidic chip module for detecting negative charge viruses, particularly norovirus, present in a food sample and a method for detecting negative charge virus.

In order to accomplish the above object, the present invention provides a method for producing negative-acting microbeads, comprising contacting a sample containing a negative-acting virus with a submergible microbead to adsorb and concentrate the negative- Vibrating the adsorbed and concentrated substratum microbeads to elute viral RNA; And detecting the eluted viral RNA.

The present invention also relates to an upper poly (methylmethacrylate) (PMMA) chamber layer comprising a sample injection section, a substratum microbead filling chamber and a viral RNA amplification chamber; An adhesive layer positioned at a lower end of the PMMA chamber layer; And a valve layer located at the lower end of the adhesive layer and having a plurality of valve holes.

The present invention relates to a kit for detecting negative-acting viruses using a transformable microbead and a detection method, and more particularly, to a simple but effective method for detecting norovirus. According to the present invention, 1) the loss of norovirus from the sample is minimized, 2) effective destruction of norovirus, and 3) simple microfluidic systems. In the present invention, microvalves capable of 1) concentration of norovirus and bead beads, 2) micro valves capable of vibrating the micro chamber space change control and bead beating were used. This simple platform technology is based on the optimization of microbeads and the simple control of the driving conditions. E. coli and salmonella It is applicable to a variety of viruses as well as other pathogenic bacteria such as.

1 shows the entire detection process of murine NoV through a microfluid chip module.
Figure 2 shows several transformer-driven microbeads.
FIG. 3 shows a form of a transformable microfluidic chip module composed of transformable microbeads.
Figure 4 shows the zeta potential of murine NoV and three other functionalized beads.
Figure 5 shows the efficiency of the functionalized microbeads. (a) the adsorption of murine NoV on pH, (b) the adsorption of RNA on pH, and (c) the adsorption of murine NoV on the amount of beads.
6 shows the eluting efficiency of bead beating. (a) the amount of total RNA eluted from murine NoV and (b) RNA degradation by bead beating.
FIG. 7 shows a sample preparation process of a murine NoV in an end oyster with respect to a microfluidic chip module composed of a transformable microbead.
Figure 8 shows the detection efficiency according to various murine NoV concentrations in oysters in connection with a microfluidic chip module composed of substratum microbeads.

Using a microfluidic sensor chip module including a transformable bead in a simple but efficient manner, the present inventors sequentially concentrated and destroyed murine norovirus in the oysters and amplified RNA to detect them (FIG. 1). Substrate microbeads are used for various purposes in the concentration and destruction of viruses in a space-changeable microchamber. The sample solution (homogenized oyster and buffer mixture) is injected into the sample pretreatment chamber (space change, valve is lifted) and the electrostatically charged action of the submerged microbeads and murine norovirus in the chamber. The viruses adsorbed on the microbeads are destroyed by bead beating in the chamber where the space has been changed (bringing the valve down and vibrating). Through the sample washing step, the desired RNA was extracted into the amplification chamber and successfully detected. Each major step was individually investigated and conditioned. Finally, the various concentrations of murine norovirus contained in the oysters were successfully assayed within 4 hours and the present invention was completed.

The present invention relates to a method for producing negative-acting viruses, comprising contacting a sample containing negative-negative virus with a transforming microbead to adsorb and concentrate the negative-acting virus on the transforming microbeads; Vibrating the adsorbed and concentrated substratum microbeads to elute viral RNA; And detecting the eluted viral RNA.

Specifically, the negative charge virus may be, but is not limited to, norovirus (NoV).

Preferably, the transformable microbeads may be represented by the following formulas (SP1), (SP2) or (SP3), but are not limited thereto.

≪ Formula 1 >

(2)

(3)

In one embodiment of the present invention, preferably the sample may have a pH of from 3 to 7. More preferably, the sample may have a pH of 5 to 7. When the sample is less than pH 5, both murine norovirus and the substratum microbeads are positively charged and can not be adsorbed to each other. When the sample is above pH 7, both murine norovirus and the substratum microbeads are negatively charged They can not be adsorbed to each other. Therefore, when the sample is at a pH of 5 to 7, the murine norovirus is negatively charged and the transformable microbeads are positively charged, so they can be adsorbed to each other by electrostatic force.

Preferably, the time of oscillation of the substratum microbeads may be from 1 to 20 minutes, the oscillation intensity may be from 1 to 100 Hz, and the step of eluting the viral RNA may be carried out such that both the murine norovirus and the substratum microbeads are negative But it is not limited thereto.

The method of amplifying and detecting the RNA of the eluted virus can be detected through Nucleic Acid Sequence Based Amplification (NASBA), but is not limited thereto.

