KR101659762B1 - A METHOD OF DETECTING PATHOGENIC MICRO-ORGANISM USING A yeast - Google Patents

Abstract

The present invention relates to a detection method and a detection kit for easily and rapidly detecting a harmful microorganism, for example, food harmful microorganisms. Specifically, the present invention relates to a method for culturing yeast cells, comprising culturing yeast; Culturing the yeast and the sample together; And a step of measuring the amount of lysosomes of the yeast cultured together with the sample, and a detection kit for the microorganism.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microorganism detection method using yeast,

The present invention relates to a detection method capable of detecting various harmful microorganisms more quickly, inexpensively and more effectively, compared to a harmful microorganism, specifically, a method for detecting microorganisms for food, more specifically, a detection method using a detection primer for a specific microorganism Detection system.

The increase in the rate of food poisoning due to environmental changes such as the increase of eating habits such as school meals and the increase of eating out opportunities and the increase of global warming and room temperature are collecting and enlarging in scale, There is an urgent need for countermeasures for securing the security. In particular, with the development of distribution and communication, food materials produced in various parts of the world are distributed to various countries, and as the period required for transportation and storage increases, the contamination of food due to food spoilage and the occurrence of food poisoning This is becoming a problem.

Most of the factors that threaten the safety of the food are microorganisms, and the influence of foodborne pathogenic bacteria is the greatest.

Specifically, more than 250 kinds of diseases that are problematic in humans due to food mediation are known, and representative diseases among them are food poisoning. Important pathogens propagating through food are Salmonella spp., E. coli O157, Listeria monocytogenes , Campylobacter jejuni, Staphylococcus aureus, and Clostridium fur presence. These pathogens have been reported to cause food poisoning in humans.

Such food poisoning is a disease accompanied by symptoms such as fever, vomiting, vomiting, diarrhea and abdominal pain, and most of them are bacterial food poisoning caused by bacteria. The bacterial food poisoning is classified into infectious type and poisonous type according to the onset type. Type of food poisoning is caused by infection with bacteria during the intake of contaminated food proliferate in the intestinal tract of food poisoning, Salmonella sp (Salmonella spp), E. coli O157 (Escherichia. Coli O157: H7, E. coli O157: H7) or Vibrio parahaemolyticus ) are the main causative bacteria. Poisonous food poisoning is a food poisoning caused by the ingestion of toxins present in food by producing toxins from bacteria in food. Therefore, in this case, even if the causative bacteria have already been killed in the food, food poisoning may occur when the toxins remain, and Staphylococcus ( Staphylococcus aureus) aureus) or Clostridium flops presence (Clostridium perfringens ) are the main causative bacteria.

According to statistics from the Centers for Disease Control and Prevention (CDC), about 5 million diseases a year are produced by foodborne medicines, of which about 4,000 are known to die. Most of them are food poisoning by Salmonella spp. To about 4 million people, of which approximately 2,000 people, or about 0.1%, are reported to have died. In addition, it is known that food poisoning caused by E. coli O157: H7 occurs about 10,000 per year and about 300 people die. In the case of Listeria monocytogenes, it is known that about 1,500 people occur annually and about 400 people die. Contamination of livestock food by bacteria was found to be serious.

In Korea, foodborne illnesses account for more than 50% of meat and meat products, and the causative agents are Salmonella and Staphylococcus in about 50% and 20%, respectively. In order to identify the precise causative microorganisms, the principle is to carry out biochemical tests by cultivating contaminated samples or foods, which lasts from a minimum of 3 days to several weeks. Although such media and biochemical tests are widely used as official testing methods, there is a high possibility that food poisoning will spread collectively before the identification of causative pathogens and completion of epidemiological investigation.

For example, currently available methods for detecting salmonellosis include: pre-culturing in buffered peptone water (BPW), culturing it in a selective medium, firstly verifying it, and then confirming it through biochemical and serological analysis And usually takes about 5 to 6 days.

Accordingly, there is a need for methods for rapidly detecting the contamination of a specific food in the food industry, that is, whether the microorganism remains in the food or whether the microorganism is contaminated by the food. In order to secure safety, the need for HACCP (Hazard Analysis and Critical Control Points) and other systems is increasing.

