KR100376917B1 - Probes for Detecting Gastrointestinal Pathogens, and DNA Chip and Detection Method Using the Same - Google Patents

Abstract

The present invention includes Escherichia coli O-157, enteritis vibrio, including diarrheal diseases, including Vibrio cholerae that causes cholera, Salmonella typhimurium, which causes typhoid, Shigella dysenteriae, S. flexneri , which causes bacterial dysentery. Probe for the rapid diagnosis of gastrointestinal bacteria including Vibrio parahaemolyticus , Staphylococcus aureus , Campylobacter jejuni, Yersinia enterocolitica, Pseudomonas aeruginosa and Listeria monocytogenes , and DNA chips using the same A detection method using a chip. According to the present invention will detect the digestive tract infection bacteria in a short time to prevent the abuse of antibiotics and to reduce the pain of patients to help to quickly and prompt prescription, food test, restaurant hygiene test that requires a large amount of sample test in addition to clinical test It can be effectively used for the purpose of quarantining imported agricultural, livestock, aquatic products and diagnosing livestock.

Description

Probes for Detecting Gastrointestinal Pathogens, and DNA Chip and Detection Method Using the Same}

The present invention relates to a detection probe for digestive infections, a detection method using the same, and a detection DNA chip.

Despite the development of medicine or food hygiene, hundreds of millions of people around the world suffer from diseases caused by contaminated food, so food poisoning is still one of the most important diseases for humans. A recent WHO report found that 300 to 350 times more food poisoning cases were reported.

In particular, developing countries suffer greatly from diseases including cholera, campylobacterosis, coliformosis, salmonella, bacterial dysentery, brucellosis and hepatitis A. Each year, 1.5 billion people have diarrhea, and more than three million children under five die. Many of the diarrheal diseases also result from food poisoning.

Despite the application of technologies such as safe drinking water supply, sound hygiene standards and pasteurization, many industrial countries have recently experienced an increase in food poisoning outbreaks. Surveys show that 5 to 10 percent of the population is infected with food poisoning each year. Among them, Listeria monocytogenes , E. coli O-157: H7, and S. Typhimurium are known as new serious threats to public health.

According to a U.S. survey, the number of cases of bacterial food poisoning in the United States is estimated at 6.5 million to 33 million, with an estimated 500 to 9,000 deaths, and an annual economic burden of about $ 2.9 billion to $ 6.7 billion. have. Canada also estimated the loss of food poisoning in 1996 to $ 240 million. Although most food poisoning symptoms are mild, they can be surprisingly severe. In addition to acute gastroenteritis symptoms such as diarrhea and vomiting, many foodborne pathogens can cause invasive diseases. For example, L. monocytogenes infections can cause meningitis or sepsis in newborns and immunoconverters, and can cause miscarriage in pregnant women. Salmonellosis can cause sepsis. Food poisoning can also cause chronic sequelae or disorders. Escherichia coli O157: H7 is a major cause of hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in children . Infections with non-typhoid Salmonella or Y. enterocolitica can cause arthritis. Since then, it has been the cause of Guillain-Barre syndrome, one of the most common causes of laxative paralysis in the United States.