Meanwhile, the amplified products can be detected by methods known in the art. For example, gel electrophoresis, enzyme-linked gel assay (ELGA), and electrochromiluminescent (ECL) . However, in this method, it is inconvenient to separately perform the detection step for each sample in order to confirm the result of the amplification reaction. Therefore, it may be preferable to use a fluorescence detection method such as a molecular beacon probe or a TaqMan probe. Particularly, in the embodiment according to the present invention, the fluorescence detection method is particularly advantageous because it can perform both concentration, amplification and detection of the sample in a single chamber.

The present invention also relates to an upper poly (methylmethacrylate) (PMMA) chamber layer comprising a sample injection section, a substratum microbead filling chamber and a viral RNA amplification chamber; An adhesive layer positioned at a lower end of the PMMA chamber layer; And a valve layer located at the lower end of the adhesive layer and having a plurality of valve holes. Specifically, the negative charge virus may be, but is not limited to, norovirus (NoV).

Specifically, the PMMA chamber layer is configured such that the sample injecting unit, the substratum microbead filling chamber, and the RNA amplification chamber are connected to each other and the sample injected through the sample injecting unit is sequentially passed through. The viral RNA, which is adsorbed and concentrated in the transformable microbeads in the substratum microbead filling chamber and eluted by the vibration, can be amplified and detected in the RNA virus amplification chamber. And the micro chamber space change control and the bead beating vibration drive are designed to be able to be driven through the micro valves connected through the plurality of valve holes provided in the valve layer.

Specifically, the adhesive layer and the valve layer may be made of polydimethylsiloxane (PDMS), but the present invention is not limited thereto.

Preferably, the transformable microbeads may be represented by the following formulas (SP1), (SP2) or (SP3), but are not limited thereto.

≪ Formula 1 >

(2)

(3)

Preferably, the size of the transformable microbeads may range from 30 to 300 [mu] m, but the numerical ranges are for illustrative purposes only and the invention is not necessarily limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples which do not limit the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

< Experimental Example  >

The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.

1. Experimental material

All compounds were purchased from Sigma-Aldrich Co. (St. Louis, MO, US). Crustostrea gigas ) were harvested at sea and purchased at local markets (Seoul, South Korea). Microbeads (100 ± 20 μm) were purchased from Daehan Sciences Co. (Korea). Polydimethylsiloxane (PDMS) Sylgard 184 and its curing agent were purchased from Dow Corning (Midland, ML, USA).

2. Murine ( murine ) Norovirus ( norovirus ; NoV ) Culture and storage

Murin NoV was received from Professor Choi Chang-Soon of the Department of Food Science at Chung-Ang University. The murine macrophage-like cell line RAW264.7 was purchased from ATCC (USA) and was cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (Gibco) And maintained in Dulbecco's modified Eagle's medium (MEM). Viruses were incubated with RAW264.7 cells in Dulbecco's MEM containing 5% FBS. Viruses were inoculated and cultured on confluent RAW264.7 cells for 3 days. To release the viruses, infected cells were frozen and thawed three times and centrifuged at 15,000 rpm at 4 ° C for 15 minutes. The supernatant was stored at -80 ° C until further use in the experiment.

3. Murine NoV Quantification of

A standard protocol using Qiagen RNA mini KIT and a real-time RT-PCR procedure were used as a basic tool to quantify the Murine NoV and eluted RNA adsorbed on the microbeads.

Real-time polymerase chain reaction (PCR) was performed with the following primer sets: F (5'-CACGCCACCGATCTGTTCTG-3 ') and R (5'- GCGCTGCGCCATCACTC-3'). Nucleic Acid Sequence Based Amplification (NASBA) was performed with the following primer sets: forward (5'-GGTCTCTGGCCGCCTTCTTTCTAA-3 ') and reverse (5'- Gt;

The NASBA reaction in the microchip was performed in a commercially available master mixture (NucliSens ^® Basic Kit, Biomerieux, France) in SYBRs Green II (Invitrogen; USA) for situ detection was added and incubated at 41 ° C for 2 hours.

