KR19990065107A - Simultaneous Detection of Multiple Genes of Escherichia Coli O157: H7 Using Polymerase Chain Reaction - Google Patents

Abstract

E. coli O157: H7 infection is a food poisoning that infects humans mainly through livestock foods, causing severe bloody diarrhea due to hemorrhagic enteritis, and in severe cases, hemolytic uremic syndrome and thrombocytopenic purpura. It is a lot of damage in Europe and other countries.

The present invention is capable of detecting four specific genes (two verotoxins, barrier-associated pathogens and enzyme-specific genes) of pathogenic E. coli (O157: H7) by applying a polymerase chain reaction (PCR) method. Invention of the world's first multiplex-PCR method. The detection efficiency of this method can be detected even if only 1-10 per 1g of meat is detected, and the detection time can be detected within 1 to 3 days, and the detection of Escherichia coli O157: H7 with a single test is performed. Toxin and barrier-associated pathogen) are also very convenient. Therefore, this method is applicable to industrial fecal and human food poisoning patients as well as pathogenic E. coli testing of meat, and is likely to be used in industry, and is expected to greatly contribute to the production of safe livestock products and public health.

Description

Simultaneous Detection of Multiple Genes of Escherichia Coli O157: H7 Using Polymerase Chain Reaction

The present invention is to apply the polymerase chain reaction (PCR) technique to livestock products, and to determine whether E. coli O157: H7 can be identified quickly and accurately by multiple gene co-detection (Multiplex-PCR) and contamination of pathogenic E. coli in meat. The invention relates to a method of early confirmation of the purpose of ensuring the safety of livestock products and to provide public health.

Pathogenic E. coli O157: H7 Food poisoning has occurred worldwide in North America, Europe, Asia, and Africa since it first occurred in 1982 in the United States, and is fatal to humans due to hemorrhagic colitis and hemolytic uremic syndrome. Therefore, the development of rapid and accurate detection method of E. coli O157: H7 in meat in terms of the safe production and supply of livestock foods has become very important in public health.

Escherichia coli O157: H7 has an attaching and effacing (eae A) factor that attaches to the intestinal cells and destroys the epithelial cells, and this eae A gene is expressed on the cell wall of E. coli O157: H7. ), And this intiminant acts as an important pathogenic factor in the attachment of barriers and destruction of epithelial cells. In addition, Escherichia coli O157: H7 is known to produce one or two verotoxins (slt I and II), which is known to induce hemolytic uremic syndrome by infiltrating into kidney tissue. In addition, since E. coli O157: H7 does not have β-D-glucuronides (MUG) activity unlike ordinary E. coli, E. coli O157: H7 can be distinguished from general E. coli using this specific enzyme gene (uidA).

Separation method of Escherichia coli (O157: H7) was developed in the 1980s and has been widely used as a final confirmation method, but it has a disadvantage in that it takes much time. In addition, there are toxin test method, enzyme immunoassay method, gene search method, etc., but the procedure is cumbersome and time-consuming disadvantages. Recently, the US Food and Drug Administration (FDA) has developed a simultaneous detection method for three genes such as verotoxin and glucuronides enzyme, but it has not been put to practical use.

Several methods have been used to detect pathogenic E. coli (O157: H7) from feces or meat from animals and humans.

First, as a standard method, the samples were enriched in enrichment medium, and then cultured in MacConkey sorbitol agar to confirm the biochemical characterization of sorbitol non-degrading bacteria. How to identify. Escherichia coli is an intestinal bacterium that is commonly found in nature or feces. More than 9,000 kinds are known according to the presence of more than 171 cell antigens (O), 80 or more flagellar antigens (H), and 56 or more capsular antigens (K). The bacteria are not easy to analyze and most are nonpathogenic. Therefore, in order to isolate and identify bacteria, various biochemical properties must be confirmed.E. Coli O157: H7 does not ferment sorbitol, unlike ordinary E. coli, and glucuronidase enzyme (β-D-glucuronidase) activity It does not have a characteristic, because it does not grow at 44.5 ℃, which is the temperature for separating the common E. coli, it requires a lot of effort and time because it needs to be confirmed. Therefore, Escherichia coli O157: H7 requires a lot of time and labor for at least 7 days until the final confirmation of bacterial diagnosis and hemodynamic identification, and detection rate is low. In addition, even if E. coli O157: H7 is isolated from the sample, in order to confirm the pathogenicity of E. coli, it is necessary to identify two verotoxins (slt I and II) and attach them to the barrier and cause the disease (attaching and effacement: eae A) Checking takes a lot of time.