Currently, the most commonly used method for measuring food poisoning in Korea is to use the feces of suspected food or suspected patients as a sample, cultivate the sample in a selective medium based on an agar medium, and then isolate a colony- This is a method to confirm the presence of food microorganisms. This method takes a long time and disadvantage is that it can not be easily measured by the general public. In addition, recently, there has been proposed a method of quantitatively searching a specific bacterium by using a specific primer for a specific bacterium or using a polymerase chain reaction (PCR) method. However, Which is a critical point.

The method of confirming food safety in food safety, especially on food field, is premised on a quick and effective test for food microorganisms, and it is intended to inhibit the intake of contaminated food in the early stage and to prevent the onset of disease The most important point of the food safety inspection at the field such as the food station is to judge whether or not the food is contaminated by food-related microorganisms or corrupt microorganisms.

Therefore, it is urgently required to develop a microorganism detection method for harmful microorganisms that can measure contamination by food microorganisms, which is fast, simple, and does not require expensive equipment.

KR 2008-0102423 A KR 2013-0039532 A

The object of the present invention is to provide a method for detecting harmful microorganisms, and a method for detecting harmful microorganisms using eukaryotic microorganisms.

It is another object of the present invention to provide a detection kit for detecting harmful microorganisms using eukaryotic microorganisms.

In order to achieve the above object, the present invention provides a method for detecting harmful microorganisms.

In order to achieve the above object, the present invention also provides a harmful microorganism detection kit.

Specifically, the method for detecting a harmful microorganism of the present invention may include a step of culturing a eukaryotic microorganism and a sample, and then measuring a lysosome of a eukaryotic microorganism.

More specifically, the method for detecting a harmful microorganism includes culturing a eukaryotic microorganism, preferably a yeast; Culturing the yeast and the sample together; And measuring the amount of lysosomes in the yeast cultured together with the sample.

The step of culturing the yeast is characterized in that the yeast is cultured when the optical density (OD) value measured at 595 nm is 0.8 to 1.4, preferably 0.9 to 1.3, more preferably 1.0 to 1.2 .

In addition, the eukaryotic microorganism is yeast, preferably Saccharomyces as MY process in (S accharomyces sp.) (( S accharomyces microorganisms, more preferably in my processes Sergio busy Saccharomyces cerevisiae .

The harmful microorganism refers to a microorganism that causes diseases or contaminates food, including animals, including microorganisms such as E. coli , Corynebacterium sp., Microorganisms, Salmonella sp. Microorganism, Staphylococcus ( Staphylococcus aureus) aureus), Bacillus cereus (Bacillus cereus , Vibrio parahaemolyticus), L. monocytogenes to Ness (Listeria monocytogenes), ten o'clock Catania Entebbe recalls Lee Utica (Y. entericolitica), Clostridium flops presence (Clostridium perfringens), Shh Gela spp (Shigella sp.), Campylobacter Jeju you (the at least one member selected from the group consisting of Camphylobacter jejuni), preferably Escherichia coli (E. coli), Corynebacterium gum (Corynebacterium glutamicum) And Salmonella enterica. ≪ Desc / Clms Page number 7 >

The harmful microorganism detection kit of the present invention may be a microorganism detection kit comprising a eukaryotic microorganism culture solution.

Specifically, the eukaryotic microorganism is yeast, preferably Saccharomyces as MY process in (S accharomyces sp.) (( S accharomyces microorganisms, more preferably in my processes Sergio busy Saccharomyces cerevisiae .

Specifically, the microorganism detection kit may include a culture medium in which yeast is cultured when the optical density (OD) value measured at 595 nm is 0.9 to 1.3.