Food poisoning is a general term of poisoning symptoms such as acute gastroenteritis and neurological disorders caused by food or water. It is caused by collectively ingesting food contaminated with food poisoning bacteria or plant and animal natural poisons and sometimes toxic chemicals. . But most of the food poisonings in our country are bacterial. The incidence of food poisoning by bacteria came to light in 1885 when Gaertner revealed that acute gastroenteritis caused by meat was caused by Salmonella. As a salmonella found is Salmonella enteritidis in eggs are spread by the recent attention around the world. Since then, various bacteria have been reported as causative agents of food poisoning, and about 17 species are currently known as food poisoning bacteria. Diarrheal Escherichia coli has the same pathogenic and infectious symptoms as Shigella (enterally invasive Escherichia coli), but because it does not cause infection from person to person, it is treated as food poisoning and not as infectious disease. In addition, cholera bacteria are serotypes (O1) of one of Vibrio cholerae , although some of them contain the same pathogenicity as cholera bacteria, but only cholera bacteria with cholera toxin production are treated as infectious diseases, and the remaining species (NAG Vibrio). ) Is treated as food poisoning. On the other hand, many cases of salmonella food poisoning caused by extremely small amounts of 100-1000 salmonella have been reported in the United States or Canada. Diarrhea caused by vibrio or toxin-like Escherichia coli has many cases of aquatic infections, but it is not considered that the beverage contains a large amount of more than 1 million bacteria, and even a small amount of aquatic infections is estimated to be established. In addition, there has been a reported case of population incidence of up to 100 campylobacteritis in the United States. On the other hand, typhoid fever, bacterial dysentery, cholera, etc., which are known to be infected from person to person, have recently been turned into food and water-borne infections. This proves that it is unreasonable to distinguish between infectious diseases and food poisoning because foodborne pathogens cannot develop humans in trace amounts, but infectious pathogens can develop even with very small amounts. As mentioned above, it is very difficult to distinguish between gastrointestinal infectious diseases and bacterial food poisoning by the amount of pathogen or infection. Because of this, there is a recent tendency to use the term food and water-borne infection, including bacterial dysentery or typhoid, for food and water-borne infections. In addition, the virus requires living cells or tissues to multiply, and because food can not proliferate, even if the disease is caused by food, viral diarrhea is not included in food poisoning.

Bacterial food poisoning can be broadly classified into infectious, toxin and mixed forms according to its onset (see Table 1). Infectious type is a bacteria that is pre-proliferated in food is ingested with food to further multiply in the small intestine, causing toxic symptoms. The representative causes are Salmonella, enteritis vibrio, campylobacter, enteropathogenic E. coli. The toxin type causes the toxins caused by the toxin as the bacteria in the food multiply and produce the toxin and consume the food. Representative toxin-type food poisoning bacteria are Staphylococcus aureus, which is caused by enterotoxin. Food poisoning has a short incubation period (average 3-5 hours), no fever, and symptoms such as vomiting, diarrhea and abdominal pain, especially severe vomiting. Infectious toxin types are bacteria that have grown in food and settle in the intestine to produce toxins, and the toxins cause diarrhea. Food poisoning caused by toxins such as Escherichia coli, gastrointestinal bacterium, and Cereus bacterium belongs to them.

Bacteria causing bacterial food poisoning Infection type Toxin type Infectious toxin type Salmonella spp Clostridium botulinum Clostridium perfringens Campylobacter jejuni Staphylococcus aureus Bacillus cereus Yersinia enterocolitica Enteropathogenic Escherichia coli Enterohemorrhagic Escherichia coli Enterotoxigenic Escherichia coli Shiga-like toxin producing Escherichia coli Vibrio cholerae Listeria monocytogenes Vibrio parahemolyticus Vibrio vulnificus Brucella abortus

Food poisoning monitoring is important to prevent food poisoning. In this regard, cooperation between laboratories, doctors and public health institutions is required. Currently, most food poisoning surveillance is epidemiological only for passive and socially problematic large-scale food poisoning. However, with a more efficient surveillance system and a faster pathogen detection system, it will be possible to detect different populations of infection over time or region and to detect outbreaks early.

Currently, the diagnostic methods used for the diagnosis of these diseases are most frequently used in the local public health centers and frontline medical institutions by culturing bacteria in the feces of patients, and then using antisera. Although the test results are relatively accurate, the bacteria must be cultured from the suspected patients, so patients with clinical symptoms such as diarrhea are not only difficult to isolate because they have already given antibiotics or other drugs, It takes a long time, such as 3 to 4 days and the sensitivity is low, many difficulties have been proposed to prevent the rapid spread by these diseases. Moreover, since the antiserum used in the aggregation reaction method depends on the total amount of imports, it is necessary to develop a diagnostic method using pure domestic technology.

