KR20040083917A - Method for rapidly detecting pathogenic microorganism - Google Patents

Abstract

PURPOSE: A method for rapidly detecting a pathogenic microorganism is provided, thereby rapidly and accurately detecting pathogenic microorganisms, and detecting low concentration of pathogenic microorganisms. Therefore, disease, such as food poisoning, caused by pathogenic microorganisms can be prevented. CONSTITUTION: The method for rapidly detecting pathogenic microorganisms comprises the steps of: (a) pulverizing a sample, adding the pulverized sample into a medium containing 0.1 to 5% (w/v) of glucose, and culturing the sample at 150 to 200 rpm to obtain a pathogenic microorganism in logarithm growth phase; (b) collecting the cultured cell pellet from the cultured medium and purifying DNA of the cultured cells by alkaline method; and (c) PCR amplifying the purified DNA as a template using a primer set, and verifying the amplification, wherein the pathogenic microorganism is any one selected from Campylobacter jejuni, E. coli 0157:H7, Bacillus cereus, Listeria monocytogenes, Yersinia enterocolitica, Salmonella sp., Staphylococcus aureus, Shigella spp., and Vibrio parahaemolyticus; and the primer set has the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2 for Campylobacter jejuni, SEQ ID NO:3 and SEQ ID NO:4 for E. coli 0157:H7, SEQ ID NO:5 and SEQ ID NO:6 for Bacillus cereus, SEQ ID NO:7 and SEQ ID NO:8 for Listeria monocytogenes, SEQ ID NO:9 and SEQ ID NO:10 for Yersinia enterocolitica, SEQ ID NO:11 and SEQ ID NO:12 for Salmonella sp., SEQ ID NO:13 and SEQ ID NO:14 for Staphylococcus aureus, SEQ ID NO:15 and SEQ ID NO:16 for Shigella spp., or SEQ ID NO:17 and SEQ ID NO:18 for Vibrio parahaemolyticus.

Description

Rapid detection of pathogenic microorganisms {METHOD FOR RAPIDLY DETECTING PATHOGENIC MICROORGANISM}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a method for rapidly detecting pathogenic microorganisms, and more particularly, to a method capable of quickly and accurately detecting whether pathogenic microorganisms are contaminated and thus preventing disease occurrence due to pathogenic microorganisms.

[Private Technology]

Despite the development of science and technology, it is still difficult to completely prevent food poisoning. Food poisoning is food poisoning caused by natural poisoning (blowfish poison, mushroom poison, etc.) based on the cause; Food poisoning due to food decay; And food poisoning by incorporation or reproduction of bacteria in food. Among these food poisonings, food poisoning caused by natural poisoning can be easily prevented by avoiding the ingestion of the foods that cause the food poisoning. Food corruption is easily detected by changes in the appearance, smell, etc. of food poisonings. Relatively easy. However, in the case of food poisoning caused by bacteria, that is, bacterial food poisoning, food poisoning is most difficult to prevent because it is generally not observed by the naked eye and is difficult to detect by professional methods. can do.

Recently, as food facilities, such as restaurants, order delivery, food centers, and fast food restaurants, are being used in large quantities, and many consumers use them, there is a risk that many people will get food poisoning in a short time when bacterial food poisoning occurs in these facilities. And because bacterial food poisoning is severe, such as severe vomiting, diarrhea, abdominal pain, and high fever, the occurrence of group food poisoning is a significant problem for both food suppliers and consumers. In addition, these food poisoning causative bacteria produce toxic substances such as endotoxins in the body of infected people, so the treatment should be performed after infection, and in many cases, the treatment is difficult. Especially, very serious such as hemorrhagic enteritis such as pathogenic E. coli O157: H7 Symptoms may be severe and lead to death in severe cases. Therefore, it can be said that there is an urgent need for early detection and prevention of the causative agent of pathogenic food poisoning.

As a method of detecting and confirming the causative agent of bacterial food poisoning, the collected sample is cultured using a selective medium, the colonies formed on the medium are visually observed, or other detection and confirmation means are used. In the test method using a polymerase chain reaction (PCR) has been developed and used.