Second, Escherichia coli O157: H7 is known to produce one or two shiga-like toxins (SLT, verotoxin). Verotoxin kills Vero cells, so it is not detected by cytotoxicity test using cell culture. It is possible. Cytotoxicity test can detect toxins in fecal or bacterial culture filtrate, but the detection rate is very low, it takes a lot of time, and also it is not possible to distinguish the type of verotoxin, and more than 57 species of E. coli producing verotoxin are produced. It is known and it is difficult to distinguish between them.

Third, as an enzyme linked immunosorbent assay (ELISA), various kits using O157 antibodies have been used, but are not yet developed in Korea. O157 antibodies are Salmonella N group (O30) and Citrobacterium bacteria. (Citrobacter freundii), Yersinia enterocolitica O9, Escherichia hermanii (Escherichia hermanii) and cross-aggregation reactions due to the low specificity is a lot of false positives, H7 antibody test has to be performed separately This method also cannot identify related pathogenic factors (slt I, II, eaeA).

Fourth, verotoxin I and II specific probes have been developed as a gene probe method that can specifically detect all E. coli producing verotoxin including E. coli O157: H7. It is difficult to distinguish 57 species of E. coli which are produced, and its specificity or sensitivity is lower than that of polymerase continuous reaction (PCR).

Fifth, it has been known to be a rapid, specific and sensitive method for the detection of E. coli Verotoxin I and II genes or barrier-associated pathogens by polymerase chain reaction (PCR). In 1995, the U.S. Food and Drug Administration (FDA) simultaneously detected three genes, glucuronidase enzyme (uidA) and verotoxin I and II, which are used to identify E. coli O157: H7 by PCR. Developed a method. This method has the disadvantage that it does not detect the attaching and effacement (eaeA), which is attached to the barrier and causes the fallout of enteric villi and hemorrhagic colitis, and the direct detection of E. coli O157: H7 in meat is still applied. I can't.

The present invention has developed a multiplex-PCR technique that complements the above-mentioned shortcomings in accurately identifying E. coli O157: H7 and can detect E. coli O157: H7 directly and rapidly in meat. .

That is, the present invention simultaneously identifies four genes (uidA, sltI, slt II, eaeA) of E. coli O157: H7 using only one test using multiplex-PCR, and directly detects E. coli O157: H7 in meat. to provide.

1 is a schematic diagram of a method for separating bacterial chromosomes (genomic DNA).

Figure 2 is a schematic diagram of the chromosome separation method of E. coli O157: H7 in meat.

3 is a schematic diagram of a method for detecting E. coli O157: H7 using a polymerase chain reaction (multiplex-PCR).

Figure 4 is an electrophoresis picture showing the results of the specific detection of E. coli O157: H7 using a polymerase chain reaction (multiplex-PCR).

Figure 5 is an electrophoresis picture showing the results of the detection sensitivity of E. coli O157: H7 in meat.

The present invention is described in detail as follows.

In order to detect E. coli O157: H7 quickly and accurately, it is necessary to prepare specific primers.The glucuronidase specific enzyme gene (uidA), which can distinguish between E. coli and pathogenic E. coli O157: H7, and hemolytic uremic disease of the disease Specific primers for four genes, including two verotoxin genes (slt I and II), which cause the late syndrome, and barrier-associated pathogens (eaeA), which cause hemorrhagic infection, were artificially synthesized and shown in Table 1.

Table 1 Sequence of specific primers for detecting E. coli O157: H7

[Isolation of Escherichia Coli in Meat]

To isolate E. coli O157: H7 from meat, 25 g of beef was ground in 225 ml of modified EC broth: novobiocin 20ug / ml, followed by enrichment culture at 37 ° C for 24 hours, and then 10 ml of the primary enrichment medium was mTSB. 20ug / ml) was inoculated into 90ml and cultured at 37 ° C. for 24 hours. The enrichment broth was treated with MacConkey sorbitol agar and Fluorocult E. coli O157 medium supplemented with 0.05 ug / ml of Cefixime and 2.5 ug / ml of potassium tellulite. ) Or directly diluted appropriately (10 ² -10 ³ ), then incubated for 24 hours at 37 ° C. For colonies that are colorless in SMAC and green in fluorocult medium, at least 5 MacConkey agar per plate. The plate was incubated for 24 hours. Agglutination of lactose-degrading bacteria with O157 antiserum was performed. Biochemical tests of isolated bacteria were performed on TSI forming A / A (yellow / yellow) indole: Indol (+), MR (+), VP (-), citric acid: Citrate (-), and urease: Urease (-), arabinose: Arabinose (+), lysine: Lysine (+) test confirmed E. coli, MUG (-), KCN (-), Cellobiose resolution (-) Biochemical and serological tests (O and H antigens) were further performed.