On the other hand, since most of the genetic engineering methods used in the past had limitations in conducting experiments with a large number of genes at the same time, the development of new technologies is urgently required in the post genome era. That's because it takes too much time to study new genetic information that reveals hundreds of a day or all genetic coded organisms by conventional methods. To overcome this problem, one of the most recently developed methods is a gene search method using DNA chips. DNA chips are made by combining mechanical and electronic technologies with existing molecular biological knowledge. Using machine automation and electronic control technology, we can put hundreds or even hundreds of thousands of DNA into tiny spaces. In other words, the DNA chip is a high-density pasted with a huge number of DNA for gene search. DNA chips that align multiple DNA molecules on a slide glass or silicon substrate in an orderly array are very useful for microarray analysis of gene expression, mutation, and polymorphism. Representative genetic engineering methods that can be replaced with such DNA chips include Southern and Northern blot, PCR, mutation detection and DNA sequencing. The main difference from the above methods is that at least hundreds of genes can be searched in a short time at the same time. Another difference is that in southern and northern blots, nitrocellulose membranes are used as the medium for attaching the genetic material, whereas DNA chips use solids such as glass. This allows DNA chips to attach very small amounts of genetic material at high densities and simultaneously retrieve a large number. It is also applicable to biomolecules other than DNA. Various chip fabrication and analysis systems are being developed and sold around the United States, but are not yet available to general researchers. In order to keep abreast in this field, Korea needs to actively develop DNA chip technology and expand its field of application. In addition, there is a need to develop DNA chips that can quickly and accurately detect bacteria causing digestive diseases in all environmental fields such as humans, animals, meat, vegetables, and beverages. The diagnostic DNA chip developed to date has a high search equipment and manufacturing cost, and it is still difficult to be commercialized. Therefore, for the practical use of DNA chips, it is possible to reduce the cost of introducing foreign technology by accumulating pure domestic technology, to lower the production cost through mass production technology, and to reduce the economic burden of medical treatment. The need to be presented.

Therefore, an object of the present invention is to provide a probe for diagnosing digestive diseases by analyzing toxic genes related to intestinal infectious diseases such as cholera, typhoid fever, dysentery, and E. coli, and analyzing specific genes for each strain.

Another object of the present invention is to provide a DNA chip using the probe, and to provide a new rapid diagnostic method using the DNA chip.

1 is a result of performing a polymerase chain reaction using the target DNA preparation primer designed in the present invention for 10 types of diarrheal disease-causing digestive organs, which can identify gene amplification products of specific regions of 10 strains. have.

Figure 2 is a result of asymmetric polymerase chain reaction of the target DNA preparation primers designed in the present invention for the strains of the digestive tract infection bacteria causing 10 diarrhea diseases.

Figure 3 is fixed 10 probes on the gel-based DNA chip. The five probes in the upper row were planted with probes for five toxins among the causative agents of diarrheal diseases, and the probes for five food-derived toxins among the causative agents of diarrheal diseases were planted below.

4 is an experimental result of hybridization with a target DNA of Vibrio parahaemolyticus on the gel-based DNA chip prepared in FIG. 3. The first probe in the top row shows strong fluorescence.

5 is an experimental result of hybridization with the target DNA of Vibrio cholerae on the gel-based DNA chip prepared in FIG. 3. The second probe in the top row shows strong fluorescence.

6 is an experimental result of hybridization with a target DNA of E. coli O-157 on the gel-based DNA chip prepared in FIG. 3. In the third probe in the upper row, strong fluorescence can be identified.

7 is an experimental result of hybridization with the target DNA of Shigella flexneri on the gel-based DNA chip prepared in FIG. In the fourth probe in the upper row, strong fluorescent material can be identified.

8 is an experimental result of hybridization with a target DNA of Salmonella typhimurium on the gel-based DNA chip prepared in FIG. 3. The fifth probe in the top row shows strong fluorescence.

9 is an experimental result of hybridization with a target DNA of Staphylococcus aureus on the gel-based DNA chip prepared in FIG. 3. In the first probe in the bottom row you can see the strong fluorescence.