Polymerase Chain Reaction (PCR) is a simple and convenient way to exponentially amplify DNA sequences of specific regions with very small amounts of DNA. Two different primers in the opposite direction and Taq DNA polymerase, which are functionally stable at high temperatures, are used in three different temperature cycles: denaturation step, annealing step, and extension step. Repeatedly amplify specific DNA. The detection method of pathogenic microorganisms using PCR is amplified by PCR reaction using primers that recognize specific genes of microorganisms to be detected and confirmed by electrophoresis, etc. Pathogenic microorganisms can also be detected.

As for the PCR method, the step of confirmatory experiments is omitted compared to the culture method using a medium, which is a general test method (more than 96 hours), or the immunoenzyme assay (Enzyme Immuno Assay: more than 48 hours), which measures antibodies to peptides. Accuracy and speed (24 hours), but it is a problem when the concentration of genes is very low. If the amount of bacterial food poisoning bacteria infected with food is small, the process of growing the bacteria should be accompanied. In this case, the bacterial growth time is 16 to 20 hours, which accounts for almost 85% of the pathogenic microorganism detection process, which is the biggest obstacle to rapid pathogenic microorganism detection.

Therefore, in addition to the development of a PCR method for detecting a variety of pathogenic microorganisms, it is also essential to develop a method capable of accurately obtaining a large amount of bacteria in a short time.

In order to solve the problems of the prior art, an object of the present invention is to provide a method capable of proliferating a large amount of pathogenic microorganisms causing food poisoning in a short time.

It is also an object of the present invention to provide a method capable of quickly and accurately detecting pathogenic microorganisms causing food poisoning.

1 is a graph showing the growth rate measured by the stirring speed of Salmonella typhimurium,

2 is a growth curve showing the growth of Salmonella typhimurium according to glucose concentration,

Figure 3 is an electrophoresis picture showing the PCR results when using different DNA purification method for the same microorganism,

Figure 4 is an electrophoresis picture confirming the detection limit of microorganisms according to the DNA purification method,

Figure 5 is an electrophoresis picture showing the results of PCR after recovering the DNA of the pathogenic microorganisms by the conventional heat extraction method,

6 is a graph showing bacterial growth when cultured at 180 rpm in a medium containing glucose,

Figure 7 is purified by DNA after heat extraction or alkaline method from the cells obtained for each incubation time, PCR

8 is a result of extracting DNA from the microorganism of 1 to 105 CFU / ml by the alkaline method and then PCR 30 times or 60 times as a template.

The present invention to achieve the above object

(a) Pathogenic microorganism detection samples are crushed and added to a culture medium containing 0.1 to 5 (w / v)% glucose, and cultured at a stirring rate of 150 to 200 rpm to be pathogenic in the initial log growth phase or log growth phase. Obtaining a microbial culture;

(b) collecting the cells from the pathogenic microbial culture and purifying DNA by alkaline method; And

(c) performing a PCR with a primer set for pathogenic microorganism specific gene amplification using the purified DNA as a template, and providing a method for detecting pathogenic microorganisms, the method comprising amplifying amplification.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention are studying a method to more accurately and quickly examine the contamination of pathogenic microorganisms, and the method of separating and propagating pathogenic microorganisms from pathogenic microbial contaminants most time for pathogenic microbial testing, and from the multiplying pathogenic microorganisms. The step of separating the DNA spends a lot of time for the pathogenic microbial test, and confirmed that it is an important part of the accurate test to solve this problem, media composition, culture conditions and DNA separation method was established respectively.

The pathogenic microorganisms of the present invention are common pathogens that cause harmful harm to animals, and are preferably microorganisms causing food poisoning. Representative microorganisms causing food poisoning are Campylobacter jejuni , E. coli O157: H7 , Bacillus cereus , Listeriamonocytogenes , Yersinian entero Yersinia enterocolitica , Salmonella sp. , Staphylococcus aureus , Shigella spp. , And Vibrio parahaemolyticus .

In order to proliferate a large amount of pathogenic microorganisms in a fast time, and then to provide a favorable medium composition and culture conditions in order to perform the DNA purification process smoothly.

The composition of the medium comprises a carbon source commonly used by pathogenic microorganisms, preferably Luria-Bertani medium (tryptone 1 (w / v)%, yeast extract 0.5 (w / v)%, sodium chloride 1) (w / v)%) to glucose. The glucose is an essential nutrient for the growth of pathogenic microorganisms and is easy to metabolize and economical at low cost. Glucose may be included in the LB medium at 0.1 to 5 (w / v)%, preferably 0.5 to 3 (w / v)%. If the glucose content is less than 0.1 (w / v)%, it may be difficult to promote the growth of pathogenic microorganisms, and if it exceeds 5 (w / v)%, the growth rate of pathogenic microorganisms may be inefficient compared to the glucose content.