The harmful microorganism refers to a microorganism that causes diseases or contaminates food, including animals, including microorganisms such as E. coli , Corynebacterium sp., Microorganisms, Salmonella sp. Microorganism, Staphylococcus ( Staphylococcus aureus) aureus), Bacillus cereus (Bacillus cereus , Vibrio parahaemolyticus), L. monocytogenes to Ness (Listeria monocytogenes), ten o'clock Catania Entebbe recalls Lee Utica (Y. entericolitica), Clostridium flops presence (Clostridium perfringens), Shh Gela spp (Shigella sp.), Campylobacter Jeju you (the at least one member selected from the group consisting of Camphylobacter jejuni), preferably Escherichia coli (E. coli), Corynebacterium gum (Corynebacterium glutamicum) And Salmonella enterica. ≪ Desc / Clms Page number 7 >

For example, the forcible microorganism detection kit may include a culture containing microorganisms belonging to the genus Saccharomyces sp. Cultured at an optical density (OD) value measured at 595 nm of from 0.9 to 1.3. The microorganism detection kit according to claim 1, wherein the microorganism is at least one selected from the group consisting of E. coli , Corynebacterium glutamicum , and Salmonella enterica .

As described above, the present invention can detect a variety of harmful microorganisms, particularly harmful microorganisms related to food, so that it is possible to easily detect infection or pollution by a microorganism in a simple manner for food or feed, And whether or not it is infected by food poisoning bacteria can be economically feasible. In particular, a method using a currently used selective medium requires a long time, requires a highly trained technique in identifying a bacterium, and recently developed methods using a PCR or DNA probe require disadvantages such as expensive equipment However, in the case of the present invention, it is expected that the present invention can be used in various industrial fields related to foods since the contamination of foods can be confirmed simply and quickly.

1 to 3 when incubated with the microorganism for the S. cerevisiae according to one embodiment of the present invention, by checking the amount of the lysosome via the degree of fluorescence in lysosomes, Figure 1 is a fluorescent expressed by C. glutamicum will showing the degree, Figure 2 shows the fluorescence would expression levels by E. coli, FIG. 3 shows the fluorescence level by the Salmonella enterica, (E. coli) Figure 1a (C. glutamicum), Figures 2a and The abscissa of the graph of FIG. 3 ( Salmonella enterica ) shows the amount of each microorganism to be added, and the ordinate shows the relative intensity of fluorescence based on the fluorescence of the control without any harmful microorganisms. The photographs of FIGS. 1B and 2B are photographed to check actual fluorescence, and the respective English marks (Control, a, b, c, and d) of the photographs correspond to FIGS. 1A and 2A.
FIG. 4 is a graph showing fluorescence intensity of ribosomes according to the cell amount of S. cerevisiae , that is, optical density (OD) value according to an embodiment of the present invention . The vertical axis represents the relative intensity of fluorescence based on the fluorescence of the control without addition of any harmful microorganisms.

The present invention relates to a method for detecting harmful microorganisms.

The present invention relates to a harmful microorganism detection kit.

The inventors of the present invention have conducted research to develop a method for detecting a microorganism of the present invention in a simple and rapid manner and to quickly and efficiently detect contamination of foodstuffs in the field. As a result, it has been found that a lysosome, When bacteria enter from the outside of the cell, they have the function of decomposing the invading external bacteria while being activated. When these functions are used, when the food is contaminated by the bacteria that do not have the harmful microorganism, lysosome, The present invention has been completed. This further advances the study, using the eukaryotic microorganism, in my processes Sergio busy in a particular yeast Saccharomyces (S accharomyces cerevisiae), the same for the micro-organisms in order according to the cell an amount of a My process Sergio busy in the saccharide against It was confirmed that the lysosomal activity, that is, the sensitivity of the detection method, can be changed. From these results, it can be seen that when the optical density (OD) value measured at 595 nm is about 1.1, The lysosome fluorescence expression level was the highest, and it was confirmed that the sensitivity of the detection method was the most excellent. Further, it was further confirmed that this detection method is applicable to various harmful microorganisms, thereby completing the present invention.

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains, unless the context clearly dictates otherwise in this specification. For example, microorganisms such as singletons And Cell Biology Dictionary (2nd ed. (1994)); Cambridge Dictionary of Science and Technology; Genetics Glossary (5TH ed.), R. Reiger, et al., Hale, Malham and Harper Collins Biotechnology Dictionary (1991).

The lysosome has a large number of hydrolytic enzymes in eukaryotic cells, and it refers to a cell organelle that plays an important role in the intracellular digestion of extraneous foreign matter by phagocytosis, in addition to the metabolism of intracellular components. Unlike the mitochondria, which are intracellular organelles, the lysosome is enclosed in a single layer structure. The inside of the lysosome is composed of a protease such as carboxypeptidase, an acidic dephosphate hydrolase, Nucleic acid hydrolase,? -Glucuritase or aryl sulfatase, and the like.

