WO1998008944A1 - Novel bacteriophage, method for screening the same, novel biobactericidal materials prepared with the use of the same, and reagent for detecting the same - Google Patents

Abstract

A bacteriophage which has an extremely high specificity for specific pathogenic bacteria and, therefore, can surely exterminate the bacteria serving as the host by the phagocytic action thereof. Novel biobactericidal materials prepared with the use of the action of this phage are usable in sterilizing anything to be protected against the infection with pathogenic bacteria, for example, foodstuffs such as fresh foods and kitchens in restaurants and schools so as to exterminate the corresponding bacteria. These biobactericidal materials may contain a mixture of two or more bacteriophages differing in properties. Such materials are highly useful, since two or more pathogenic bacteria can be exterminated thereby at the same time. Not human being, etc. but the pathogenic bacteria serving as the host are exclusively infected with the above-mentioned phage, which makes the phage highly safe. Moreover, it has an extremely potent efficacy. The bacteriophage having a high specificity exclusively for specific pathogenic bacteria and thus being an extremely safe biobactericidal material usable in anything to be protected against the infection with pathogenic bacteria, for example, foodstuffs such as fresh foods and kitchens in restaurants and schools; a method for easily screening the phage; a process for producing the phage; a stabilizer for stabilizing the phage or a preservative therefor; and a reagent and a reagent kit for conveniently detecting the pathogenic bacteria within a short period of time by using the phage.

Description

 P 7/02957

Description New bacteriophage, its screening method, new biocide material using it, and detection ffl reagent technology

 The present invention relates to a novel bacteriophage, a screening method thereof, a novel bio-sterilizing material using the same, and a detection reagent. More specifically, the present invention relates to a novel pacteriophage having 髙 ぃ specificity to various pathogenic bacteria including pathogenic Escherichia coli, a method for screening the same, and the use of such a pacteriophage alone or as a mixed force vector. The present invention relates to a novel biocidal material, a stabilizing agent and a reagent or test kit for detecting pathogenic bacteria using a bacteriophage. Background technology

 There are many types of fine sprout in the natural world, many of which carry out important life phenomena such as decay and fermentation. Among these bacteria, there are so-called pathogenic bacteria, which infect animals such as humans, pets such as dogs and cats, as well as various plants. There are many known bacteria that give rise to the origin of diseases such as diseases and pathological symptoms, and invade and proliferate in body tissues of such animals and plants to cause diseases.

 One such pathogenic bacterium, Escherichia coli, is ubiquitous in nature and is also a resident bacterium in the intestine of humans and mammals. Almost all such Escherichia coli usually do no harm to humans and babies. However, so-called pathogenic Escherichia coli, such as enterohemorrhagic Escherichia coli, exists in Escherichia coli, and this pathogenic Escherichia coli can infect humans and mammals and have serious consequences.

To prevent infection from pathogenic bacteria such as pathogenic Escherichia coli or to treat when infected with bacteria such as Escherichia coli, more types of fungicides are used than before. It is used. For example, iodine-containing germicides such as eodo tincture and chlorhexidine-containing maquilon are used to disinfect wounds. In addition, benzoic acid and the like are often used as preservatives that can be added to foods. Various effective antibiotics are used to prevent or treat bacterial infections.

 If a recent outbreak of pathogenic Escherichia coli, or enterohemorrhagic Escherichia coli, could cause infection with raw beef levers, or infection of enterohemorrhagic Escherichia coli through vegetables such as radish and lettuce, etc. In some cases, infection through sashimi or even through rare steaks may be suspected. However, when it comes to pesticides that can be used to protect against infections from foodstuffs such as fresh foods, iodine tinctures and macirons cannot be used at all for such purposes due to toxicity concerns. In addition, antibiotics used for the prevention or treatment of bacterial infections cannot be used at all for the purpose of protecting foodstuffs such as pathogenic E. coli.

 Even if a safe bactericide is present, if it is used in fresh food, it is difficult to spray it evenly, so a large concentration difference will be created, and local unevenness of the bactericidal effect will occur. This is significant because it implies a germicidal effect.

 Further, there is known a substance having a so-called bacteriostatic action, such as polylysine, which can suppress the growth of bacteria without having a bactericidal action. Must be used for straining. When used for foodstuffs such as fresh foods, taste change is expected, it is expensive, and uneven bacteriostatic action is expected.

 As described above, there is no bactericidal agent that is harmless to humans and can be used for foods such as fresh foods.Therefore, sterilization of foods such as fresh vegetables in school lunches requires disinfection with boiling water after all. It is currently being carried out. Therefore, there is a demand for the development of a sterilizing material that can be used for foods such as fresh foods and is safe for humans.

By the way, a bacteriophage (hereinafter sometimes simply referred to as “phage”) is composed of only proteins and nucleic acids, each of which is a medium between an organism and an inanimate organism that can grow only in a specific bacterium using a specific bacterium as a host. It is very small and can only be observed with an electron microscope. Pacteriophages are bacteria It is known that the disease spreads only to infected bacteria, and that the number of progeny is increased by eating out the infected bacteria. At present, a number of pacteriophages using each cell as a host are known. For example, Salmonella has a specific phage using Salmonella as a host, and Vibrio has a specific phage using Vibrio as a host. Have been. In addition, many phages that destroy food by using Escherichia coli as a host are also known. So far, about 300 phages have been known, and many phages exist in the air. Either of these pacteriophages infects E. coli, but is completely harmless to humans and other mammals. Similarly, phages that use bacteria other than E. coli as hosts are harmless to humans and other mammals at all.

 However, such pacteriophages act only on bacteria, and have high specificity for host bacteria, and act only on one particular bacterium or only a small number of a plurality of particular bacterium. It is known. For example, phage acting on Escherichia coli are no exception, they cannot act on all or many different types of Escherichia coli, and it is also known that the well-known T2 phage cannot act on Escherichia coli C . Thus, although pacteriophages have been used only as research targets and have made great progress in gene research, on the other hand, their specificity is too high, so that pacteriophages are used industrially. It has not received much attention so far. Therefore, there has never been any idea of using karoku pacteriophage as a bactericide.

 As described above, a large number of pacteriophages using various bacteria as hosts are known, but no pacteriophage using pathogenic bacteria such as enterohemorrhagic Escherichia coli as a host has been known so far.