4. Micro glass Bead  Functionalization

Three different glass beads were prepared using the following method (Fig. 2). Since it is difficult to measure the surface charge of heavy microglass beads with a zeta-potential measurement (Malvern, Zetasizer NanoZS, GB), the present inventors measured the surface charge of nanoparticles treated by the same surface treatment method. Total surface activation of the glass beads was performed by adding a slight modification to the previously reported method (Ozmen et al., 2009; Zengin et al., 2006). Prior to silanization, the glass beads were etched in 4M NaOH. Then, to remove the base, the beads were rinsed several times with DI water. Finally, the beads were rinsed with anhydrous ethanol and dried under vacuum. Silica nanoparticles (SiNPs) were prepared by the reverse microemulsion method according to the previously reported method (Bagwe et al., 2004). For the synthesis of 100 nm SiNPs, 1.77 g of Triton X-100 and 1.6 ml of hexanol-1 were mixed with a stir bar in a glass vial containing 7.5 ml cyclohexane. After 10 minutes, 300 μl of ammonium hydroxide was added to the mixture and 200 μl DI water was added with continuous stirring at 1000 rpm. Then 100 μl of tetraethyl orthosilicate (TEOS) was added and stirring was continued for 24 hours. The reaction was stopped by the addition of ethanol. The SiNPs were washed 3 times and stored in ethanol. To prepare glass bead coated (3-aminopropyl) triethoxysilane (APTES), the glass beads were dissolved in a mixture of toluene, APTES and N, N-di Was refluxed for 4 hours with a mixture of N, N-diisopropylethylamine (DIEA). After silanization, the glass beads were rinsed twice with toluene, rinsed twice with ethanol, and then dried under vacuum.

To prepare the substratum glass beads (SP1), the APTES coated microbeads were suspended in 40 ml of acetonitrile, 10 g of bromoacetic acid (97%) and 5 ml of DIEA mixture. The suspension was refluxed at 100 &lt; 0 &gt; C for 8 hours. The residue was washed twice with acetonitrile and washed twice with ethanol. The glass beads were then dried under vacuum conditions and stored at room temperature for later use. To prepare transformed frosted glass beads (SP3), APTES coated microbeads were suspended in 40 ml of acetonitrile, 5 g of succinic anhydride (97%) and 5 ml of DIEA mixture. The suspension was refluxed at 100 &lt; 0 &gt; C for 8 hours. The residue was washed twice with acetonitrile and washed twice with ethanol. The glass beads were then dried under vacuum conditions and stored at room temperature for later use. The substratum particles (SP2) were prepared using the sequential use of SP1 and SP3.

5. Murine NoV  Manufacture of added oyster samples

Oyster samples were prepared by applying a slight modification to the previous report (Kingsley, 2007). Live oysters were individually suspended in 10 ml of sea water containing murine NoV of various concentrations [10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ plague forming units (PFU) And cultured for 16 hours (overnight). Digestive glands (stomach and digestive diverticulum) of a single oyster were cut and finely cut using a razor blade. The digested glands and residual solution were transferred to a clean tube and 7.5 ml of 0.25 M glycine-0.14 M NaCl (Sigma) buffer (pH 5) was added. Oyster samples were resolved again, centrifuged at 10,000 g for 30 min at 4 ° C, and the supernatant was collected.

6. Microfluidic chip manufacturing

As shown in FIG. 3, the chip device is divided into chambers having an upper microfluidic chamber (including beads), a lower valve channel and a valve membrane. PDMS valve channels and chambers were fabricated with slightly modified methods previously reported (Unger et al., 2000). The upper chamber is composed of 1 mm high poly (methylmethacrylate) (PMMA).

< Example  1> Micro Bead  Function analysis

In the detection process shown in FIG. 1, the microbeads in the microchip are utilized for three different purposes. 1) From the sample, murine norovirus is concentrated by electrostatic adsorption on the surface of the microbeads. 2) Nucleic acid is extracted from murine norovirus adsorbed to microbeads by bead beating. 3) The nucleic acid is eluted from the sample pretreatment chamber. In order to perform the above three different functions, it is necessary to use a composite valve capable of spatially deforming the micro-channels as well as the micro-channels from the channel shape (300 ㎕) to the chamber shape (500 ㎕) and performing the bead beating. In the present invention, surface charge of each of murine norovirus and microbeads was investigated, and the adsorption and destruction process of murine norovirus was optimized, and the nucleic acid could be eluted by the surface function of the transformable microbeads.

< Example  2> Murine Nov  And functionalized SiNPs Charge of

FIG. 4 shows that the potential of functionalized silica nanoparticles (SiNPs) according to the murine norovirus and pH is negatively charged at pH 4. As can be seen, the surface charge of SP1, SP2 and SP3 changes from positive charge to negative charge at pH 7 ~ 9, 5 ~ 7 and 3 ~ 5 respectively. Electrostatic adsorption of murine norovirus at the surface of the microbeads requires microbeads with charge opposite to that of the murine norovirus surface. SP1 shows positive charge at pH 5 and pH 7, SP2 at positive pH 5, and murine Norovirus negative charge at pH 4. Therefore, murine norovirus (negative charge) will be attached to SP1 and SP2 by electrostatic force at pH 5-7. Basically, this range of pH (pH 5-7) is important because the sample containing murine norovirus has a pH of 5-7.