In the present specification, the term "culture" means that a specific microorganism is cultured in a culture medium or a culture solution, and the culture may include a culture containing the specific microorganism or a culture of a microorganism which has been inactivated by the microorganism. The concentrate of the culture or culture is not limited in its formulation, for example, the formulation may be liquid or solid.

In this specification, the medium may be a mixture of a nutritional material required by a cultured object, that is, a microorganism to be cultured, and a substance added for a special purpose, in order to cultivate the specific microorganism. The medium is also referred to as an incubator or a culture medium, and is a concept including both natural medium, synthetic medium and selective medium.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for detecting harmful microorganisms. Since the harmful microorganisms including the food-harmful microorganisms do not have the cell organelles such as lysosomes, the eukaryotic microorganisms, specifically the yeast, more specifically the microorganisms of the genus Saccharomyces sp. When cultured together with a microorganism bacterium, the lysosome of the eukaryotic microorganism can be activated and the microorganism can be easily detected in the case of confirming the activation of the lysosome.

The eukaryotes are largely composed of flea, which includes fungi such as yeast, Opisthokonta and Amoebozoa, including mammals such as yeasts and artisans, and twins including both multicellular animals and plants It may be a eukaryotic microbial or eukaryotic animal cell, in particular, a concept embracing it. In view of the speed and economy of the detection method, the eukaryote may preferably be a eukaryotic microorganism, and the eukaryotic microorganism may be a yeast, preferably a microorganism of the genus Saccharomyces sp. , And more preferably Saccharomyces cerevisiae .

The method for detecting a harmful microorganism of the present invention may include the step of measuring a lysosome of a eukaryotic microorganism after the eukaryotic microorganism and the sample are cultured.

Specifically, the method for detecting a harmful microorganism includes: culturing a eukaryotic microorganism; Culturing the eukaryotic microorganism and the sample together; And measuring the lysosome amount of the yeast cultured together with the sample.

The eukaryotic microorganism may be a yeast, preferably a microorganism belonging to the genus Saccharomyces sp., More preferably Saccharomyces cerevisiae .

The step of culturing the eukaryotic microorganism is preferably performed such that when the yeast has an optical density (OD) value measured at 595 nm of 0.4 to 1.9 or 0.8 to 1.4, preferably 0.9 to 1.3, more preferably 1.0 to 1.2, .

The step of culturing the eukaryotic microorganism may be performed using a eukaryotic microorganism culture medium. For example, when the eukaryotic microorganism is yeast, it may be performed using YPD medium or the like.

The medium or culture medium means that the microorganism is cultivated in the eukaryotic microorganism or the like, and the microorganism or the culture medium is made of a substance required for the growth of the cultured microorganism. The medium is used for the survival and growth of eukaryotic microorganisms, Materials that can supply the necessary water and nutrients may be included.

The nutritive substance may be a carbon source, a nitrogen source, an inorganic salt, and a growth factor (vitamins). The nutrient may be a substance capable of detecting both the eukaryotic microorganisms and the harmful microorganisms, which are means for detecting a harmful microorganism. Examples of the carbon source include polysaccharides such as disaccharides such as glucose, fructose, mannose, ribose and xylose, disaccharides such as sucrose, melobiose, cellobios and lactose, and sugar alcohols such as sorbitol, mannitol and glycerol, And the like, but is not limited thereto. As the nitrogen source, inorganic nitrogen such as ammonium salt or nitrate, or organic nitrogen such as amino acid or peptone may be used. The inorganic nutrients may be sodium phosphate, potassium phosphate, magnesium sulfate, magnesium chloride, etc. which can provide P, Na, Cl, K, Mg, S, Ca, Fe, Mn, . The nutritional ingredients may be potato juice, yeast extract, peptone, or the like, which are not individual ingredients.