On the other hand, among the bacteria hosting the pacteriophage, Escherichia coli, in particular, has a very strong growth potential, and divides once every 20 to 30 minutes. Grows to 1 gram per day. As mentioned above, Escherichia coli is a bacterium that is ubiquitous in nature and also in the human intestine of mammals, and usually has no harm to humans or human milk excretions. Has no effect. Utilizing such a property of Escherichia coli, many so-called biomedical products are produced by culturing Escherichia coli. As mentioned above, although there is no known bacteriophage hosting a pathogenic cell such as clothing ^ hemorrhagic large gland 'as a host, screening with a conventional bacteriophage screening method is also required. It can be said that a method for efficiently and reliably screening such bacteriophages has not yet been established. Therefore, there is also a need to develop a screening method for efficiently and surely screening such pateriophages.

 In addition, it can be said that there is no effective bactericide that can be used directly in foods such as fresh foods. Therefore, it has been proposed to develop one product that can be used directly for foodstuffs such as fresh food.

 In developing such a disinfectant, if the disinfectant is used directly in foodstuffs such as fresh food, etc., a stabilizer is required so that it can be stored for a long time while ensuring its safety. Alternatively, development of preservatives is also required.

 Conventionally, as a phage storage solution, Tris-HCl buffer, phosphate buffer, and the like are well known, but these have been used only for research so far, and are used for fresh foods and other foods. It cannot be used directly. In addition, no phage preservation solution that can be sprayed on fresh food and drinkable has been known at all.

 In the past, when food poisoning occurred due to infection with pathogenic bacteria, it took a certain number of days to identify the infectious pathogenic bacteria that caused the outbreak. Therefore, in the case of food poisoning caused by infection with pathogenic bacteria such as enterohemorrhagic Escherichia coli, the infection bacteria must be quickly identified in order to promptly cure. is necessary. Therefore, there is a demand for the development of a detection reagent and a reagent kit that can detect pathogenic bacteria such as enterohemorrhagic Escherichia coli in a short time and reliably. Disclosure of the invention

In order to respond to the above request, the present inventors have made intensive efforts focusing on the above-mentioned specific effects of pacteriophages, and as a result, a specific pacteriophage specifically kills pathogenic Escherichia coli. And the use of the bactericidal material containing the pacteriophage in fresh foods and other foodstuffs to kill such pathogenic E. coli. It has been found that bacteria can be germ-free and the safety of food products such as fresh food can be ensured.

 Therefore, an object of the present invention is to provide a bacterium phage exhibiting extremely high specificity only for pathogenic bacteria.

 Another object of the present invention is to provide a screening method that can efficiently and reliably screen for such highly specific pateriophages.

 Furthermore, the present invention relates to a biocidal material containing a pacteriophage that can stabilize a phage for a long period of time while ensuring extremely high safety even when the pacteriophage is used directly in foodstuffs such as fresh food. It is intended to provide

 Furthermore, the present invention provides a method for stabilizing phage for a long period of time in such a biocidal material containing bacteriophage while ensuring extremely high safety even when the phage is used for foodstuffs such as fresh food. The purpose of the present invention is to provide a preservative for phage that can be used.

 In addition, the present invention provides a reagent for detecting a bacterial phage capable of efficiently, quickly and reliably detecting a host pathogenic bacterium by utilizing the phagocytic action of pateriophage. The purpose is to provide the reagent kit.

As used herein, the term "pathogenic bacteria" refers to bacteria that infect animals such as pets such as humans, dogs, and cats and give them a source of disease or pathological symptoms. In addition, it means a bacterium that becomes a host of a bacteriophage and is phagocytosed and destroyed by the bacteriophage, and should be understood as a generic term for pathogenic bacteria including pathogenic E. coli including enterohemorrhagic E. coli. It is. In this specification, Escherichia coli, which is a representative bacterium, will be described as a pathogenic bacterium, but Escherichia coli is merely a representative of a pathogenic bacterium and is described as an example of a pathogenic bacterium. It should be understood that the present invention is not limited to such an example. In order to achieve the above-mentioned object, the present invention provides pacteriophages having high specificity only against pathogenic bacteria.

 The present invention also provides, as a preferred embodiment thereof, a Pacteriophage having high specificity only against a specific pathogenic Escherichia coli.

 In addition, the present invention provides a novel bacteriophage having high specificity only for a specific type of Escherichia coli, and thereby provides a novel bacteriophage containing various pathogenic Escherichia coli. It will be possible to respond to a wide range of demands using bacterium phage useful for bacteria.

 Furthermore, the present invention provides a screening method for efficiently and reliably screening for pateriophages having 髙 ぃ specificity only for pathogenic bacteria.

 The present invention provides a novel bio-sterilizing material containing pateriophage having high specificity only for pathogenic bacteria. Such a novel bio-sterilizing material has the advantage that its bacteriophages can destroy pathogenic microbes that can be infected without causing any adverse effects on the human body. It can also be used directly on products.

 In addition, the novel biocidal material provided by the present invention includes such a bacteriophage consisting of a cocktail of bacteriophages having two or more different properties so that it can simultaneously cope with a plurality of different pathogenic bacteria. ing.

 Further, the novel bio-sterilized material contains a stabilizing agent or preservative for a culture solution of bacteriophage which can stably store the paterio phage for a long period of time. Disinfecting materials can be stored for long periods of time, while ensuring their safety and stability.

 The present invention also provides a method for producing a culture medium of Pacteriophage, which comprises obtaining a culture medium of Pacteriophage by infecting and growing a bacterium, which is a host, in which Pacteriophage is cultured. Is what you do.

In a preferred embodiment of the present invention, a medium for culturing pathogenic bacteria is made to contain calcium ions, so that a pacteriopha having high specificity for pathogenic bacteria is obtained. —Provides a manufacturing method that allows the propagation of di.

 This ^ ijlj provides L1I with a stabilizer or preservative for the culture of bacteriopacteriophage, which can save bacteriophage solution in 7iZ and store it. Another aspect of the present invention is a pathogen capable of detecting the presence of a pathogenic bacterium easily and in a short time by using a specific pateriophage. We provide kits for detection of sex bacteria. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing the bactericidal effect of the cross streak method on the plate, and shows that the bacteria that have come into contact with the test bacterium are completely killed.

 Figure 2 shows the effect of this drug in a liquid medium. In the figure, (1) shows the growth curve of bacteria without this drug, and (+) shows the MOI When added at = 10, it indicates that the growth of bacteria was suppressed.