Culture conditions are to increase the agitation speed to increase the growth rate of the microorganism in a short time to improve the air contact rate, or to supply oxygen, preferred culture conditions are the initial logarithm to 35 to 38 ℃ at a stirring speed of 150 to 200 rpm It is cultured in the growth phase or logarithmic growth phase. Stirring speed is a factor that determines the oxygen supply rate, the growth of pathogenic microorganisms may be lowered if the stirring speed is out of the range. The culture temperature is a factor that affects the growth of pathogenic microorganisms, and the growth of pathogenic microorganisms may be delayed or lowered when it is out of the temperature range, and the most preferable culture temperature is 37 ° C.

Pathogenic microorganisms show differences in DNA recovery according to growth stages. Microorganisms in the early log phase are easy to lysis afterwards, and microorganisms in the log phase are active in DNA replication, but it is difficult to recover a large amount of DNA due to the low degree of microbial growth before the early log growth period. Microorganisms that have reached a stagnant or stagnant period are also unable to recover large amounts of DNA due to natural death. Therefore, it is preferable to culture the microorganisms in the initial log phase state or log phase state.

The pathogenic microorganism detection sample can be cultured by adding 0.001 to 50 g per 100 mL of culture medium, the culture medium and the culture conditions are carried out as mentioned above to prepare a pathogenic microbial culture. The cells are then purified from the pathogenic microbial culture to purify the DNA.

DNA purification from pathogenic microorganisms is a common alkaline or ultrasonic grinding method. Alkaline method has a significantly higher DNA recovery compared to the commonly used heating method (Boiling method), and is easy to use because the DNA separation kit or column using the alkaline method is commercialized. Ultrasonic pulverization method can be usually carried out by the method of grinding the cells, there is an advantage that can be used easily and simply without the use of other extraction solvent.

In the purification of DNA, the conventional method is hot water extraction of 1 ml of microbial culture. However, the present invention may further comprise the step of concentrating the microbial culture in 0.01 to 0.1 by volume in order to increase the DNA recovery. The concentration method is to use the cells obtained by centrifugation to dissolve the cells in a solvent of 1/100 to 1/10 volume of the microbial culture.

The purified DNA is subjected to PCR with a primer set for pathogenic microorganism specific gene amplification as a template and confirms amplification.

PCR can be carried out by a conventional method, and the primer set for amplifying a pathogenic microorganism specific gene is a conventional pair of primers capable of detecting a pathogenic microorganism specific gene. As an example of a primer set for amplifying a pathogenic microorganism specific gene, a base pair shown in SEQ ID NOs: 3 and 4, a pair of primers for amplifying a gene of Campylobacter jejuni having the sequences shown in SEQ ID NOs: 1 and 2 A pair of primers for gene amplification of E. coli O157: H7 having E. coli O157: H7 , a pair of primers for gene amplification of Bacillus cereus having the nucleotide sequences shown in SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8 Primer primer pair for gene amplification of Listeria monocytogenes having the nucleotide sequence shown in Fig. 9, and primer pair for gene amplification of Yersinia enterocolitica having the nucleotide sequence shown in SEQ ID NOs: 9 and 10, SEQ ID NO: A primer pair for amplifying a gene of Salmonella sp. Having the nucleotide sequences shown in 11 and 12, the nucleotide sequences shown in SEQ ID NOs: 13 and 14 Primer pairs for gene amplification of Staphylococcus aureus having a gene, primer pairs for gene amplification of Shigella spp. , Having the nucleotide sequences shown in SEQ ID NOs: 15 and 16, and SEQ ID NOs: 17 and 18. It is a primer pair for gene amplification of Vibrio parahaemolyticus having the nucleotide sequence shown.

The primer pairs of SEQ ID NOS: 1 and 2 can specifically detect Campylobacter jejuni , and the size of the amplification product is 127 bp.

The primer pair of SEQ ID NO: 3 and 4 can specifically detect E. coli O157: H7, the size of the amplification product is 208 bp.

The primer pairs of SEQ ID NOs: 5 and 6 can specifically detect Bacillus cereus, and the size of the amplification product is 305 bp.