Since the medium for culturing the eukaryotic microorganism may include all substances capable of culturing eukaryotic microorganisms, the medium is not limited in its composition or properties. In this respect, the medium may be a synthetic medium consisting of natural components such as components extracted from an organism or natural substances such as seeds or fruits of plants, or a part of the components of the synthetic medium are mixed with peptone or yeast extract And a semi-synthetic medium in which the chemical composition is not known.

The sample means a part of the food or material or a workpiece of the food or the object suspected of being contaminated with the harmful microorganism. In this respect, the sample can be prepared by a method comprising collecting a sample suspected of being contaminated with a harmful microorganism and subjecting it to sterilized water or physiological saline, e.g., 0.85% saline solution.

The microorganisms for E. coli (E. coli), the genus Corynebacterium (Corynebacterium sp.) Microorganisms, genus Salmonella (Salmonella sp.) Microorganism, Staphylococcus aureus (Staphylococcus aureus), Bacillus cereus (Bacillus cereus , Vibrio parahaemolyticus), L. monocytogenes to Ness (Listeria monocytogenes), ten o'clock California Entebbe recall Lee urticae (Y. entericolitica), Clostridium flops presence (Clostridium perfringens , Shigella sp., and Camphylobacter jejuni, and more specifically, E. coli , Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium glutamicum ), And Salmonella enterica .

The step of culturing the eukaryotic microorganism and the sample together may be carried out under conditions in which both the eukaryotic microorganism and the suspected microorganism suspected to be contained in the sample can grow and reproduce, and are not limited by other conditions. As an example, the culturing step may be performed at 10 캜 to 35 캜 or 15 캜 to 30 캜 or 20 캜 to 27 캜.

The step of measuring the lysosome amount of the cultured eukaryotic microorganism is not limited as long as it is a method capable of identifying lysosomes of a eukaryotic microorganism. For example, the amount or activity of lysosome in the eukaryotic microorganism can be confirmed by fluorescence or the like, or a method of measuring through the amount of enzyme in the lysosome.

Since the method for measuring harmful microorganisms according to the present invention has a technical effect of rapidly and economically measuring, the step of measuring the lysosome amount of the eukaryotic microorganism in this aspect is a method of treating a lysosome-related fluorescent substance, You can do this by checking.

The fluorescence measurement can be performed using a conventional fluorescence microscope or the like in order to qualitatively measure the degree of fluorescence and no special additional device is required. However, when it is desired to quantitatively compare the degree of fluorescence, A separate device which can be checked can be additionally included.

The device that can be further included to confirm the degree of fluorescence may be, for example, a spectrometer or a device capable of verifying the degree of fluorescence.

In the case of a device capable of confirming the degree of fluorescence, it may be a fluorescence analyzer utilized in an ELISA (Ezyme-linked immunosorbent assay) method or the like. For example, the eukaryotic microorganism and the sample can be contained in a transparent container and a device capable of confirming the degree of fluorescence of the container, that is, a light source, for example, an LED light source and a fluorescence detection device.

In order to confirm the degree of fluorescence, in order to confirm the degree of fluorescence when ELISA (Ezyme-linked immunosorbent assay) immuno-precipitation method or the like is applied, a specific region of a ribosome such as a ribosome, An antibody capable of recognizing the ribosome or an antibody capable of recognizing the ribosome and a secondary antibody capable of recognizing the antibody.

In addition, the spectrometer may be a monochromator or a spectrophotometer. The spectrophotometer is also called a photoelectric spectrophotometer, and is a device for measuring the spectrum of light emitted or absorbed by a substance, and includes an apparatus for analyzing the energy of a particle beam such as an electron beam including an electromagnetic wave. Since the spectroscope can measure the spectrum of light emitted or absorbed by a specific substance, when the lysosome is activated or the amount of lysosome is increased, the degree of the change can be quantitatively measured. Depending on the degree of the change, Can be used for quantitative detection of microorganisms.

For example, measuring the lysosomal volume of the cultured eukaryotic microorganisms can be carried out by using a LAMP, LIMP, LysoBrite, CytoPainter Lysosomal Staining Kit, Lyso-ID ^® Green detection kit, Cell Navigator ^TM Lysosome Staining Kits like.

The present invention also relates to a kit for detecting a microorganism.

The malignant microorganism detection kit may include a eukaryotic microorganism culture solution.