FIG. 3 is a diagram showing a culture state of bacteriophage. In FIG. 3, a) shows a petri dish in which only the agar medium was cultured, and a) shows a petri dish in which enterohemorrhagic 0157 was cultivated on the agar medium. It turns out that the petri dish is white. (Ii) shows a culture obtained by adding an undiluted phage solution (2 × 10 ^{1 D} / ml) on an agar medium, which phagocytoses and destroys only pathogenic adenobacterium 0157 together with enterohemorrhagic 0157. In this figure, it is clear that all enterohemorrhagic 0157 has been destroyed by phagocytosis. 3) shows a culture obtained by adding a liquid (2 × 10 V ml) obtained by diluting the phage 1,000-fold with a new phage stock on an agar medium together with enterohemorrhagic 0157. The figure shows that all enterohemorrhagic 0157 is phagocytosed. E) is a solution obtained by diluting the phage by a factor of 1,000 with a new phage stock solution (2 x 10 ⁷ / ml) and a 20-fold dilution with tap water together with enterohemorrhagic 0157 on an agar medium. The results obtained by adding and culturing the latter are shown, and this figure shows that all enterohemorrhagic 0157 is destroyed by phagocytosis. On the agar medium together with enterohemorrhagic 0157, a 1,000-fold dilution of the phage with a new phage stock solution (2 x 10 ⁷ / mU after further diluting with tap water 100-fold) In this figure, it is shown that all the enterohemorrhagic 0157 are destroyed by phagocytosis.

 (Bacteriophage)

 Bacteriophages are present in the excrement of many animals, such as domestic animals such as sea lions, pets such as dogs and cats, birds such as cattle and crows, and poultry such as chickens, and sewage. It is widely known that it can be separated from such excrement and sewage.

 Regardless of the origin, any bacteriophage that can be used in the present invention achieves the object of the present invention as long as it has high specificity against pathogenic bacteria such as pathogenic Escherichia coli including enterohemorrhagic Escherichia coli. It is not limited to a particular type of phage. In other words, as the pateriophage, any pacteriophage capable of adsorbing on the host pathogenic bacterium and specifically lysing and destroying the bacterium to achieve the object of the present invention can be used. can do. Examples of such bacteriophages include, for E. coli, T-system phage (Τ1, Τ2, Τ3, Τ4, Τ5, Τ6, Τ7, etc.), X-174, 、, Χ Χ80, Q β In addition to PI, PI, etc., naturally, a novel bacteriophage screened by the screening method of the present invention can also be mentioned. In addition to Bacillus subtilis, bacteriophages such as SZP01 and SP02 are also available against Bacillus subtilis.

Among such bacteriophages, in particular, SEQ ID Nos. 1-1 to 1 to 4, SEQ ID Nos. 2-1 to 2-10, SEQ ID Nos. 3-1 to 3-5 or SEQ ID No. 4- shown in the following sequence listing Each of the Pacteriophages having DNA containing a fragment having the nucleotide sequence represented by each of 1 to 4-5 has specificity against enterohemorrhagic Escherichia coli 0157. Of these bacteriophages Each DNA sequence was identified by the method described below.

 (Bacteriophage screening method)

 Many pacteriophages conventionally used for molecular biology research cannot dissolve the germ cells targeted by the present invention, especially germ cells such as enterohemorrhagic thighs. It has been found.

 It is also possible to screen for the highly specific pacteriophage according to the present invention by the conventional method for screening pacteriophage. However, a screening method that can screen for pacteriophage having 髙 ぃ specificity against pathogenic bacteria, particularly pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli, has not been established yet. Therefore, in order to increase the efficiency and certainty of the screening, we devised the following bacteriophage screening method, and have a high specificity from nature, especially for pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli. It has been found that pacteriophage that can be lysed sexually can be screened.

In the present invention, in order to screen for bacteriophages having high specificity for pathogenic bacteria considered to be present in various kinds of animal excrement and sewage, the samples are collected. First, the sample is infected with, for example, Escherichia coli Β, Β, and C strains, and the positive enzyme (m ⁺ ) is modified with the restriction enzyme minus (ι · —) to amplify the phage present in the sampzole. I do. This lysate is plated on pathogenic bacteria, a single plaque is separated, and such an operation is repeated several times to perform several rounds of amplification to obtain a Takata ita stock. Of these, only clear plaques that were almost completely lysed were selected, and the next step was to expose them to EDTA at high temperature (5565'C) for several hours, and to use phage containing naturally occurring deletion DNA. Select only By repeating this operation several times, it is possible to select a phage that is stable and has only the essential gene.

 (Culture of bacteriophage)

The method for culturing a novel pateriophage according to the present invention will be described by taking, as an example, the case of cultivating pateriophage using Escherichia coli as a host. First, Escherichia coli is cultured in a medium containing polypeptone, yeast extract or the like at room temperature or by heating at room temperature or in a jar armature until the number of large HSiMis reaches a predetermined number. Incubate the vessel with ffl. At the stage when the number of E. coli cells reaches a predetermined number, for example, 2-3 × 10 ⁸ / m (for example, ΔD = 0.2), pacteriophage is added to the medium. If the cultivation is continued under the same conditions as before, after adding the pacteriophage, the pacteriophage will infect the large intestine, and the pacteriophage will grow in the E. coli cells and completely destroy the E. coli. If the ligation and cultivation are continued for a predetermined time, Escherichia coli is no longer alive in the culture solution, and a culture solution of pacteriophage is obtained. This culture solution is purified according to a conventional method, and a residue of Escherichia coli is removed according to a conventional method such as centrifugation to obtain a culture solution of bacteriophage.

 Further, the pacteriophage according to the present invention can be cultured using an agar medium containing, for example, polypeptone, yeast extract and the like. In this case, it is also possible to add a large concentration of cocoon and phage to a medium containing a large number of ifi nutrient medium, and culture the mixture at room temperature or in a heated medium. It can be cultured. The phage cultivated in this manner is dissolved, for example, by adding the natural medium to the phage preservation solution described above and dissolving the mixture, and separating the mixture from solids such as culture residue by centrifugation or the like. And the supernatant can be used as a stock solution. In other words, on the surface of the large intestine す る, there is a receptor structure that adsorbs pacteriophage, and the pacteriophage adsorbs to the large intestine via this receptor structure, and is the gene of the pacteriophage itself. And RNA, etc., into E. coli cells. When such DNA or RNA is injected into the cells, the pacteriophages phagocytose the colon in a short time and grow in the cells. When the bacterium is propagated in the body, the Escherichia coli is completely destroyed, and as a result, the bacterium is propagated in the culture solution to form the bacterium solution. This mechanism can be said to be the same when killing pathogenic bacteria using the bio-sterilizing material of the present invention including pateriophage.

