KR20190017572A - Device for Collection and Lysis of Microorganisms - Google Patents

Abstract

The present invention relates to a microorganism collecting and crushing apparatus, which can collect microorganisms floating in air and simultaneously crush them to extract nucleic acid, so that floating microorganisms can be detected.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a device for collecting microorganisms,

The present invention relates to a device capable of collecting microorganisms floating in air and simultaneously extracting nucleic acids by breaking microorganisms.

In recent years, fear of infectious microorganisms spreading through the air has spread not only in Korea but also worldwide. This is not only for people, and if diseases like swine fever and avian influenza occur, hundreds of thousands of livestock are buried and social and economic damage is very serious. Because of this situation, it is a reality that it is necessary to take measures against microorganisms currently infecting the air, for example, viruses or bacteria. Especially, it is absolutely necessary to have a technique for early identification of disease-causing microorganisms.

In the case of the technology for detecting or measuring microorganisms, techniques for detecting microorganisms in samples such as soil and water have already been commercialized, but the technology for detecting microorganisms present in the air has not yet reached a basic level. The method of detecting microorganisms in the air can be divided into a method using filtration and a method using inertia and gravity in principle. The method using filtration is a method using a porous membrane filter. The air sucked by the pump is passed through a filter, and then the filter is transferred to an aqueous phase for analysis, or the presence of microorganisms is determined through culture. The inertial and gravitational methods can be largely classified into impactor, centrifugal, and impinger methods. The collision method is a method in which microorganisms are collected by contact with liquid and solid medium under the force of inertia, centrifugation is performed by separating the particles by size using centrifugal force, and then the separated material is cultured in a liquid and solid medium . Impin is a method of putting an aqueous solution into a dust collector with a tube and passing air through the aqueous solution to collect microorganisms in the air.

Recently, many studies have been conducted to detect microorganisms present in the air by using a small chip. Studies have been conducted on identifying bacteria trapped in a chip using an antigen-antibody reaction to a specific bacterium, or inserting a solid medium in a chip and passing air through the chip to culture the collected bacteria.

The present inventor has completed a device capable of extracting nucleic acids by simultaneously collecting and disrupting suspended microorganisms in the air in order to solve the disadvantage that it takes a long time in the process of confirming and collecting the collected microorganisms.

1. Korean Patent No. 10-1579590

SUMMARY OF THE INVENTION An object of the present invention is to provide a bead accommodating portion in the form of a channel for accommodating beads; An air inflow portion connected to the bead accommodating portion to supply outside air into the bead accommodating portion; And a channel-shaped bead beating portion having an upper end opened and a lower end blocked with a porous film, wherein the bead beating portion moves in a vertical direction of the bead accommodating portion, and the bead accommodating portion And a method for detecting floating microorganisms using the apparatus.

One aspect of the present invention provides a bead assembly comprising: a bead receiving portion in the form of a channel for receiving a bead; An air inflow portion connected to the bead accommodating portion to supply outside air into the bead accommodating portion; And a channel-shaped bead beating portion having an upper end opened and a lower end blocked with a porous film, wherein the bead beating portion moves in a vertical direction of the bead accommodating portion, and the bead accommodating portion Thereby sealing and closing the microorganisms.

In another aspect of the present invention, there is provided a microorganism collecting and crushing apparatus for collecting suspended microorganisms in air, And disrupting the collected microorganisms. The present invention also provides a method for detecting floating microorganisms.

The apparatus for collecting and crushing microorganisms according to an embodiment of the present invention can collect microorganisms suspended in the air and simultaneously crush them to extract nucleic acids, so that microorganisms in the air can be detected.

1 is a schematic view showing the structure of a microorganism collecting and crushing apparatus according to an embodiment of the present invention.
2 is a view showing an operation of a microorganism collecting and breaking apparatus according to an embodiment of the present invention.
FIG. 3 is a graph showing the results of checking the microbial collection efficiency according to the presence or absence of beads and the amount of beads in the microorganism collecting and crushing apparatus according to an embodiment of the present invention.
FIG. 4 is a graph showing the results of checking the collection efficiency of microorganisms according to the air flow rate in the apparatus for collecting and crushing microorganisms according to an embodiment of the present invention.
FIG. 5 is a graph showing the results of confirming the nucleic acid extraction efficiency of microorganisms according to the amount of beads used in the apparatus for collecting and crushing microorganisms.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Like parts are designated with like reference numerals throughout the specification.

Hereinafter, a microorganism collecting and crushing apparatus according to an embodiment of the present invention will be described in more detail with reference to the accompanying drawings.

FIG. 1 schematically shows the structure of a microorganism collecting and crushing apparatus according to an embodiment of the present invention.

The main body 10 of the microorganism collecting and crushing apparatus includes a bead receiving portion 110, an air inlet portion 120, and a bead beating portion 210.