The eukaryote may be a eukaryotic microorganism in terms of the detection of a microorganism that is rapid, simple and economical, which is an effect of the present invention, and the eukaryotic microorganism is a yeast, preferably a microorganism of the genus Saccharomyces sp. , And more preferably Saccharomyces cerevisiae .

The eukaryotic microorganism culture solution is obtained by culturing the eukaryotic microorganisms in a medium. The medium or culture medium means that the eukaryotic microorganism is prepared from a substance required for growth of the microorganism, such as the microorganism, , And the medium may include materials capable of supplying water and nutrients necessary for survival and development, that is, growth of eukaryotic microorganisms and the like.

The nutritive substance may be a carbon source, a nitrogen source, an inorganic salt, and a growth factor (vitamins). The nutrient may be a substance capable of detecting both the eukaryotic microorganisms and the harmful microorganisms, which are means for detecting a harmful microorganism. Examples of the carbon source include polysaccharides such as disaccharides such as glucose, fructose, mannose, ribose and xylose, disaccharides such as sucrose, melobiose, cellobios and lactose, and sugar alcohols such as sorbitol, mannitol and glycerol, And the like, but is not limited thereto. As the nitrogen source, inorganic nitrogen such as ammonium salt or nitrate, or organic nitrogen such as amino acid or peptone may be used. The inorganic nutrients may be sodium phosphate, potassium phosphate, magnesium sulfate, magnesium chloride, etc. which can provide P, Na, Cl, K, Mg, S, Ca, Fe, Mn, . The nutritional ingredients may be potato juice, yeast extract, peptone, or the like, which are not individual ingredients.

Since the medium for culturing the eukaryotic microorganism may include all substances capable of culturing eukaryotic microorganisms, the medium is not limited in its composition or properties. In this respect, the medium may be a synthetic medium consisting of natural components such as components extracted from an organism or natural substances such as seeds or fruits of plants, or a part of the components of the synthetic medium are mixed with peptone or yeast extract And a semi-synthetic medium in which the chemical composition is not known.

The eukaryotic microorganism culture solution is characterized in that the eukaryotic microorganism has an optical density (OD) value of 0.4 to 1.9 or 0.8 to 1.4, preferably 0.9 to 1.3, more preferably 1.0 to 1.0, When it is 1.2, it may be cultured. In view of this, the culture solution may be diluted until the eukaryotic microorganism has an optical density (OD) value of 0.8 to 1.4, preferably 0.9 to 1.3, more preferably 1.0 to 1.2 as measured at 595 nm And may be a cultured culture.

The microorganisms for E. coli (E. coli), the genus Corynebacterium (Corynebacterium sp.) Microorganisms, genus Salmonella (Salmonella sp.) Microorganism, Staphylococcus aureus (Staphylococcus aureus), Bacillus cereus (Bacillus cereus , Vibrio parahaemolyticus), L. monocytogenes to Ness (Listeria monocytogenes), ten o'clock California Entebbe recall Lee urticae (Y. entericolitica), Clostridium flops presence (Clostridium perfringens , Shigella sp., and Camphylobacter jejuni, and more specifically, E. coli , Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium glutamicum ), And Salmonella enterica .

The malignant microorganism detection kit may further include a reactor for reacting the eukaryotic microorganism and the sample, for example, a reaction tube. Further, the eukaryotic microorganism culture solution or the eukaryotic microorganism culture solution may further include a main body or a body including divided spaces in which a plurality of the eukaryotic microorganism culture solutions can be placed.

In addition, the malignant microorganism detection kit may further include a device capable of identifying or measuring the ribosomes of the eukaryotic microorganism. With respect to identifying the ribosomes, a device using color development or fluorescence may be added to quantify the amount of ribosomes.

The device for identifying or measuring the lysosome of the cultured eukaryotic microorganism is not limited as long as it is an apparatus capable of identifying a lysosome of a eukaryotic microorganism. For example, the amount or activity of lysosome in the eukaryotic microorganism can be confirmed by fluorescence or the like, or a method of measuring through the amount of enzyme in the lysosome.