In this method of culturing pac-teriophages, when culturing pac-teriophages using Escherichia coli as a host with pathogenic large intestine の such as enterohemorrhagic E. coli, It is preferable to add a small amount of metal such as magnesium, manganese, or calcium to a medium for cultivating Escherichia coli, and it may be necessary to make calcium ions particularly present in the medium. Any drug can be added as long as it releases calcium ions in the culture medium for culturing the colon, etc., and does not adversely affect the Escherichia coli or pacteriophage to be cultured. Examples of such a drug include calcium chloride.

 (Stabilization of phage stock)

 In order to stably store the pacteriophage according to the present invention cultured as described above for a long period of time, it is necessary to use a buffer containing salts in the culture and a trace amount of metal such as Mg, Mn, or Ca. Can also be added. For further stabilization, glycerol can be added at about 0.001-5%, preferably about 0.1-1%. Furthermore, sugars such as polysaccharides such as maltose and glucose, amino acids such as glycine, arginine and lysine, ethylparaben and polylysine can also be added. The novel phage preservation solution according to the present invention uses an amino acid such as glycine, arginine, lysine, or the like, and preferably glycine, in order to stabilize bacteriophage contained therein while ensuring high safety for food. Good to do. In this case, the amino acid is prepared by adjusting a buffer containing 10 mM to 丄 M, preferably 50 mM to 500 mM to pH 6 to 8, preferably pH 6.5 to 7.5, and adding 5% of sodium chloride as necessary. Preferably, calcium chloride can be added in the range of 0.03% -1%, and calcium chloride in the range of 10 mM, preferably 0.1-lniM.

 The novel phage preservation solution according to the present invention as described above does not inactivate the phage even when diluted up to about 100-fold with ordinary tap water, and can exert a sufficiently strong phagocytic-destroying effect. The same applies when diluted with household alkaline water or acidic water. The phage contained is not inactivated, and a sufficient phagocytic destruction effect is exhibited.

 (New bio-sterilizing material)

The novel biocidal material using the pacteriophage according to the present invention can be prepared as follows. That is, the culture solution of Pacteriophage cultivated as described above is separated from the culture residue by a conventional method such as centrifugation, and the culture solution is used as the novel bio-orchid material of the present invention. Prepare as stock solution. In the novel bio sterilizing food according this ¾fjm this, Park bacteriophage, for example, 10 ² cells / ml to 10 ¹² cells / ml, at any concentration preferably in the range of 10 ³ cells / ml to 10 ⁸ cells / ml I just need.

 In addition, the novel bio-sterilizing material according to the present invention can contain such a bacterium phage alone or in a cocktail of two or more kinds. The combination can be appropriately changed according to the purpose of use. In this case, of course, the pathogenic bacteria hosting each of the combined bacteriophage cocktails can be simultaneously phagocytosed.

 The stock solution of the novel biocidal Si material obtained in this manner is used after diluting the stock solution to an appropriate ratio because the concentration of the phage is usually too high. In this case, dilution with the phage preservation solution described above is preferable, but dilution with tap water or the like is also possible.

In addition, in the present invention, the use of a cocktail of two or more bacteriophages having different properties can suppress the emergence of resistant bacteria due to high frequency use. For example, in the present invention, in order to suppress the emergence of resistant bacteria during frequent use in the sterilization of pathogenic Escherichia coli, for example, when a cocktail in which equal amounts of T2 phage and λ phage are mixed is used as a sterilizing material. it is that the each of the resistant bacteria frequency is 10 ^6, resistant bacteria frequency in the case of using the above cocktail 10- ¹² becomes also the emergence of resistant bacteria in theory experimentally also Almost nothing can be achieved.

 Further, in order to stabilize the novel bio-sterilizing material according to the present invention, a bactericide for food, for example, benzoic acid is added in an amount of 0.002%. It can be added at a rate of about 2%, preferably about 0.1% to 0.3%.

Furthermore, since the novel bio-sterilizing material according to the present invention has no smell or taste, when used with foods, a flavor such as lemon may be added. The mechanism of the production of the killing material according to the present invention will be described by taking, as an example, the origin of bacterial Escherichia coli. In the case of culturing bacteriophage, the mechanism is the same as that when the phage grows while eating Escherichia coli. is there.

 In other words, there is a receptor structure on the surface of Escherichia coli that adsorbs Pacteriophages, through which the Pacteriophages adsorb to the bacteria and inject their own genes, such as DNA and RNA, into the E. coli cells. I do. When such DNA or RNA is injected into the cells, the pacteriophages phagocytose Escherichia coli and grow in the cells within a short time. When the Bataterio phage multiplies in the cells, E. coli is completely destroyed and, as a result, killed. Therefore, the germicidal material according to the present invention has a completely different mechanism of action from other conventionally used germicides.

 The pacteriophage used in the present invention is relatively strong in air, but is weak against stomach acid, etc. because it does not have a cell membrane unlike E. coli, and is lost in gastric juice when it enters the body orally. It is alive and subsequently digested and absorbed. Therefore, the bactericide according to the present invention containing the pacteriophage can be said to be a germicidal material or bactericidal food having a bactericidal action due to phagocytosis, unlike the conventional bactericide. Based on this property, the sterilizing material according to the present invention was named as a novel bio-sterilizing material containing pacteriophage.

 Since the novel biocidal material of the present invention is bactericidal by the action mechanism of flii, it is phagocytosed and proliferates, so it is not affected by concentration unevenness. Can be sterilized by phagocytosis over time. Therefore, there is an advantage that the material can be used at an extremely low concentration. (Reagents for detecting pacteriophage and their kits)

The pacteriophage according to the present invention can be used as a reagent for detecting a pathogenic bacterium serving as its host. Since this phage can phagocytose and destroy the host pathogenic bacterium, a reagent for detecting the host pathogenic bacterium using the phagocytic action of the phage is used. Can be prepared. There are various types of reagent kits using this phage.For example, an agar medium capable of culturing a phage is injected into a container such as a petri dish. Reagent kits in different formats. When a reagent kit of this type is used, a sample containing a test substance that may contain the pathogenic cell to be detected is applied to the surface of the culture medium, and an appropriate temperature τ culture is performed. Then, if the phage contained in the agar medium is present in the presence of the iogenic force of the host, the phage will be phagocytosed. The traces of this phagocytosis cause the medium to be lysed and become transparent or translucent. If the agar medium is in such a state, the sample examined contains pathogenic bacteria corresponding to the phage included in the agar medium as a detection reagent, and the corresponding pathogenic bacteria are not detected. It has been detected.