10 is an experimental result of hybridization with the target DNA of Campylobacter jejuni on the gel-based DNA chip prepared in FIG. A strong fluorescence can be seen in the second probe in the bottom row.

FIG. 11 is an experimental result of hybridization with a target DNA of Yersinia enterocolitica on the gel-based DNA chip prepared in FIG. 3. Stronger phosphors can be seen in the third probe in the bottom row.

12 is an experimental result of hybridization with the target DNA of Pseudomonas aeruginosa on the gel-based DNA chip prepared in FIG. In the fourth probe in the lower row, strong fluorescence can be identified.

FIG. 13 is an experimental result of hybridization with a target DNA of Listeria monocytogenes on the gel-based DNA chip prepared in FIG. 3. In the fifth probe in the lower row, strong fluorescence can be identified.

FIG. 14 shows an oligo DNA chip in which 10 identification probes for detecting gastrointestinal infections on a gel-based DNA chip were planted 5 × 5.

The present invention is one of the most common gastrointestinal infectious agents including Vibrio cholerae, which causes cholera, Salmonella typhimurium, which causes typhoid fever, Shigella dysenteriae, S. sonnei, and S. flexneri . To detect the extracellular bacteria of the genes for the detection of extracellular bacteria such as Escherichia coli O-157, the causative agent of diarrheal disease, Vibrio parahaemolyticus , which is caused by enteritis vibrio, Staphylococcus aureus , which causes bacterial food poisoning, Campylobacter jejuni, Yersinia enterocolitica, Pseudomonas aeruginosa, and Listeria monocytogenes . exotoxin production genes, lipopolysaccharide (LPS) production genes that make up the outer membrane of bacteria, various pathogenic genes and antibiotic resistance genes that cause disease when infected by humans Analyzed Results Designed a probe around, and subsequently detecting a fire extinguisher pathogen DNA chips fixing the probe in the production, and rapid time to target from the digestive pathogen (target DNA) is obtained to perform a hybrid (hybridization) reaction in the production of DNA chips. According to the present invention, various types of digestive infection bacteria can be diagnosed at high speed with one DNA chip.

More specifically, five kinds of toxins such as Vibrio parahaemolyticus, Escherichia coli O-157, Shigella dysenteriae, Salmonella typhimurium , Staphylococcus aureus , Campylobacter jejuni, Yersinia enterocolitica, Pseudomonas aeruginosa, and Listeria monocytogene species DNA chip for high-speed diagnosis of digestive tract infection bacteria that can be detected in one chip, probe for identification, and polymerase chain reaction amplification product to be used as target DNA of chip A specific oligonucleotide primer is provided for obtaining.

The probe of the present invention and the DNA chip using the same are DNA chips for high-speed diagnosis of the digestive tract infection bacteria, and are first detected as oligo chips for clinical diagnosis to detect the digestive tract infection bacteria in a short time to prevent the abuse of antibiotics, It can help to relieve pain and make quick prescriptions.It can also be used for food test, restaurant hygiene test, imported agricultural, livestock, marine product quarantine, and livestock diagnosis that require large amount of sample test besides clinical test. Can be.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1 Identification of Probe Design for Detecting 10 Gastrointestinal Infections

In order to detect 10 selected digestive infections, identification probes are required to be planted in DNA chips. The identification probe was attached to a gel prepared by adding glycidyl methacrylate to be planted by attaching an amine-linker at the 5 'end of the oligonucleotide probe to bind the oligonucleotide probe. Nucleotide probes were synthesized in a DNA synthesizer. The synthesis of this amine oligonucleotide probe was produced and supplied by Bioneer. The nucleotide sequence of the amine oligonucleotide probe used is as follows.