The primer pairs of SEQ ID NO: 7 and 8 can specifically detect monocytic listeria, and the size of the amplification product is 454 bp.

The primer pairs of SEQ ID NOs: 9 and 10 can specifically detect Yersinia enterocytica, and the size of the amplification product is 551 bp.

The primer pairs of SEQ ID NOs: 11 and 12 can specifically detect Salmonella, and the size of the amplification product is 678 bp.

The primer pairs of SEQ ID NOs: 13 and 14 can specifically detect Staphylococcus oreus, and the size of the amplification product is 264 bp.

The primer pair of SEQ ID NO: 15 and 16 can specifically detect Shigella, the size of the amplification product is 959 bp.

The primer pairs of SEQ ID NOs: 17 and 18 can specifically detect enteritis vibrio, and the size of the amplification product is 375 bp.

PCR reaction can be carried out using one PCR primer or multiple polymerase chain reaction (M-PCR) using a primer set including two or more PCR primers to quickly and accurately detect several pathogenic microorganisms at once. can do.

The pathogenic microorganism detection method of the present invention can prevent disease occurrence caused by food contaminated with pathogenic microorganisms in advance by detecting whether pathogenic microorganisms are contaminated more quickly and accurately than conventional methods, and can detect even low concentration microorganisms. Detection efficiency is very high.

Hereinafter, in order to more clearly show the efficiency of the pathogenic microorganism detection method of the present invention, it will be described by comparing the difference between the known detection method and the test time, for example. Table 1 below shows the difference between the known method and the method for detecting pathogenic microorganisms of the present invention, and Table 2 shows the time required for the audit, and the method of the present invention shortens the test time by about one-half compared to the conventional method. It can be made.

Notice Method The present invention Sample + LB medium -Homogenized sample by crushing + LB medium containing glucose Incubate with stirring at 37 ° C., 16-22 hours, up to 80 rpm 37 ° C., 4-6 hours, 180 rpm stirred culture -1 ml of culture solution-12000 rpm, centrifugation for 2 minutes-Pellet 200 ul dissolved in sterile water and mixed-100 ℃, heat extraction for 10 minutes-12000 rpm, centrifugation for 10 minutes-supernatant 10 ul -Centrifuge 100 ml of the culture solution at 10000 rpm for 2 minutes-Dissolve the pellet in 200 ul sterile water and mix-Obtain DNA dissolved by 200 ul by alkaline method (AccuPrep ^™ GMO DNA Extraction kit or Quazen kit) -PCR reaction -10 ul DNA PCR -Electrophoresis on 2% agarose gel -Electrophoresis on 2% agarose gel

division Notice The present invention Specimen (microbial culture) manufacturing 1290 minutes 450 minutes DNA extraction 17.5 minutes 30 minutes PCR analysis 123 minutes 123 minutes Electrophoresis and determination 103 minutes 103 minutes Sum 24 to 26 hours 12 hours

In order to prevent the occurrence of disease caused by pathogenic microorganisms, it is very important to confirm whether the pathogenic microorganisms are contaminated quickly. Therefore, the existing inspection time of the present invention is significantly reduced to obtain accurate results in a short time. In addition, the pathogenic microorganism detection method of the present invention can easily detect low concentration microorganisms.

Concentration with a generally sikjungdokgyun may cause food poisoning in animals is one different depending on the strain, and about 10 ⁴ ~ 10 ⁶ cfu / ml level, in the case of Bacillus cereus from about 10 ⁶ cfu / ml, Staphylococcus brother below mouse In the case of 10 ⁴ cfu / ml, such pathogenic microorganisms should not be detected in food.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example 1

1-1. Growth rate of pathogenic microorganism according to agitation speed

The growth of the Salmonella typhimurium ATCC14023 was measured according to the stirring speed. Salmonella typhimurium ATCC14023 was inoculated in LB medium, and a portion of the culture was taken every hour while stirring at 180 rpm at 37 ° C. to measure O.D at 600 nm, and bacterial growth was measured while stirring at 80 rpm as a control.

1 is a graph showing the growth rate according to the stirring speed of Salmonella typhimurium, ◆ is a bacterial growth curve at 180 rpm, ■ is a bacterial growth curve at 80 rpm. Experimental group that increased the air contact rate at agitation speed of 180 rpm can be confirmed that the logarithmic growth phase is started faster than the control group, so that a large amount of cells can be obtained even with a short incubation time.