 For example, if the pathogenic bacterium to be detected is pathogenic Escherichia coli, the Escherichia coli divides and proliferates in a very short time. About 30 minutes after the sample is applied to the agar medium serving as the detection reagent kit, it can be determined whether or not Escherichia coli to be detected is present. In other words, using the phage according to the present invention, for example, when detecting Escherichia coli, it can be detected in a short time of about 30 minutes, which is extremely convenient. In addition, the use of this detection kit or its reagent kit makes use of the phagocytosis of phage, even if the pathogenic bacteria to be detected are not extremely profitable in the sample to be tested. This is very useful because it can be reliably detected. By using phage having two or more different properties as a test vein or a reagent kit for such detection, different types of pathogenic bacteria can be simultaneously detected, which is extremely useful and convenient. .

Therefore, the reagent for detecting a protozoan bacterium or the reagent kit thereof according to the present invention has such an extent that a specimen to be tested is brought into contact with a petri dish containing an agar medium containing a phage and the pathogenic bacterium can grow. Since the presence or absence of the pathogenic bacterium can be determined simply by leaving it in an appropriate place at a temperature, no other measuring instruments or devices for detection are required, so the method of use is extremely simple. Therefore, the reagent for detecting a pathogenic bacterium and the reagent kit thereof according to the present invention can be easily used, for example, for primary screening for detecting a pathogenic bacterium f. The novel biosterile material according to the present invention can also be applied in various ways. The novel biocidal material according to the present invention can be used for any place, such as a place where the immature microbial iMi can exist, or an article that can be infected by pathogenic bacteria. Can be protected from infection with pathogenic bacteria, or can be tested for contamination by such pathogenic bacteria.

 In other words, the novel bio-sterilizing material according to the present invention can be directly used, for example, by spraying food products such as fresh foods, dipping the food products therein, or washing it. Therefore, this novel bio-sterilizing material can be used at any stage of preservation and cooking of foods such as fresh foods so that the foods and the like are not contaminated by pathogenic bacteria.

 In addition, the novel bio-sterilizing material according to the present invention must be kept clean, for example, in places where cleanliness should be maintained, such as kitchens, kitchens, storage rooms, hospitals, and clothing such as aprons and white coats. By spraying directly on items that should not be used, it is possible to prevent contamination of the place or items by pathogenic bacteria. Furthermore, the novel bio-sterilizing material can be used as a washing solution to prevent pathogenic bacteria from entering the body or infecting others through the body such as hands. Furthermore, this novel bio-sterilizing material can be used, for example, in the cultivation or production process of fresh foods and the like. For example, spraying on foodstuffs such as fresh food, mixing and adding to cultivation vegetables for cultivated vegetables such as kale radish, spraying on cultivated vegetables such as kale radish, or especially in hydroponics It can be used by putting a certain concentration in the water.

 The new bio-sterilizing material can also be used for sterilization purposes, for example, in fish markets, meat processing plants and other places where fresh food is processed, and cattle barns and other livestock breeding grounds.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these embodiments are described only for the purpose of illustrating the present invention by way of example, and do not limit the present invention in any way. All modifications and alterations are intended to be included within the scope of the present invention. Should.

Example

 (Example 1)

Bacteriophage screening

Samples were collected from animals such as pigeons, crows, sea lions, dogs, cats and poultry, and from sewage. Approximately 1 g of each sample or 5 to 丄 0 ml in the case of sewage was dissolved in B medium, and the residue was precipitated by centrifugation to obtain a supernatant. Phage, which is presumed to be present in a small amount in this supernatant, was amplified by infecting E. coli K strains, B strains and C strains with the restriction enzyme minus () to the modified enzyme positive (m ⁺ ) host. . This lysate was plated on 01 (37, and plaques were separated. After several rounds of amplification, a high-yield Yuichi stock was obtained. In the next step, only phages containing naturally-occurring deleted DNA were selected by exposing them to 5-10 mM EDTA for several hours at high temperature (55Τ65Τ :). By repeating this operation several times, it was possible to select a phage that is stable and has only essential genes.

 A large number of phages were isolated by screening in this manner. As a result of examining the effects of these phages on multiple types of enterohemorrhagic 0157, about four types of phages having strong effects were selected, and these were selected as Pacteriophages # 1, # 2, # 3 and # 4 identified. The DNA sequence for these phages was determined as follows.

 (Example 2)

Preparation of pateriophage

Escherichia coli is cultivated in a 1-liter jar fermenter using an E. coli culture medium (Elbros) containing polypeptone, a list extract, etc., and the absorbance becomes 0.2 (the number of E. coli cells 2-3 X lOVml). At this stage, a phage that specifically destroys only the pathogenic Escherichia coli 0157 obtained by screening in Example 1 was added so as to obtain Moi 20 (M〇I = 20) (Japanese Patent Application No. Hei 8- 261 132) and 37 for 4 hours or more. When the culture broth became slightly transparent, add a few drops of black-mouthed form, culture at 37 for 10 minutes, then 8,000 The supernatant was obtained by centrifugation at 30 rpm for 30 minutes. The obtained supernatant was filtered through a 0.45 micron Millipore-filtration filter to obtain a phage which specifically destroys only the pathogenic large intestine ligament 0157.

 (Example 3)

 (Preparation of new phage stock solution)

(1) glycine 15 g, 0.5 mM of _{CaC t 2 'H 2 0,} 0.5% of NaC 〖, by adding glycerol to be 0.02%, NaOH at pH 6.8 - adjusted to 7.0. Thereafter, the volume was adjusted to 1,000 ml with pure water, and the mixture was sterilized with an autoclave for 121 ^ for 15 minutes to prepare a new phage stock solution.

 (2) After adding 15 g of glycine, 0.5 · of NaCl, and 0.02% of glycerol, adjust to ρΗ6.8-7.0 with NaOH, and adjust to l, 000 ml with pure water. A new phage stock was prepared by sterilization at 121 in an autoclave for 15 minutes.