Probe Identification for Gastrointestinal Infections No. Target bacteria Identification probe (5'amine) Length One Listeria monocytogenes aagacgagcaaaacagcaacgatag ttttgattatcggatccaaccactaatca (SEQ ID NO: 1) 54mer 2 Staphylococcus aureus atgttttcaaaagcggttgtggatt cgataatgatagcaacaaaggc (SEQ ID NO: 2) 47mer 3 E.coli O-157 ttcgataggctggggaaactaggta aattaattccacgccaacca (SEQ ID NO: 3) 45mer 4 Vibrio cholerae ttttggggtgcttgatgaacaatta catcgtaataggggctacagaga (SEQ ID NO: 4) 48mer 5 Pseudomonas aeruginosa ggctatgtgttcgtcggctacc acggcaccttcctcgaagcggcgcaaag (SEQ ID NO: 5) 50mer 6 Shigella flexneri gtggcaaggttcttt cttaaggctcctttttggtcgctaa tccctgaatg (SEQ ID NO: 6) 50mer 7 Camphylobacter jejuni agttttggtttt ggtggcaaaaaggaaaaagctgtag aagaagttgctgatac (SEQ ID NO: 7) 53mer 8 Yersinia enterocolitica tattcctgctcagtgtgaactgctg cgctgttcgttgcaggaaggtatggat (SEQ ID NO: 8) 52mer 9 Vibrio parahemolyticus ccgtacaaagatgtttat ggtcaatcagtattcacaacgtcagg tactaaatgg (SEQ ID NO: 9) 54mer 10 Salmonella typhimurium aagttcgaagaacactgcctctcc tacagtgggattagacagcgccattgt (SEQ ID NO: 10) 51mer

Example 2 Preparation of Polyacrylamide Gel with Added Glycidyl Methacrylate

Acrylamide solution was made to a final concentration of 8%. The ratio of acrylamide and N, N'-methylene bis acrylamide was set to 99: 1, and glycidyl methacrylate was mixed with a mixer by adding 0.1% to acrylamide solution. In the polyacrylamide gel reaction, N, N, N ', N-tetramethylethylenediamine was polymerized by adding 1/100 and 10% ammonium persulfate by 1/20.

Example 3: Gel Immobilization on Glass Slide Surface

Place the microscope slide (76 x 26 mm) in the wash solution overnight and then rinse with distilled water. The slide surface was then treated with Binding solution (5 microliters, 3- (Trimethoxysilyl) propyl methacrylate; 5 microliters, Acetic acid; 990 microliters, Ethylalcohol) and dried. And the glass plate on the other side was treated with coating solution and dried for 2 hours. 30 ul of the polyacrylamide gel solution synthesized on the surface of the glass plate was added, and the slide treated with the glass coat solution was covered thereon and reacted for 30 minutes. The covered slides were separated to form polyacrylamide gels with glycidyl methacrylate added to a thickness of 20 micrometers or less.

Example 4 Immobilization of Amine Probes on Gels

In order to fix the probe on the gel, the amine probe solution was mixed 1: 1 with 0.1 M sodium borate pH 9.3 and spotted on the slide gel surface with a diameter of 2 × 2 mm to react at room temperature for 30 minutes. In this way, the amine group of the probe and the glycidyl group of glycidyl methacrylate on the gel surface react to fix the probe on the gel with a stable bond.

Of the 10 identification probes for detecting gastrointestinal infections on gel-based DNA chips, the five probes in the top row are five toxins among the causative agents of diarrheal diseases ( Vibrio parahaemolyticus, Vibrio cholerae, E. coli O- 157, Shigella flexneri and Salmonella typhimurium ), and below are probes for five food-derived toxins ( Staphylococcus aureus, Campylobacter jejuni, Yersinia enterocolitica , Pseudomonas aeruginosa and Listeria monocytogenes ) among the causative agents of diarrheal diseases. I did it.

Example 5 primer design for preparing target DNA of digestive infection bacterial DNA chip

After selecting 10 representative gastrointestinal infectious agents that cause diarrhea diseases, exotoxin production genes among these genes, lipopolysaccharide (LPS) production genes constituting the outer membrane of bacteria, By using a primer design program called 'Primer 3', oligonucleotides that can diagnose digestive diseases can be diagnosed based on the analysis of the pathogenic genes and antibiotic resistance genes that act to cause illness when a person is infected. Design was made (see Table 2).