The bead receiving part 110 may serve to increase the contact time of the air and the aqueous solution by receiving micro-sized beads into the space through which the air supplied from the air inlet passes.

The air inlet 120 may include a porous membrane 130 connected to the bead receptacle 110 and a mesh having a size of 30 to 50 μm may be present in the porous membrane 130 It is possible to prevent the bead from flowing out from the inside of the bead accommodating portion to the air inflow portion.

In addition, the air inlet 120 may be connected in a diagonal direction with respect to a central axis of the bead receiving portion 110 in the form of a channel. Since the air inlet 120 is connected in the oblique direction, the air supplied through the air inlet can be rotated in the form of a whirl in the bead receptacle 110, and the time for the air to stay inside the bead receptacle is increased, It is possible to raise the possibility.

The bead beating part 210 may have a channel shape in which the upper end is opened to allow air to flow into the bead receiving part 110 and the lower end is blocked with the porous film 130. In addition, the bead beating portion 210, which is in the form of a channel, has a smaller diameter than the channel diameter of the bead accommodating portion, and can move in the up and down direction of the bead accommodating portion. The bead beating portion may have the pressure dispersing portion 220 to prevent the aqueous solution inside the bead receiving portion from flowing out to the open upper end of the beating portion when the bead beating portion moves in the up and down direction.

In addition, since the bead beating unit hermetically seals the inside of the bead accommodating unit, air can be flowed out only through the opened upper end, and the rubber ring 230 can be provided on the outer side of the lower end closed with the porous film for sealing.

FIG. 2 is a photograph showing an operation of a microorganism collecting and crushing apparatus according to an embodiment of the present invention.

2A is a photograph showing the microorganism collection process. First, the bead accommodating portion is filled with a collection aqueous solution and micrometer-sized beads. Next, the upper end of the bead beating section is connected to the vacuum pump through a pipe, and when the vacuum pump is operated, the outside air flows into the bead accommodating section through the air introducing section. The introduced air passes through the inside of the bead accommodating portion filled with the bead and the collected aqueous solution, and suspended microorganisms in the air are collected in the collected aqueous solution during this process.

FIG. 2B is a photograph showing a process of breaking up the microorganisms collected in the bead accommodating portion. When the microbial collection process is completed, the operation of the vacuum pump is terminated and the bead beating section is moved to the upper side. When the bead beating part is moved to the upper side and the vacuum pump is operated, collision between the beads in the bead accommodating part, that is, bead beating is caused by the force of the air introduced from the outside. When the bead beating occurs, the collected microorganisms are disrupted and the nucleic acid is eluted into the capture aqueous solution in the bead accommodating portion. By detecting the nucleic acid, the presence of microorganisms floating in the air and the kinds of microorganisms can be confirmed. The airborne floating microorganisms may be viruses or bacteria.

The method for detecting the nucleic acid of the microorganism can be performed by a conventional method known in the art, for example, a polymerase chain reaction (hereinafter referred to as PCR), a real-time PCR have. When the nucleic acid is detected using PCR, the presence of the microorganism and the kind of the microorganism can be easily confirmed by using a specific microorganism-specific primer sequence.

As described above, the present invention has been described with reference to particular embodiments, such as specific constituent elements, and limited embodiments and drawings. However, the present invention is not limited to the above- And various modifications and changes may be made thereto without departing from the scope of the present invention.

Hereinafter, one or more specific examples will be described in more detail through experimental examples. However, these examples are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these examples.

Experimental Example  One: Bead  Presence and absence Bead  Microorganism by volume Capture  Check efficiency

In order to confirm the collection efficiency depending on the presence or absence of beads and the amount of beads in a microorganism collecting and crushing apparatus (hereinafter referred to as collecting apparatus), the following experiment was conducted.

First, in order to produce air (aerosol) containing pathogenic bacteria, Salmonella typhimurium (hereinafter referred to as S. typhimurium ), adenovirus and Staphylococcus aureus Hereinafter referred to as S. aureus ) were mixed and sprayed in a specified range using an InnoSpire Essence Nebulizer Compressor. At the same time as the aerosol was sprayed, air was sucked by a vacuum pump to allow the aerosol present on the outside to pass through a collecting device containing various amounts of micro glass beads (0, 1, 5, 10, 15 and 20 g). In order to compare the efficacy, experiments were carried out simultaneously with a known microorganism collecting device, impinger. To equilibrate the solution into the impinger and the collecting device, 10 ml of the capture solution (10 mM Tris-HCl, pH 9) was added to the impinger and 15 ml to the collecting device, respectively. The flow rate of inhaled air was maintained at 10 LPM (L / min) using a rotameter.

When all of the aerosols sprayed from the nebulizer were inhaled, the bead beating portion was moved upward to perform bead beating, and a part of the collected aqueous solution was collected. PCR was performed with the collected aqueous solution to confirm the collection efficiency and the collection efficiency of microorganisms. The collection efficiency was confirmed by calculating the amount of microorganisms determined by PCR with respect to the amount of microorganisms.