(3) glycine 15 g, 0.5 mM of CaC l ₂ · H ₂ 0, was added such that the glycerin port Ichiru to 0.02%, after preparation in PH6.8-7.0 with NaOH, 1,000 ml with pure water I made it. Thereafter, sterilization treatment was performed in an autoclave for 121 and 15 minutes to prepare a new phage stock solution.

(4) 15 g of glycine, 0.5 mM of CaCl ₂ · H ₂₀ , and 0.5% of NaCl were added, and the mixture was adjusted to pI-16.8-7.0 with NaOH, and then adjusted to 1,000 ml with pure water. Thereafter, the mixture was sterilized in an autoclave at 121 TC for 15 minutes to prepare a new phage stock solution.

_{(5) CaC l 2 · Η} 2 ϋ the 0.5niM, 0.5% of NaCl, was added glycerol to be 0.ϋ2%, after preparation to pH6.8-7.0 with NaOH, 1,000 ml with pure water I made it. Thereafter, the mixture was sterilized in an autoclave at ι ₂₁ for is minutes to prepare a new phage stock solution.

(6) 7.5 g of glycine, 0.5 mM of CaCl ₂ 'H ₂₀ , 0.5% of NaCl, and 0.02% of glycerol were added, and the pH was adjusted to 6.8-7.0 with NaOH. Thereafter, the volume was adjusted to 1,000 ml with pure water, and sterilization treatment was performed in an autoclave at 121 for 15 minutes to prepare a new phage stock solution. (7) Arginine 0.2M, CaCl ₂ -H ₂₀ (J.5mM, NaCi ϋ.5? Ό, glycerol 0.02%) were added, and ρΗ6.8-7.0 was adjusted with NaOH. Then, the volume was adjusted to 1,000 ml with pure water, and sterilized with an autoclave at 121 ° (: 15 minutes) to prepare a new phage stock solution.

(8) Lysine 0.2M, CaCl ₂ · H ₂₀ were added so that Ο.δηιΜ. NaCl was 0.5% and glycerol was 0.02%, and ρΗ6.8-7.0 was adjusted with NaOH. Then, the volume was adjusted to 1,000 ml with pure water, and sterilized by autoclaving for 121 and 15 minutes to prepare a new phage stock solution.

 (Example 4)

Preparation method of new bio-sterilizing material

 The pacteriophage prepared in Example 1 above was propagated by a known method using host Escherichia coli, and an increase of about 100-fold was measured in each step. That is, Escherichia coli that reached the logarithmic growth phase was infected with bacterio phage by MOI (Multiplicity of Infection) 10 and lysed about 100 minutes later, and then clonal form was added. Then, bacterial debris (DNA, RNA, protein, cell membrane, etc.) was removed by centrifugation to prepare a crude product of Pacteriophage. The resulting crude preparation had an infectivity of about 10 V ml.

Glycerol is added to the obtained bacteriophage sample in a concentration of 0.1 to 0.01%, and stabilizers such as lauroyl sarcosine salt and benzoate are added, and Mg, Mn, or Ca is added as gold to add new biotin. A sterilizing material was prepared. When this sterilized material was stored in a low-temperature room (4 ± 1), the bacterium of Pacteriophage could be stored stably without inactivation for 6 months. In preparing the new bio-sterilized material, the bacterio phage was basically prepared using two different species so that no residual bacteria appeared. For example, when using the T2 file temporary and λ phage, the frequency of appearance of resistant ^{bacteria, 10- 6 X 10- 6 = 10-} 12 becomes theoretically be no problem at all in the experimental Natsuta. As a result, harmful bacteria including pathogenic Escherichia coli could be completely eliminated by using the bio-sterilizing material of this example. In addition, there was no reduction in the eradication effect due to frequent use. (Example 5)

Preparation method of new bio-sterilizing material

Bacteriophages were propagated in a known manner using host E. coli and measured at about a 100-fold increase at each step. That is, Escherichia coli that reached the logarithmic growth phase was infected with bacteriophage by MOI (Multiplicity of Infection UO), and after about 100 minutes of lysis, clostic form was added. Then, bacterial debris (DNA, RNA, protein, (Cell membranes etc.) were removed by centrifugation, and the supernatant was purified for pateriophage.Purification was performed by precipitation with polyethylene glycol and density gradient centrifugation with CsCI to give an infectious sample of about 10 ¹² / ml. I got

 0.1-0.01% of the obtained bacteriophage standard, stabilizers such as lauroyl sarcosine salt, benzoate, etc. are added, and Mg, Mn or Ca is added as a metal. Was prepared. When this sterilized material was stored in a low-temperature room (at 4 ± 1), it was possible to store the pacteriophage in a stable manner without inactivation for 6 months. The biosterile material was prepared in the same manner as in Example 1. As a result, the same operation and effect as those of the first embodiment were obtained.

 Depending on the purpose of use, the bacterium was used after adjusting the concentration of bacterio phage to a MOI of 10. These operations and preservation were performed in a cold room (4 in soil 1) in principle.

 (Example 6)

Preparation method of new bio-sterilizing material

 Escherichia coli was added to an L-broth medium composed of 10 g of polypeptone, 3 g of extract extract, and 2.5 g of NaC1 for s and cultured at 37 × in a jar armen overnight. At the stage when ΔD = 0.2 in the spectrophotometer, pacteriophage was added (MOI: 20). At this stage, the number of E. coli cells was about 2-3 x 10 V ml.

After adding the bacteriophage, the cells were cultured for 4 hours at 37 ° C. in a jar amen while aeration was performed, and a few drops of black-mouthed form were dropped when the culture solution became slightly transparent. Then, the mixture was centrifuged at 8,000 n) m for 30 minutes to remove the residue, and the supernatant was obtained. This supernatant was used as a stock solution for the preparation of a sterilizing material. (Example 7)

 (1) River kit for detection of pathogenic Escherichia coli

 A 10% agar medium having the following composition was heated to 451: and completely dissolved, uniformly poured as a lower layer medium on a petri dish, and allowed to stand at room temperature to solidify.

 10% polypeptone

 3% first extract

 2.5% NaCl

 0.1% glucose

5 Mm CaCl ₂

This 10% agar lower layer media surface, 5% agar 3ml of E. coli 0157 having the same composition prepared separately: H7 ® strain (ϋ 'Number Body: 4xl0 ⁷ /0.2m0 solution 0.1 which contains the The mixture was heated to 45 ': and completely dissolved, uniformly injected as an upper layer medium, allowed to stand at room temperature and solidified to prepare a double agar medium.