Example 5 Preparation of Target DNA of Gastrointestinal Infectious Bacteria Detection DNA Chip

After selecting 10 representative digestive organisms causing diarrhea diseases, genomic DNA was extracted from these cultured bacteria. 50 ng to 100 ng of each group were used as templates, and 20 pmoles of specific primers of each strain were added thereto, followed by PCR using AccuPower PCR PreMix (Bioneer corp.) Kit. At this time, the amplification conditions were performed for 5 minutes at 94 ° C. for initial denaturation, followed by denaturation at 94 ° C. for 1 minute, annealing at 55 ° C. for 1 minute, and extension at 72 ° C. for 1 minute. The last extension was carried out for 5 minutes. After completion of the reaction, the PCR amplification product was subjected to electrophoresis on 1.8% agarose gel and confirmed by staining with ethidium bromide (EtBr). The results are shown in Figure 1 and the size of the amplification products are shown in Table 1. In FIG. 1, lane 1 is a 100 bp marker DNA size marker, lane 2 is amplified product of 104 bp amplified from the genomic gene of Listeria monocytogenes (KCTC3569), lane 3 is amplified from the genomic gene of Staphylococcus aureus (KCTC1928) 99 bp amplification product, lane 4 is amplified product of 98 bp amplified from the genomic gene of E. coli O-157 (KCTC2419), lane 5 is 109 bp amplified from the genomic gene of Vibrio cholerae (KCTC2715) Amplification product, lane 6 is a 118 bp amplification product amplified from the genomic gene of Pseudomonas aeruginosa (KCTC1637), lane 7 is a 95 bp amplification product amplified from the genomic gene of Shigella flexneri (KCTC2008), lane 8 is Campylobacter 112 bp amplified product amplified from genomic gene of jejuni (KCTC0000), lane 9 is 129 bp amplified product amplified from genomic gene of Yersinia enterocolitica (ATCC9610), lane 10 is Vibrio parahaemolyti 95 bp amplified product amplified from the genomic gene of cus (ATCC17802), lane 11 shows a 117 bp amplified product amplified from the genomic gene of Salmonella typhimurium ( KCTC2057). After the PCR product thus identified was purified, asymmetric PCR was performed using this as a template. In this method, a primer having a base sequence complementary to that of a probe among a pair of primers designed for the production of a target DNA is added to the probe 100 times higher (primer 1; 20 pmoles, primer 2; 200 fmoles) than the other primer. It is possible to make more of the single-chain amplification products that can be combined with (Fig. 2). Each lane in FIG. 2 is the same as FIG.

PCR primers for diagnosing digestive infections No. Target strain name Primer for PCR (5 '-> 3') Amplification Product Size One Listeria monocytogene aagacgagcaaaacagcaacgatag (SEQ ID NO: 11) attgctcgtgtcagttctgggagta (SEQ ID NO: 12) 104 bp 2 Staphylococcus aureus atgttttcaaaagcggttgtggatt (SEQ ID NO: 13) accgataacgtgaacgttcttacca (SEQ ID NO: 14) 99 bp 3 E.coli O-157 cgatgccaatgtactcggaaaaata (SEQ ID NO: 15) ttcgataggctggggaaactaggta (SEQ ID NO: 16) 98 bp 4 Vibrio cholerae ttttggggtgcttgatgaacaatta (SEQ ID NO: 17) gaaacctgccaatccataaccatct (SEQ ID NO: 18) 109 bp 5 Pseudomonas aeruginosa ggctatgtgttcgtcggctacc (SEQ ID NO: 19) gatatagaaaccgcgccagatcg (SEQ ID NO: 20) 118 bp 6 Shigella flexneri tcaagcattactggagcagtcacac (SEQ ID NO: 21) ctaaggctcctttttggtcgctaa (SEQ ID NO: 22) 95 bp 7 Camphylobacter jejuni ggtggcaaaaaggaaaaagctgtag (SEQ ID NO: 23) cgcaaccattttcatctaacaaagc (SEQ ID NO: 24) 112 bp 8 Yersinia enterocolitica tattcctgctcagtgtgaactgctg (SEQ ID NO: 25) agaagttaatcctgcccagatcgtc (SEQ ID NO: 26) 129 bp 9 Vibrio parahemolyticus ggtcaatcagtattcacaacgtcagg (SEQ ID NO: 27) gacaccgctgccattgtatagtctt (SEQ ID NO: 28) 95 bp 10 Salmonella typhimurium aagttcgaagaacactgcctctcct (SEQ ID NO: 29) ccacctcatcgatggaatttaaaca (SEQ ID NO: 30) 117 bp