< Example  3> Surface Substation  Micro Bead  Prize Murine NoV And RNA Of elongation

The adsorption% (adsorbed amount / put-in amount) of the murine norovirus adsorbed on SP1 and SP2 microbeads was investigated by various pHs (Fig. 5). Murine norovirus was well adsorbed on SP1 at pH 5-7 and SP2 was well attached at pH 5. Through this, it was confirmed that the electrostatic force acts mainly between the particle and the murine Norovirus. Interestingly, at pH 3, about 60% of the murine norovirus was adsorbed to SP1 even though both positively charged murinovirus and SP1. This is because it has a larger positive charge than SP2. This implies that the force with which the murine norovirus is adsorbed on the solid surface is also acted upon by other forces, such as hydrophobic interaction, van der Waals, and dispersion, as well as electrostatic forces. The elution percentage of RNA (eluted amount / put amount%) is an important factor in the role of microbeads that adsorb and destroy murine Norovirus. This is because the RNA must be transferred to the amplification chamber with minimal loss. We determined the elution buffer pH to pH 9 with a maximum electrostatic repulsion between the solid surface (SP1 or SP2) and the RNA. The% RNA elution value from the micro chamber containing SP1 or SP2 shown in Fig. 5 (b) was Tris buffer (100 mM). RNA was not eluted from the micro chamber containing SP1. It is presumed that the initial amine structure residues of SP1 are not all reacted to acetic bromate (FIG. 2). These results also demonstrate that the total charge of SP1 is positively charged at pH 7, even though the third amine has a pKa value of 7 to 8, as measured by the pKa calculation program. In conclusion, RNA is well adsorbed on SP1. On the other hand, in the case of SP2, the% RNA elution gradually increases as the amount of microbeads decreases. Therefore, the beads optimized in the present invention were selected as 0.2 g SP2 microbeads according to the RNA extraction effect. However, as shown in FIG. 5 (c), the adsorption rate of murine norovirus using 0.2 g of SP2 at a relatively high rate (1 ml / min) is insufficient at 54%. Nonspecific adsorption of murine norovirus by electrostatic attraction on a solid surface is affected by the rate ratio and the surface area of the microbeads (amount of microbeads). Therefore, to achieve effective adsorption of murine norovirus at 0.2 g of SP2, the sample injection rate with adsorption buffer should be lower than 0.5 ml / min.

< Example  4> Bead Beating ( bead beating ) optimization

Bead beating is a well known method of breaking down bacteria or other cells by physical force. Typical bead beating is by hand shaking or using an automatic vibrator. In the present invention, a pneumatic microvalve was used to change the sample preprocessing chamber space and a method of vibrating the beads using valves was used. For RNA stability, TE buffer solution (pH 9) was used without using reagents such as NaOH. When 0.2 g of microbeads were shaken gently for 1 to 2 minutes, the murine norovirus was destroyed by about 30% by bead beating sample destruction. This result has a complex inherent control over the amount of microbead, high vibration velocity, or long vibration time. In the present invention, since the vibration speed is limited in the microchip, the microbead condition capable of increasing the% destruction is optimized with optimization of the vibration time except for the speed. In the present invention, the maximum amount of 0.4 g based on the microbead size (100 mu m) was used. In addition, 0.2 g of ordinary glass beads without any surface treatment were added. Based on 0.4 g of microbeads, the percent destruction of murine norovirus was investigated according to vibration time and bead size. The extracted RNA% in Fig. 6 was calculated from the amount of RNA eluted using Qiagen Viral RNA kit and the percentage of eluted RNA from bead beating. Two interesting phenomena were found at this time. 1) No observations were found in the early observations, but the virus destruction after 10 minutes was excellent in large size beads. 2) It was found that there is an optimization value of vibration time according to each bead size. These results imply that not only the RNA elution but also the capsid of the virus is broken down by the bead beating method. The RNA breakage due to bead beating was confirmed as shown in Fig. 6 (b). The RNA was broken better in smaller sized beads. Although large-sized beads contain fewer RNA fragments, more viruses are destroyed than small-sized beads. Therefore, the optimum bead size and bead beating time optimization point must be selected. As a result of comparison with commercially available viral RNA extraction kit, beeting for 3 minutes using a bead having a size of 100 μm confirmed that approximately 80% of the RNA was extracted from the virus. As a result, in the present invention, driving of 10 HZ vibration for 3 minutes using 0.4 g of microbeads (0.2 g SP2 and 0.2 g general glass beads) of 100 mu m in size was carried out for optimization of adsorption and breakdown of murine norovirus and optimization of RNA elution .