1-2. Verification of growth rate of pathogenic microorganism according to culture medium

Salmonella typhimurium ATCC14023 was inoculated in a medium containing glucose at 0, 1, 3 and 5 w / v%, respectively, and grown at 37 ° C. to confirm the growth.

Figure 2 is a growth curve showing the growth of Salmonella typhimurium according to glucose concentration, ◆ is a control culture in LB medium, ■ is an experimental group incubated in LB medium containing 1 w / v% glucose, (Triangle | delta) is an experimental group incubated in the LB medium containing 3 w / v% glucose, and x is an experimental group incubated in the LB medium containing 5 w / v% glucose.

When cultured in a medium containing glucose, the microorganism quickly escaped the lag phase and entered the logarithmic growth phase in 2 hours of cultivation, but in the glucose-free medium, the induction phase slowly entered the logarithmic growth phase for 4 hours. Comparing the growth rate of microorganisms in the logarithmic growth period, the most active group in the glucose 1 and 3 w / v% addition group.

Therefore, by culturing pathogenic microorganisms in a culture medium containing glucose, it is possible to shorten the induction period of the microorganisms and at the same time induce rapid cell growth in the logarithmic growth phase.

1-3. Verification of microbial detection effect by concentration

The conventional method recovers DNA by heat extraction of 1 ml of pathogenic microbial culture, and PCR is performed using the template. In this experiment, while reducing the incubation time of the microorganisms, the cultured microbial culture solution was concentrated to 100 to 500 times in order to increase the accuracy of the test, it was confirmed by the same method.

As a result, when the microbial culture was concentrated and used as a sample 100 to 500 times, the microorganisms could be detected up to 104 CFU / ml. On the other hand, when 1 ml of microbial culture was used, only about 105 to 106 CFU / ml of microorganisms could be detected.

Therefore, the concentration method can be used to shorten the microbial incubation time and improve the microbial detection limit.

1-4. Verification of microbial detection effect according to DNA purification method

Conventional DNA purification method is a heat extraction (Boiling) method. This is a method of destroying the bacteria by applying heat to the enriched culture, wherein the extracted gene is collected. This method may not be 100% lysis (lysis) cells, in the present invention was confirmed the DNA recovery rate using the alkaline method or ultrasonic crushing method.

Salmonella typhimurium ATCC14023 at 104 to 106 CFU / ml each inoculated in LB medium containing 1 g / 1% of the glocos, incubated at 37 ℃ and 100 ml in 0.91 (107 CFU / ml) O.D600nm Centrifugation gave cells.

Cells were hemolyzed by heat extraction, alkaline method and ultrasonic crushing method, respectively, and DNA was isolated, and each DNA was PCR-templateed to confirm PCR amplification.

Thermal Extraction Method: Centrifuge 100 ml of the culture solution at a speed of 12,000 rpm for 5 minutes, remove the supernatant, suspend the collected cells in 200 µl of sterile distilled water, and heat the mixture at 100 ° C. for 10 minutes. After the treatment, centrifuged at 12,000 rpm for 10 minutes, and then the supernatant is collected as a heat extract sample.

Alkaline Method: Add 1 mL of the cell Buffer Buffer and Proteinase K, and then add the Binding Buffer to the column and use it as a heat extract sample.

The detailed process is as follows.

1. Take 1 ml of the prepared sample.

2. Into a 5 ml E-tube, add 400 ul of plant hemolysis buffer (PL). Add 20 ul (20 mg / ml) proteinase K and mix, and then react for 10 minutes while mixing once at 60 ℃.

3. Centrifuge the reaction tube at 12,000 rpm for 5 minutes and transfer the supernatant to a new tube.

4. Add 100 ul of isopropanol, mix lightly for 5 seconds, mix, and spin down for 10 seconds to drop the solution on the lid.

5. Transfer the sample to the combined column where the sample is bound to the 2 ml collection tube so that the lid does not get wet.

6. Close the lid and centrifuge for 1 minute at 8,000 rpm. If after centrifugation the solution does not completely exit the column, once again centrifuge for 1 minute at 12,000 rpm to empty the column completely.

7. After centrifugation, transfer the binding column to a 2 ml collection tube. Add 500 ul of Wash Buffer 1 (W1) to prevent the edges from getting wet, close the lid and centrifuge for 1 minute at 8,000 rpm.