[Test of Verotoxin for Escherichia Coli]

Verotoxin test for Escherichia coli was carried out by a general cytotoxicity test using a Vero cell line, and the bacteria were inoculated in 20 ml of trypticase soy broth and shaken at 37 ° C for 20-24 hours. The culture solution was centrifuged at 7,000Xg for 30 minutes, the supernatant was filtered using a 0.45um membrane filter, and the filtrate was diluted twice. Vero cell lines were cultured at 37 ° C. and 5% CO ₂ in EMEM (Eagle's minimun essential medium) medium containing 5% calf fetal serum and antibiotic gentamicin (50ug / ml). . The culture supernatant filtrate was added to each 20μl per well of the microplate was incubated for 3 days in 37 ℃, 5% CO ₂ incubator to observe the cytopathic effects (CPE) daily.

A final dilution factor of 50% killing (CD ₅₀ ) after 3 days of culture was determined.

Strains used in the present invention were 15 strains of E. coli O157: H7, which were sold at Pennsylvania State University, and 9 strains of E. coli, Salmonella cholerasuis, S. dublin, Klebsiella pneumoniae, Clostridium perfringens, Yersinia enterocolitica, A total of 31 species, including Edwardsiella tarda, were used.

[Isolation of Chromosomes from Bacteria]

Separation of genomic DNA from bacteria was carried out by cetyltrimethyl ammonium bromide (CTAB) -DNA precipitation (Fig. 2). That is, the bacteria were inoculated in 10 ml of LB medium and shaken overnight, and then 1.5 ml of the culture solution was taken to centrifuge the cells to remove the media components.

Resuspend by adding 567 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0) to the precipitate, and then 3 μl of 30 μl of 10% SDS and 20 mg / ml of proteinase K dissolved in distilled water. The cell membrane was destroyed by treatment at 37 ° C. for 1 hour. To remove cell components, LPS and protein components, 100 μl of 5M NaCl solution and 80 μl of CTAB / NaCl solution (10% CTAB in 0.7M NaCl) were added and reacted at 65 ° C. for 10 minutes, followed by the same amount of phenol. : Chloroform: Chloroform: Isoamylalcohol 25: 24: 1 was added and centrifuged at 12,000 rpm for 10 minutes to remove protein components. The supernatant containing the chromosome (DNA) was transferred to a new test tube and 0.6 times of isopropanol was added to precipitate the chromosome. The chromosome precipitate was washed with 70% ethanol and dried, suspended in TE buffer (RNase: 20 µg / ml), and used after measuring the chromosome concentration in a chromosome mass analyzer (Pharmacia LKB).

[Isolation of Escherichia Coli Chromosome from Beef]

Separation of genomic DNA of E. coli present in beef was carried out by crushing 25 g of meat into 225 ml of modified EC broth (novobiocin 20 µg / ml) as in the above bacterial separation method, followed by primary enrichment culture, and then 10 ml of culture medium to mTSB medium. (novobiocin: 20 ㎍ / ml) was inoculated in 90ml after the secondary enrichment culture was isolated by the heating (boiling) (Fig. 3).

Centrifuge the 10ml enrichment broth at 12,000rpm for 5 minutes, remove the media, resuspend with 500µl of TE buffer (10mM Tris-HCl, 1mM EDTA pH 8.0), and boil at 100 ℃ for 10 minutes, and then centrifuge supernatant. Was separated. The same amount of phenol: chloroform: isoamylalcohol (25: 24: 1) was added and centrifuged at 12,000 rpm for 5 minutes to remove protein components. The supernatant containing the chromosome was transferred to a new test tube and 0.6 times of isopropanol was added to precipitate the chromosome. The precipitate was washed with 70% ethanol, dried and suspended in 20 ul of TE buffer (pH 8.0).

[Amplification of Gene by PCR]

In order to determine the conditions of denaturation, annealing and extension in the Multiplex-PCR, preliminary experiments were conducted at different temperatures and times, and then 2.5mM MgCl ₂ , 50mM KCl, From the reaction solution of 10 mM Tris HCl (pH8.3), 0.2 mM dNTPs (deoxynucleotide triphosphates), 50 pmole primers (uidA, slt I, II, eaeA) and E. coli O157: H7 or enterobacteriaceae used in the experiment About 10ul of the isolated chromosome was added and treated for 5 minutes at 94 ° C. Then, Taq DNA polymerase 2.5 units were added to make the final solution 100ul, followed by PCR amplification. PCR amplification was repeated 35 times in PCR equipment (GeneAmp, Perkin Elimer Co.) under conditions of 94 ° C / 1 min for denaturation, 60 ° C / 1 min for annealing, and 72 ° C / 1 min for extension. Extension was performed for 3 minutes. After the DNA amplification, 10 μl of each amplified sample was subjected to electrophoresis for 100 V for 1 hour in a 1.0% agarose gel containing ethidium bromide, followed by specific genes under ultraviolet irradiation. The amplification was confirmed and photographed (Figure 4).