 A solution containing 0.1 μm 1 of phage was coated on the surface of this double agar medium in a strip shape, and cultured at 37 for 7 hours to grow enterohemorrhagic Escherichia coli E. coli 0157: H7. The number of holes (plaques) due to phagocytosis was counted and the phage number was calculated accordingly. As a result, when the target phage was present in the test sample, plaques were formed on the agar medium in accordance with the number of phages, and the target phage was detected.

 (2) Kit for detecting pathogenic E. coli

 An agar medium having the following composition was prepared.

 10% polypeptone

 3% yeast extract

 2.5% NaCl

 0.1% glucose

5 Mm CaCl ₂

Enterohemorrhagic E. coli 0157: H7 strain (number of bacteria: 4 × 10 ⁷ /0.2 ml) and phage of each degree were mixed in 0.1 ml of a 5% cold solution having the same composition prepared separately. Add 3 ml of natural medium, warm this mixture to 45, dissolve completely, inject uniformly as the upper medium, and immediately incubate at 37: 7 for 7 hours. E. coli 0157: H7 was grown, and the rate of cell growth inhibition by the phagocytosis of phage was observed. When a low concentration of phage was inoculated, the number of holes (plaques) due to the phagocytosis of the phage was counted, and the phage number was calculated accordingly.

 As a result, it was possible to detect E. coli 0157.-H7 by using the phagocytosis of phage specific to E. coli 0157.-H7. Also, the number of phages (titer) can be assayed by this method.

The results obtained above are shown in the table below. In this case, the titer of the stock solution was 4.0 × 10 ¹⁰ / ml.

 * Calculation method: number of plaques X dilution ratio / O.lmi

(Fage titration: dl method)

 The growth of Escherichia coli having reached an absorbance of 0.2 and the diluted sample of the phage were mixed, layered on an agar medium as described above, and the phage titer was assayed.

 The effect of the bacteriophage according to the present invention and the biocidal material using the same will be further described.

(Bactericidal effect on solid medium) The bactericidal effect of the germicidal material was examined in consideration of the case where pathogenic bacteria, which are harmful bacteria, adhered or bleed on the 151-shaped object.

As shown in 1-11, person Ui¾i was applied to 桢 in one line. The ¾ fabric before or bio sterilized materials prepared after coating in Example 1, 10- ΙϋΛ 1 (the gamma ³ coated with the diluted solution to the vertical (the method is referred to as a cross-scan Bok leakage method) After culturing overnight at 37, all of the 菡 that came into contact with Bacteriophage were completely lysed, and no colony formation was observed, indicating a strong effect of the bactericidal material.

 (Bactericidal effect in liquid medium)

 Bacteria causing food poisoning often proliferate in food liquids (juices, soups) or in hydroponic sprouts or radish. In such a case, it is effective to give this drug when the bacterial concentration is low per unit volume, so the following experiment was conducted to confirm the effect.

 Escherichia coli was planted in a commonly used commercially available medium for hydroponic culture, and about 1/10 of a bouillon medium was added to measure proliferation. A few hours later, the culture was started at 37, and this fungicide was added. After 12 hours, 菡 completely died.

 Even when the harmful bacteria proliferated, the sterilized material was used at Μ〇Ι: 10-50, and the harmful bacteria proliferated could be completely eliminated.

 (Sterilization effect by spray)

The bactericidal effect of this drug was examined using chopping boards used at home for about one year. First, 10,000 Escherichia coli were dissolved in about 10 ml of a medium (bouillon medium), and this solution was applied to both sides of a cutting board, and after 30 minutes, excess water was removed. The chopping board was then divided into two equal parts, one of which was used as a test for the application of this drug and the other was used as a control. The agent was sprayed all over the experimental chopping board. On the other hand, the same amount of water was sprayed on the control cutting board. After storage overnight 37, the number of remaining bacteria adhering to both cutting boards was examined by washing the cutting boards with 500 ml of medium and counting the number of bacteria. As a result, although no large intestine was detected from the experimental cutting board, about 1 to 2 million E. coli cells were detected from the control ffl cutting board. (Effect on Escherichia coli attached to meat *)

 Using 10 beef steaks, 丄 0 slices were sprayed with Escherichia coli, which had an average of about 100 colonies. These were divided into two groups, an experimental group and a control group. The experimental group was sprayed with a spray containing the drug and the control group was not sprayed. After 12 hours at 37, a portion of this steak was randomly cut (about 10 cuts), replicated on an agar medium, and further cultured overnight, and the number of colonies generated in both samples was calculated. As a result, no colonies appeared on the steaks of the experimental group sprayed with this drug, whereas 10 steaks were found in the steaks of the control group without spraying this drug. This result indicated that the bactericide according to the present invention exhibited an excellent bactericidal effect.

 (Bacteriophage salt S sequencing method)

 Among the pacteriophages according to the present invention, the nucleotide sequences of the DNAs of bacteriophages # 1, # 2, # 3 and # 4 isolated by the above screening were determined in accordance with a conventional method.

 (1) Propagation of pacteriophage

 Infect a liquid medium with Escherichia coli N60, incubate to a OD of 0.2 with a spectrophotometer, add bacteriophage so that the MOI is theoretically 10 and leave on ice for 30 minutes, then for about 20 hours Shake at 37. Then, one drop of black-mouthed form was added and shaken for 5 minutes. The culture was centrifuged at 4,500 卬 m, 4 at 20 minutes for 20 minutes, and only the supernatant was transferred to a sterilized bottle and spread on an L-agar medium to measure the concentration. As a result, 500 ml of pacteriophage having a lxli ^ / ml degree was obtained.

 (2) Pakteriophage constriction

20% PEG'2M NaCl was added to the phage culture solution obtained above, and left on ice for 1 hour. After centrifugation at 12,000 rpm for 4 and 20 minutes to remove water, 1 ml of SM solution was added to the precipitate, drowned well, and transferred to a microcentrifuge tube. To confirm the concentration, the mixture was spread on an L-agar medium and the concentration was measured. As a result, the concentration was 1.3 × 10 ² Vml. (3) Preparation of phage DNA

 10% SDS 0.5M EDTA was added at 0, 1% and 5 mM, respectively, to the shrinking culture solution (1/1 {0}; each), (38'C, 丄 5 minutes [10 heating. Extracted with phenol, then extracted with chloroform, and extracted again with chloroform. An equal amount of isopropanol was added to the obtained aqueous layer, and the mixture was cooled to -70 and released for 10 minutes. After that, centrifugation was performed at 15,000 rpm at 4 for 15 minutes, the supernatant was removed, and the precipitate was washed with ethanol, and dried by suction. Was dissolved in a TE solution.