As a result, it was confirmed that the highest collection efficiency was obtained when the amount of beads was 15 g in all the microorganisms used in the experiment. In addition, when the beads are present, the collection efficiency is increased compared with the case where the beads are not present. As a result, the presence of the beads increases the collection efficiency by increasing the contact time between the air passing through the collector and the aqueous solution.

FIG. 3 is a graph showing the results of checking the microbial collection efficiency according to the presence or absence of beads and the amount of beads used in the microorganism collecting and crushing apparatus.

Experimental Example  2: microorganisms according to air flow rate Capture  Check efficiency

In order to confirm the optimum flow rate for collecting pathogenic bacteria in the air, 15 g of beads were filled in the bead receiving part of the collecting device manufactured, and S. typhimurium and adenovirus were mixed in the same manner as in Example 1, Respectively. After the injection, air was sucked at a rate of 5 LPM, 10 LPM, and 20 LPM, respectively. When the aerosol inhalation was completed, the bead beeting was performed, and the efficiency of the collection was confirmed by PCR.

As a result, it was confirmed that both microorganisms had the highest collection efficiency when the air flow rate was 10 LPM. In particular, S. typhimurium exhibited a collection efficiency of about twice that of impinger. As the LPM increases, the contact time between the aqueous solution and the air is reduced. Therefore, it is considered that the collection efficiency does not increase more than 10 LPM.

FIG. 4 is a graph showing the results of checking the collection efficiency of microorganisms according to the air flow rate in the apparatus for collecting and crushing microorganisms. It can be seen that the collection efficiency is the best when the air flow rate is 10 LPM. In comparison with the impinger, It can be confirmed that the microorganism collecting efficiency of the disclosed collecting apparatus is remarkably excellent. 4, DLC is an abbreviation for Device for Collection and Lysis of Microorganisms according to an embodiment of the present invention.

Experimental Example  3: Bead  Determination of nucleic acid extraction efficiency by amount

In order to determine the optimum amount of beads for extracting nucleic acid from the collected bacteria and viruses using a collecting device, the following experiment was conducted.

Beads of 0, 1, 5, 10, 15 and 20 g were filled in the bead receptacles of the collecting device and 15 ml of the collecting aqueous solution was added. S. typhimurium , adenovirus, and S. aureus were added to the beads and bead beeting was performed for 3 minutes, and the collected aqueous solution was recovered and PCR was performed. Microbial capture and extraction efficiency was calculated by comparing the amount of recovered nucleic acid relative to the amount of nucleic acid in bacteria and viruses of known concentration. Further, in order to confirm the stability of the nucleic acid during the bead beating, the experiment was carried out by adding purified double strand DNA.

As a result, in the case of adenovirus, the nucleic acid is extracted only by the movement of the aqueous solution, but the bacteria S. typhimurium and S. aureus can be extracted when the amount of the beads is 10 g or more. However, since the stability of double - stranded DNA is decreased in beads of 10 g or more, it is considered that about 15 g of beads are most useful in terms of microbial collection and disruption considering both collection efficiency and nucleic acid extraction efficiency.

5 is a graph showing the results of confirming nucleic acid extraction efficiency according to the amount of beads used in the microorganism collecting and crushing apparatus.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

10: Microorganism collection and crushing device
110: bead accommodating portion
120: air inlet
130: Porous membrane
210: channel
220: pressure distributor
230: Rubber ring

Claims (9)

A channel-shaped bead receiving portion for receiving the bead;
An air inflow portion connected to the bead accommodating portion to supply outside air into the bead accommodating portion; And
The bead beating portion includes a channel-shaped bead beating portion having an upper end opened to allow the air to flow into the bead accommodating portion to escape and a lower end closed with a porous film, wherein the bead beating portion moves in the vertical direction of the bead accommodating portion, The microorganism collecting and crushing device.
The apparatus of claim 1, wherein the air inlet comprises a porous membrane at the distal end that is connected to the bead receptacle.
The apparatus of claim 1, wherein the air inlet is connected diagonally with respect to a central axis of the bead receptacle.
The apparatus of claim 1, wherein the bead beating portion further comprises a pressure distributor.
The apparatus of claim 1, wherein the beads are micrometer (mu m) in size.
The apparatus of claim 1, wherein the bead is a material selected from the group consisting of glass, ceramic, metal, and polymer.
Collecting the airborne floating microorganisms in the bead accommodating portion of the microorganism collecting and crushing apparatus according to any one of claims 1 to 5; And
And crushing the collected microorganisms.
[8] The method of claim 7, wherein the step of crushing the trapped microorganisms comprises moving the bead beating unit up and down in the microorganism trapping and crushing apparatus to perform beads beating.
8. The method of claim 7, wherein the detecting method further comprises detecting nucleic acid of the microorganism that has been disrupted after the step of disrupting the collected microorganism.

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