< Example  5> In the oyster sample using microfluidic chip system Murine NoV  analysis

Using the microfluidic sensor module, it was possible to detect murine norovirus (various concentrations: 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , and 10 ⁶ PFU / single oyster) added to oyster samples. Details are shown in Fig. Sequentially, murine norovirus was adsorbed to SP2 microbeads in the adsorption step. Unpredictable proteins or greasy substances were rinsed and removed from the microfluidic chip. Murine norovirus attached to SP2 was destroyed by bead beating (six valve oscillations). The RNA was transferred to the NASBA reaction chamber and amplified for 2 hours. Referring to Figures 7 and 8, RNA was well prepared for the quantitative determination of murine norovirus contained in the oysters and was detected according to the NASBA detection method in the total analysis microfluidic chip module. Also, murine norovirus in oysters was fully detected up to 10 ² PFU.

In recent years, the detection of norovirus in health promotion, food industry, agriculture and marine industry is very important because most of the world food poisoning is caused by Norovirus. As is well known, the basis for detecting nucleic acids such as PCR or NASBA in the detection of bacteria or viruses is regarded as a fundamental method. However, this method always requires a sample pretreatment process. Norovirus is one of the pathogens that are difficult to separate and destroy from the sample because it is an RNA virus encapsulated in a capsid. In general, the process of pre-treating RNA from Norovirus contained in a food sample takes 24 hours, and the rate of successfully obtaining Norovirus from the sample is only 3-5%. Therefore, the present invention has developed a simple but effective method for detecting norovirus. In the present invention, 1) the loss of norovirus from the sample is minimized, 2) effective destruction of norovirus, and 3) simple microfluidic systems. In the present invention, microvalves capable of 1) concentration of norovirus and bead beads, 2) micro valves capable of vibrating the micro chamber space change control and bead beating were used. This simple platform technology is based on the optimization of microbeads and the simple control of the driving conditions. E. coli and salmonella It is applicable to a variety of viruses as well as other pathogenic bacteria such as.

Claims (13)

After injecting a sample containing Norovirus into a space-changeable microchamber, the microvolume is adjusted to reduce the space of the microchambers so as to form a zigzag shape so that the sample is brought into contact with the substratum microbeads to transfer the Norovirus to the transformable microbeads Adsorption and concentration;
Adjusting the microvalve to widen the space of the microchamber to vibrate the adsorbed and concentrated substratum microbeads to elute the noroviral RNA; And
And detecting the eluted noroviral RNA. delete The method for detecting norovirus according to claim 1, wherein the transformable microbeads are any one selected from the group consisting of the following Chemical Formulas (1), (2) and (3)
&Lt; Formula 1 >

(2)

(3)

The method for detecting norovirus according to claim 1, wherein the sample has a pH of 3 to 7. 2. The method according to claim 1, wherein the oscillatory time of the transformable microbead is 1 to 20 minutes, and the oscillation intensity is 1 to 100 Hz. The method for detecting norovirus according to claim 1, wherein the step of eluting the norovirus RNA is performed at a pH of 7 to 9. The method according to claim 1, wherein the step of detecting the RNA virus is performed through a nucleic acid sequence based amplification (NASBA). An upper poly (methylmethacrylate) (PMMA) chamber layer including a sample injection section, a transformable microbead-filled chamber, and a Norovirus RNA amplification chamber;
An adhesive layer positioned at a lower end of the PMMA chamber layer; And
And a valve layer located at the lower end of the adhesive layer and having a plurality of valve holes,
The microvalves connected through the plurality of valve holes provided in the valve layer are adjusted to reduce the space of the micro chamber so as to have a zigzag shape to adsorb and concentrate the norovirus on the substratum micro beads, To elucidate and quantify Norovirus RNA by vibrating the substratum microbeads. 9. The method according to claim 8, wherein the PMMA chamber layer is connected to the sample injection unit, the substratum microbead filling chamber, and the RNA amplification chamber so that the sample injected through the sample injection unit is sequentially passed through. Kits. The kit for detecting norovirus according to claim 8, wherein the adhesive layer and the valve layer are made of polydimethylsiloxane (PDMS). delete 9. The norovirus detection kit according to claim 8, wherein the transformable microbeads are any one selected from the group consisting of the following Chemical Formulas (1), (2) and (3)
&Lt; Formula 1 >

(2)

(3)

9. The norovirus detection kit according to claim 8, wherein the size of the transformable microbeads is 30 to 300 mu m.

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