Since the method for measuring harmful microorganisms according to the present invention has a technical effect of rapidly and economically measuring, the step of measuring the lysosome amount of the eukaryotic microorganism in this aspect is a method of treating a lysosome-related fluorescent substance, You can do this by checking.

The fluorescence measurement can be performed using a conventional fluorescence microscope or the like in order to qualitatively measure the degree of fluorescence and no special additional device is required. However, when it is desired to quantitatively compare the degree of fluorescence, A separate device which can be checked can be additionally included.

A device added to confirm the activation of the ribosome, the ribosome or the amount of the ribosome may be, for example, a spectrometer or a device capable of confirming the degree of fluorescence.

In the case of a device capable of confirming the degree of fluorescence, it may be a fluorescence analyzer utilized in an ELISA (Ezyme-linked immunosorbent assay) method or the like. For example, the eukaryotic microorganism and the sample can be contained in a transparent container and a device capable of confirming the degree of fluorescence of the container, that is, a light source, for example, an LED light source and a fluorescence detection device.

In order to confirm the degree of fluorescence, in order to confirm the degree of fluorescence when ELISA (Ezyme-linked immunosorbent assay) immuno-precipitation method or the like is applied, a specific region of a ribosome such as a ribosome, An antibody capable of recognizing the ribosome or an antibody capable of recognizing the ribosome and a secondary antibody capable of recognizing the antibody.

In addition, the spectrometer may be a monochromator or a spectrophotometer. The spectrophotometer is also called a photoelectric spectrophotometer, and is a device for measuring the spectrum of light emitted or absorbed by a substance, and includes an apparatus for analyzing the energy of a particle beam such as an electron beam including an electromagnetic wave. Since the spectroscope can measure the spectrum of light emitted or absorbed by a specific substance, when the lysosome is activated or the amount of lysosome is increased, the degree of the change can be quantitatively measured. Depending on the degree of the change, Can be used for quantitative detection of microorganisms.

For example, the above-described hazardous microorganism detection kit may comprise a microorganism belonging to the genus Saccharomyces sp., Wherein the microorganism has an optical density (OD) of 0.9 to 1.3 as measured at 595 nm. The present invention may be a detection kit for the detection of one or more selected from the group consisting of E. coli, Corynebacterium glutamicum, and Salmonella enterica.

Hereinafter, examples of the present invention will be described. The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

< Example 1 > Saccharomyces cerevisiae Identification of harmful microorganisms using

In this example, S. cerevisiae s2805, a eukaryotic microorganism used, was purchased from KRIBB (Korea Research Institute of Bioscience and Biotechnology) and YPD medium was used for culture of S. cerevisiae s2805. The YPD medium was used in a composition of 1% yeast extract (Becton Dickinson Co.), 2% peptone (Becton Dickinson Co.) and 2% glucose (Amresco Inc.).

The S. cerevisiae s2805 was inoculated on YPD medium and cultured at 30 ° C and 160 rpm until the OD value measured at 595 nm was 1.114.

The OD ₅₉₅ After culturing S. cerevisiae until the value became 1.114, the culture medium was removed by centrifugation, and only the strain was obtained. Then, the strain was inoculated with YPD medium and C. glutamicum (ATCC 13032), E. coli. E. coli (ATCC 10536) and Salmonella enterica (ATCC 13076) were cultured for 4 hours at 20 ° C and 160 rpm.

The medium for culturing C. glutamicum and E. coli was LB medium, and the medium for culturing Salmonella enterica was 0.5% NaCl LB medium. The LB medium was used in a composition of 0.5% yeast extract (Becton Dickinson Co.), 1% tryptone (Becton Dickinson Co.) and 1% NaCl (Sigma Aldrich Co.) (Becton Dickinson Co.), 1% tryptone (Becton Dickinson Co.) and 0.5% NaCl (Sigma Aldrich Co.).