8. After centrifugation, transfer the binding column to a new 2 ml collection tube. Add 500 ul of wash buffer 2 (W2) so that the edge of the tube does not get wet, close the lid and centrifuge for 1 minute at 8,000 rpm.

9. Centrifuge once more for 1 minute at 13,000 rpm to remove ethanol completely. At this time, check that there are no drops of water on the bottom of the coupling column. If wash buffer 2 remains in the column, this can be a problem for subsequent experiments.

10. Transfer the identified binding column to a 1.5 ml collection cube, add 200 ul of elution buffer, and let it stand at room temperature for about 1 minute so that the column is sufficiently wetted.

11. Centrifuge at 8,000 rpm for 1 minute to elute.

Ultrasonic pulverization method-The ultrasonic pulverization using a JAC ultrasonic (ultrasonic), 60 ℃, 10 minutes.

PCR reaction solution (50 μl total reaction solution)

10 μl DNA (1-50ng / μl)

5 x 10 μl reaction buffer

5 μl of primer (SEQ ID NO: 7/8) mixture

1 μl of Taq DNA polymerase (1U / μl)

Residual sterile distilled water (50 μl total)

The PCR reaction was performed for 2 minutes at initial denaturation at 94 ° C, followed by denaturation (30 seconds at 94 ° C), annealing (30 seconds at 60 ° C) and extension (30 seconds at 72 ° C). ) Was performed a total of 30 times.

After completion of the reaction, PCR products were developed by electrophoresis on 2% agarose gel to confirm the formation of strain-specific gene amplification products.

Figure 3 is an electrophoresis picture showing the PCR results when using different DNA purification method for the same microorganism, lane 1 is a 100 bp size marker, lane 2 is a negative control group, lane 3 is recovered by ultrasonic grinding method PCR result using a DNA as a template, lane 4 is a PCR result using the DNA recovered by ultrasonic crushing method from a microbial culture diluted 10-fold, lane 5 is a PCR using the DNA recovered by heat extraction method as a template The result is lane 6 as a result of PCR using the DNA recovered by heat extraction method from the microbial culture diluted 10-fold, lane 7 is the PCR result as a template from the DNA recovered by alkaline method, lane 8 is 10 times PCR results obtained by using DNA recovered from the diluted microbial culture medium as a template.

In Figure 3, it can be seen that the alkaline recovery method and the ultrasonic pulverization method is higher in DNA recovery than the heat extraction method, especially when viewed in the thickness of the band it can be seen that the alkaline method is most effective for DNA recovery.

In addition, the microbial detection limit according to the DNA purification method was confirmed when different microbial bacteria.

Figure 4 is an electrophoretic picture confirming the detection limit of microorganisms according to the DNA purification method, DNA is recovered from the cells of 104 CFU / ml by heat extraction or alkaline method PCR picture as a template. Lanes 1 and 6 are 100 bp size markers, lane 2 is a positive control, lane 3 is a column of 1 ml culture cultured at 104 CFU / ml (OD 0.14) in LB medium containing 5 w / v% glucose. Lane 4 is an alkaline method of 1 ml of the culture medium incubated at 104 CFU / ml (OD 0.14) in LB medium containing 5 w / v% glucose, and lane 5 is 5 w / v% glucose. Concentrated 100 ml culture medium incubated in 104 CFU / ml (OD 0.14) in LB medium containing as it was carried out by the alkaline method.

In Figure 4, it can be seen that whether the PCR amplification, that is, whether the detection of microorganisms vary according to the DNA purification method, when the DNA purification by the alkaline method was able to detect the microorganisms at 104 CFU / ml. On the other hand, the heat extraction method was difficult to detect low concentration of microorganisms compared to the alkaline method.

Therefore, DNA purification in the detection of pathogenic microorganisms increases the sensitivity of microbial detection using alkaline methods.

Comparative example

Inoculate Salmonella typhimurium ATCC14023 into LB medium at 103, 104, 105, 106, 107, 108 and 109 CFU / ml, respectively, take 1 ml, transfer to a 1.5 ml centrifuge tube, centrifuge at 12,000 rpm for 5 minutes, and then remove the supernatant. The collected cells were suspended in 200 µl of sterile distilled water and then heated at 100 ° C. for 5 minutes. After the heat treatment was centrifuged for 10 minutes at 12,000 rpm and the supernatant was recovered to obtain a heat extract sample.