Example 1 Specificity for Escherichia Coli O157: H7 using the Primer of the Present Invention

Four primers (primers: uidA, slt I, II, eaeA) created in the present invention were used to confirm specificity for E. coli O157: H7 by Multiplex-PCR. Each chromosome extracted from 15 E. coli O157: H7, 9 general E. coli, and 7 non-E. Coli bacteria was amplified by Multiplex-PCR method, followed by electrophoresis on 1.0% agarose gel. As a result, all of the E. coli O157: H7 strains showed 252 bp amplification products, which were specific enzyme genes (uidA), but the other species did not show the amplification products of this enzyme gene. In addition, the 1087bp gene amplification product of the barrier-associated pathogen (eaeA) was confirmed in all strains of Escherichia coli O157: H7. According to the E. coli O157: H7 strain, one or both of the verotoxin genes slt I 348bp and slt II 584bp were confirmed, and the results were consistent with the properties of the belotoxin production strains (FIG. 4). As a result, the Multiplex-PCR method was able to identify E. coli O157: H7 quickly and accurately, as well as to identify pathogens related to the bacteria.

In order to investigate the sensitivity of the present invention, Multiplex-PCR was performed by artificially inoculating Escherichia coli O157: H7 bacteria from 10 ⁰ to 10 ⁸ g per meat. As a result, up to 10 ⁴ can be detected per g of meat in the first enrichment, but up to 10 ⁰ can be detected in the second enrichment (Figure 5). This result is a very sensitive reaction that can detect up to 1-10 bacteria in the meat.

Example 2 Comparative test of the present invention and Vero cell toxicity test

Comparison between the veratoxin production of Escherichia coli and the verotoxin gene detection method using the present invention showed that verotoxin was produced in 13 strains of E. coli O157: H7 and 1 strain of E. coli O111, and 2 strains of E. coli O157: H7 and other common Escherichia coli. Did not produce verotoxin, which was in perfect agreement between the vero cytotoxicity test and Multiplex-PCR (Table 2). It was found that the present invention can not only replace the vero cytotoxicity test but also identify the verotoxin type.

[Table 2] Comparative test of Vero cytotoxicity test of Multiplex-PCR method

Example 3 Detection of Escherichia Coli O157: H7 in Beef

As a result of direct examination of 40 cases of beef by the method of the present invention, four E. coli producing verotoxin I and II were detected, but the uidA gene and eaeA gene, which is a barrier-associated pathogen, were specifically recognized in E. coli O157: H7. Was not detected, and it was confirmed that E. coli O157: H7 was not. The serotypes were O6, O112, O115, and O139, respectively, and E. coli O157: H7 was not isolated at all, and the results of the Multiplex-PCR method and the standard separation method were consistent with each other (Table 3).

[Table 3] Detection of pathogenic E. coli in meat using Multiplex-PCR

As can be seen from the above experimental results, the method of the present invention not only detects E. coli in a sample in a short time from 1 day to 3 days, but also enables rapid and accurate detection of E. coli O157: H7 in a single test. Pathogenic factors (2 verotoxins, barrier-associated pathogens) can also be identified, which is a very convenient method, and can be applied to not only meat but also feces of animals or food poisoning patients.

Claims (3)

In detecting E. coli O157: H7, simultaneous detection of multiple genes of E. coli O157: H7 by polymerase chain reaction, characterized by simultaneous use of four specific enzyme genes such as primers uidA, slt I, slt II, and eaeA Way. The method of claim 1, Primer is a multi-gene simultaneous detection method of E. coli O157: H7 by the polymerase chain reaction, characterized in that consisting of the nucleotide sequence of four specific enzyme genes as follows. The method of claim 1, The extracted chromosome (genomic DNA) was added to the enzyme continuous reaction solution with four kinds of primers (uidA, slt I, slt II, eaeA), and then denatured and amplified by amplifying the gene. E. coli O157: H7 multi-gene simultaneous detection method using multiple polymerase continuous reaction, characterized in that for identifying and reading the gene.