After the cultivation, LysoTracker (Lysotracer DND99, Invitrogen, USA), which specifically reacted only with lysosomes, was dissolved in distilled water at a concentration of 1 / 10,000, and the mixture was reacted at 30 ° C for 5 minutes. After the reaction, fluorescence was confirmed at a wavelength of 543 nm using an electron microscope (confocal laser-scanning microscope, LSM 510 META, Japan) and imaged using a Zeiss LSM image browser. The degree of expression of Rizosome in S. cerevisiae s2805 was observed. The results of the observation and the fluorescence intensity observed are compared with the fluorescence intensity of a control (control) in which no microorganism was treated, as shown in FIG. 1 to FIG. With respect to the graph, in order to confirm the activation of the lysosome as the degree of activation of lysosome per individual cell irrespective of the cell number, the fluorescence expression level was calculated as the fluorescence-exposed area relative to each cell surface area, Are shown in Figs. 1A, 2A and 3.

As shown in FIG. 1 to FIG. 3, as a result of treating the above-mentioned harmful microorganisms at different concentrations, the degree of fluorescence increases according to each treatment amount. As a result of confirming the degree of fluorescence, And it was confirmed that it was possible to quantify the biotoxic substances by using the standard curve.

< Example 2 > Saccharomyces cerevisiae Identification of optimal conditions for microorganisms

Based on the findings from Example 1 above, further experiments were conducted to determine the optimal amount of eukaryotic microorganisms to identify the harmful microorganisms according to the amount (activation) of the lysosomes, that is, the degree of fluorescence.

The seeds of S. cerevisiae s2805 were inoculated on YPD medium and cultured at 30 ° C and 160 rpm. The OD values measured at 595 nm were 7 (OD value, 0.043, 0.148, 0.428, 1.114, 1.946, 2.103, 2.311). After culturing was stopped in each of the above-mentioned divided conditions, seven experimental groups were prepared, and the respective OD ₅₉₅ By centrifugation for the group cultured for S. cerevisiae until a value of about remove the culture and then the resulting strains only, the concentration of 100,000 CFU / ml of E. coli in YPD medium with the first embodiment to the strain , And then cultured at 20 ° C and 160 rpm for 10 minutes.

After the incubation, LysoTracker was treated in the same manner as in Example 1, fluorescence was confirmed at a wavelength of 543 nm using an electron microscope, and the degree of fluorescence was observed by imaging using a Zeiss LSM image browser. FIG. 4 is a graph showing the degree of fluorescence observed in comparison with the fluorescence intensity of a control (control) in which no harmful microorganism was treated. In order to confirm the activation of lysosome according to the cell size of the eukaryotic microorganism as well as the activation level of lysosome per individual cell irrespective of the number of cells of the eukaryotic microorganism, the fluorescence expression level was calculated as the fluorescence-expressed area relative to each cell surface area, One value is shown in Fig. 4, respectively.

As shown in FIG. 4, fluorescence intensity was increased with increasing cell number, ie, OD value, of S. cerevisiae s2805 at the beginning, but it gradually decreased after the OD value exceeded 1.1. From the above results, it was confirmed that when the OD value is too low, the cell amount is too small to be used as a detector. If the OD value is too high, the cell amount is too large, , And the cell amount of S. cerevisiae s2805 used for the detection of the microorganism from the above results is 0.8 to 1.4 based on the optical density (OD) value measured at 595 nm Were evaluated as preferable.

Claims (6)

Culturing the Saccharomyces sp. Microorganism until the Optical Density (OD) value measured at 595 nm is 1.114; Culturing the cultured yeast and a sample together; And measuring the amount of lysosomes in yeast cultured with the sample;
Containing
Escherichia coli (E. coli), the genus Corynebacterium (Corynebacterium sp.) Microorganisms, genus Salmonella (Salmonella sp.) Microorganism, Staphylococcus aureus (Staphylococcus aureus), Bacillus cereus (Bacillus cereus), Vibrio para H. Vibrio parahaemolyticus , Listeria monocytogenes , Y. entericolitica , Clostridium perfringens , Shigella sp., And the like. A method for detecting a harmful microorganism against at least one harmful microorganism selected from the group consisting of Camphylobacter jejuni . delete delete delete delete The present invention relates to a microorganism detection kit comprising a culture obtained by culturing a Saccharomyces sp. Microorganism in a branch medium when the optical density (OD) value measured at 595 nm is 1.114,
Wherein the harmful microorganism is at least one selected from the group consisting of E. coli , Corynebacterium glutamicum , and Salmonella enterica .

Images