PCR reaction solution (50 μl total reaction solution)

10 μl heat extract sample

5 x 10 μl reaction buffer

5 μl of primer (SEQ ID NO: 1) mixture

1 μl of Taq DNA polymerase (1U / μl)

Residual sterile distilled water (50 μl total)

The PCR reaction was performed for 2 minutes at initial denaturation at 94 ° C, followed by denaturation (30 seconds at 94 ° C), annealing (30 seconds at 60 ° C) and extension (30 seconds at 72 ° C). ) Was performed a total of 30 times and 60 times, respectively.

After completion of the reaction, PCR products were developed by electrophoresis on 2% agarose gel to confirm the formation of strain-specific gene amplification products.

Figure 5 is an electrophoretic picture showing the results of PCR after recovering the DNA of the pathogenic microorganisms by the conventional heat extraction method, you can confirm the PCR product when the strain is used at 105 CFU / ml or more. Therefore, it is difficult to detect strains of less than 105 CFU / ml by conventional heat extraction methods.

Example 2

2-1. O.D was measured at 600 nm by inoculating Salmonella typhimurium ATCC14023 into LB medium containing 1 w / v% glucose, taking a portion of the culture every hour with stirring at 180 rpm at 37 ° C. In addition, bacterial growth was measured while Salmonella typhimurium ATCC14023 was cultured in the same conditions in LB medium.

Figure 6 shows the growth of bacteria when cultured at 180 rpm in a medium containing glucose, the graph on the left is the absorbance graph, the graph on the right is the viable cell count, ◆ is cultured in LB medium, ■ is glucose In cultured medium.

Pathogenic microorganisms grow faster in algebraic growth stages compared to conventional culture media and culture conditions when the agitation rate is rapidly cultured in a medium containing glucose, and then, the appropriate bacterium for detecting pathogenic microorganisms is 105 CFU / ml (OD 0.5). It can be obtained in 4 hours.

2-2. The microbial detection efficiency was confirmed when the DNA extraction method was improved from the conventional heat extraction to the alkaline method.

7 is purified by DNA extraction or heat extraction or alkaline method from the cells obtained by incubation time, lanes 1 and 13 are 100 bp size marker, lane 2 is a positive control group, lane 3 is a negative control group , Lane 4 is an alkaline method for the cells cultured for 0 hours, lane 5 is an alkaline method for the cells cultured for 2 hours, lane 6 is a 4 hour culture, purified DNA through the alkaline method, Lane 7 is culture 6 hours, alkaline, lane 8 culture 0 hours, heat extraction, lane 9 culture 2 hours, heat extraction, lane 10 culture 4 hours heat extraction, lane 11 culture 6 hours, heat Lane 12 was for 8 hours of cultivation and heat extraction.

In Figure 7 it was confirmed that the PCR product is increased in proportion to the incubation time when the DNA is purified by alkaline method, microorganisms are detected in all the incubation time, whereas the conventional heat extraction method microorganisms only in culture 2 hours and 6 hours It was difficult to detect and get stable results.

2-3.

When the DNA was purified by alkaline method, the detection limit of pathogenic microorganisms was measured.

8 is a result of extracting DNA from the microorganism of 1 to 105 CFU / ml by the alkaline method and then PCR 30 times or 60 times as a template. Lane 1 is a 100 bp size marker, lane 2 is a positive control, lane 3 is a negative control, lane 4 is a microbial sample of 1 CFU / ml, lane 5 is a microbial sample of 10 CFU / ml, and lane 6 is A microbial sample at 102 CFU / ml, lane 7 is a microbial sample at 103 CFU / ml, lane 8 is a microbial sample at 104 CFU / ml, and lane 9 is a microbial sample at 105 CFU / ml.

8, it can be seen that PCR can detect up to 10 ⁴ CFU / ml of microorganisms.

As mentioned above, the pathogenic microorganism detection method of the present invention can more quickly and accurately check whether the pathogenic microorganisms are contaminated than the conventional methods, thereby preventing the occurrence of diseases caused by food contaminated with the pathogenic microorganisms, and Microorganisms in concentration can also be detected and the detection efficiency is very high.