Example 6 Hybridization, Washing, and Search

After immersing the gel slide in which the probe is immobilized in sterile distilled water and washing for 3 minutes, in order to further suppress the epoxidation reaction by the amine group, it was reacted for 30 minutes by adding 1M ethanolamine, a blocking solution, and then washed three times with water for 3 minutes. It was. The slide was immersed in a 1 x Hybridization solution (1M NaCl, 1mM EDTA, 1% Tween 20, 5mM Sodium phosphate, pH 7.0) for 10 minutes, and then mixed with a mixed solution and 10 target DNA (Asymmetric PCR). Using a single chain PCR solution prepared by using a 95 ℃, heat treatment for 5 minutes) to 20: 1 was mixed on the surface of the gel and covered with a cover glass to perform a hybrid reaction for 1 hour each.

After the reaction, wash each time with 1 x hybrid solution and 0.1 x hybrid solution, and then prepare an ethidium bromide solution with a final concentration of 10 ug / ml in 0.1 x hybridization buffer and dye the solution for 10 minutes by adding it to the slide. And washed several times with water. The washed slides were dried at room temperature and then searched and analyzed using a scanner (GenePix 400A Microarray scanner, Axon instrument, U.S.A). The analysis results are shown in FIGS. 4 to 13.

According to the probe of the present invention, and a DNA chip and a detection method using the same, the microorganisms causing digestive diseases in all environmental fields such as humans, animals, meat, vegetables, and beverages can be detected quickly and accurately from a very small amount of samples. It can be useful for hospital, import / export quarantine, water quality inspection, hygiene evaluation of food hospitality, quarantine and food inspection of farm, livestock, marine products or food, restaurant sanitation, import farm, livestock, marine product quarantine and livestock diagnosis. In addition, the time required for the existing test can be shortened to about 3 hours, which can be useful for prescription and contribute to the improvement of public health. In addition, the existing diagnostic DNA chip has a high retrieval equipment and manufacturing cost, it has been difficult to commercialize yet, and because of the high cost due to the introduction of foreign products, the present invention can lower the production cost through mass production technology. By reducing the economic burden, the diagnosis of genetic diseases and the detection of pathogen infections will be more common.

Claims (4)

delete In the method of detecting digestive infection bacteria, Derived from digestive infections, SEQ ID NO: 1 for detecting Listeria monocytogenes , SEQ ID NO: 2 for detecting Staphylococcus aureus , SEQ ID NO: 3 for detecting E. coli O-157 , SEQ ID NO: 4 for detecting Vibrio cholerae , SEQ ID NO: 5 for detecting Pseudomonas aeruginosa , and Shigella dysenteriae Using a probe selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, for detecting Campylobacter jejuni , SEQ ID NO: 8 for detecting Yersinia enterocolitica , SEQ ID NO: 9 for detecting Vibrio parahaemolyticus , and SEQ ID NO: 10 for detecting Salmonella typhimurium , How to detect digestive infections. delete Derived from digestive infections, Detection of digestive organs Listeria monocytogenes, Staphylococcus aureus, E. coli O-157, Vibrio cholerae, Pseudomonas aeruginosa, Shigella dysenteriae, S. sonnei, S. flexneri, Campylobacter jejuni, Yersinia enterocolitica, Vibrio parahaella , DNA chip to which at least one probe selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10 is immobilized.