<110> SAMSUNG EVERLAND INC. <120> METHOD FOR RAPIDLY DETECTING PATHOGENIC MICROORGANISM <130> dpp030142 <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Campylobacter jejuni <400> 1 attttcgctg attttgattt tagg 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Camplylobacter jejuni <400> 2 tattgatatc tttggtggag gcga 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> PCR primer for E. coli O157: H7 <400> 3 gacagcagtt ataccactct gcaa 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> PCR Primer for E. coli O157: H7 <400> 4 gacgaaattc tctctgtatc tgcc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> PCR Primer Bacillus cereus <400> 5 gactacattc acgattacgc agaa 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Bacillus cereus <400> 6 ctatgctgac gagctacatc cata 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> PCR Primer Listeria monocytogenes <400> 7 ctggcacaaa attacttaca acga 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Listeria monocytogenes <400> 8 aactactgga gctgcttgtt tttc 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer Yersinia enterocolitca <400> 9 cctgtttatc aattgcgtct gtta 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Yersinia enterocolitica <400> 10 ataaatgggg ttcacttcac tcag 24 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> PCR primers for Salmonella sp. <400> 11 gaatcctcag tttttcaacg tttc 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> PCR primers for Samonella sp. <400> 12 tagccgtaac aaccaataca aatg 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Staphylococcus aureus <400> 13 aatttaacag ctaaagagtt tggt 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Staphylococcus aureus <400> 14 ttcattaaag aaaaagtgta cgag 24 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> PCR primer for Shigella sp. <400> 15 gacttgttaa gtataaagga aggc 24 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> PCR primer for Shigella sp. <400> 16 aagattttta ttatctgaat tggg 24 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Vibrio parahaemolyticus <400> 17 ctcatttgta ctgttgaacg ccta 24 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer for Vibrio parahaemolyticus <400> 18 aatagaaggc aaccagttgt tgat 24

Claims (5)

(a) Pathogenic microorganism detection samples are crushed and added to a culture medium containing 0.1 to 5 (w / v)% glucose, and cultured at a stirring rate of 150 to 200 rpm to be pathogenic in the initial log growth phase or log growth phase. Obtaining a microbial culture; (b) collecting the cells from the pathogenic microbial culture and purifying DNA by alkaline method; And (c) performing PCR with a primer set for pathogenic microorganism specific gene amplification using the purified DNA as a template and confirming amplification Pathogenic microorganism detection method comprising a. The method of claim 1, The pathogenic microorganism is Campylobacter jejuni (Campylobacter jejuni), Escherichia coli O157: H7 (E.coli O157: H7), Bacillus cereus (Bacillus cereus), Monocytic listeria (Listeria monocytogenes), Yersinia Enterocholicica (Yersinia enterocolitica), SalmonellaSalmonella sp.), Staphylococcus oreus (Staphylococcus aureus), Shigella (Shigella spp.) And Vibrio parahamoliticus (Vibrio parahaemolyticusAnd at least one microorganism selected from the group consisting of: The method of claim 1, wherein step (b) further comprises the step of concentrating the pathogenic microbial culture to 0.01 to 0.1 by volume. The method of claim 1, The pathogenic microorganism specific gene amplification primer set is a Campylobacter jejuni having a base sequence shown in SEQ ID NO: 1 and 2 pairs of amplification primers, E. coli O157 having a base sequence shown in SEQ ID NO: 3 and 4 : A7 primer pair for gene amplification of E. coli O157: H7 , a pair of primers for gene amplification of Bacillus cereus having base sequences shown in SEQ ID NOs: 5 and 6, bases shown in SEQ ID NOs: 7 and 8 Primer primer pairs for gene amplification of Listeria monocytogenes with sequences, primer pairs for gene amplification of Yersinia enterocolitica having the nucleotide sequences shown in SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12 A pair of primers for amplifying a gene of Salmonella sp. Having the nucleotide sequence shown in Fig . 13, having the nucleotide sequences shown in SEQ ID NOs: 13 and 14 A pair of primers for gene amplification of Staphylococcus aureus , a pair of primers for gene amplification of Shigella spp. Having the nucleotide sequences shown in SEQ ID NOs: 15 and 16, and shown in SEQ ID NOs: 17 and 18 Vibrio parahaemolyticus having a nucleotide sequence ( Vibrio parahaemolyticus ) method comprising a primer pair selected from the group consisting of a primer pair for amplification of the gene. The method of claim 1, wherein the PCR is a multiplex-polymerase chain reaction using a primer set comprising primer pairs for gene amplification of two or more different pathogenic microorganisms.