 (4) Confirm cleavage by electrophoresis

 To this aqueous DNA solution 41 was added 2 U of M buffer, sterile water 13 I restriction enzyme Hind 1 / zl, and the mixture was shaken at 37'C for 1 hour. One-tenth volume of the sample overlay was added, and one electrophoresis was performed on a 0.7% agarose gel at 100 V for 2 hours. The agar-mouth gel was left in a solution of 20 a1 of ethidium ore in 400 ml of TAE solution for 2 hours, and the band was confirmed with a UV lamp.

 (5) Dephosphorylation of vector plasmid DNA

 Next, plasmid 21 (pkk223) 3 iU buffer 21, sterile water 14 n 1 and restriction enzyme Hind ffl 11 were added, and the mixture was shaken at 37 for 1 hour. Hind 111 was deactivated for 15 minutes in a water bath at 70 to deactivate. To perform dephosphorylation, this vector 181, buffer 1 / l, BAP 0.5 II 1. sterilized water 26.51 was added, and shaken at 37 for 2 hours. After that, extraction was performed twice with phenol-cloth form, ethanol precipitation was performed, and the resultant was dissolved in TE solution 80H1. To confirm the presence or absence of the vector, 1/10 volume of the reagent for superimposing the sample was added, and electrophoresis was performed at V0.7% agarose gel at 100 V for 2 hours. Contamination and the presence of the vector were confirmed.

 (6) Deactivation of enzyme

 To perform the ligase reaction, add Μbuffer-21, sterile water 13a1, and the restriction enzyme HindΠΙ1 ^ 1 to the aqueous phage DNA solution 41, shake for 1 hour at 37, and lose Hind HI. Left in a hot water bath at 70 for 15 minutes to alive.

(7) Ligase reaction This phage DNA aqueous solution and the vector dephosphorylated in the manner described above were allowed to stand in a 60 t: hot water bath for 10 minutes. (1) Phage DNA digested with HindIII, (2) Phage DNA digested with I-IindIII and dephosphorylated vector, (3) Dephosphorylated vector, (4 ) As a vector cut with Hind III, add 10x buffer each, 10 / zl of aqueous DNA solution and sterile water to make the total volume 1001, and further add Ε. Coli DNA ligase 2 I.し た I left it.

 (8) Transformation

 Add 5 / x1 to each of the above (1), (2), (3) and (4) to competent Cell (HBIOI) 50 I, leave on ice for 30 minutes, then leave at 42 for 45 seconds. And left on ice for another 2 minutes. Then, a liquid medium was added to a volume of 0.5 ml, and the mixture was shaken at 37 for 1 hour, and then spread on an L-; medium containing ampicillin.

 (9) Culture of recombinant clone

 The colony of the above (2) was taken in 500 ml of L-liquid medium supplemented with ampicillin, and cultured for about 24 hours. The supernatant was removed by centrifugation of the medium at 6,000 rpm for 4 and 15 minutes.

 (10) Plasmid purification

The plasmid containing the phage DNA was purified using a plasmid purification kit (Funakoshi). First, 10 ml of P1 and 10 ml of P2 were added to the pellet and stirred slowly. After standing at room temperature for 5 minutes, 10 ml of cooled P3 was added, and the mixture was stirred slowly, and then left on ice for 20 minutes. After that, centrifugation was performed at 2,000 g for 4 and 30 minutes, and the supernatant was collected. This supernatant alone was further centrifuged at 20,000 g for 4 ^ for 30 minutes. The column was washed by adding 10 ml of QB. The supernatant was added to the column, and the column was washed twice with 30 ml of QC. Then, it was purified with 15 ml of QF, and 0.7 volume of isopropanol was added to the purified solution. This mixture was centrifuged at 15,000 g and 4 at 30 minutes for 30 minutes, and the obtained precipitate was washed with 70% ethanol, dried for 5 minutes and dissolved in 1 ml of sterilized water. This plasmid was cut with HindIII and subjected to electrophoresis. The nucleotide sequence of the thus obtained fragment was analyzed according to a conventional method. As a result, the bacteriophages # 1, # 2, # 3 and # 4 according to the present invention had the following sequences, respectively. A fragment having a partial sequence as shown in SEQ ID NOs: 1-1 to 1-4, SEQ ID NOs: 2-1 to 2-10, SEQ ID NOs: 3-1 to 3-5 and SEQ ID NOs: 4-1 to 4-5 It was confirmed that each had the contained DNA. Industrial potential

 The present invention provides bacteriophages having high specificity for a specific kind of pathogenic bacteria, and uses such bacteriophages to produce foodstuffs such as foodstuffs such as fresh foods. The place where food is stored, cooked, processed, and the like, as well as the birds handling such foods can be reliably prevented from being infected with the pathogenic bacteria, and can be easily used for a wide range of applications. Extremely useful. In addition, the use of the phage according to the present invention disinfects not only the infected place but also the place and the article which are expected to be infected, thereby easily and surely sterilizing the infectious bacteria. It can be performed in a short time and is extremely useful. At the same time, the use of such a phage makes it possible to easily, reliably, and in a very short time detect the host pathogenic bacterium, and to quickly determine the presence or absence of infection and the source of the infection. This has the great advantage that infection prevention and treatment can be carried out quickly.

In addition, the present invention provides bacterio phages having high specificity for pathogenic Escherichia coli such as enterohemorrhagic Escherichia coli among pathogenic bacteria of a specific kind, thereby providing food products such as fresh foods. To prevent the infectious pathogenic Escherichia coli, especially intestinal bleeding Escherichia coli, from preserving, cooking, and processing such foods, etc., and humans handling such foods. In addition, in the event of infection or a place where it is expected to be infected, the pathogenic Escherichia coli can be removed by disinfecting infected items and places or suspected infection. It is surely easy to kill in a short time and is extremely useful. In addition, the phage can be used to quickly and reliably reconstitute the corresponding pathogenic colon II in a short time. Since it can be detected in a short time, it is very easy and reliable to determine the presence or absence of the infection and the source of the infection.

 In addition, the present invention provides a novel pacteriophage, thereby increasing the number of pathogenic bacterial rice plants to be used as host cells by 3%, and can cope with more rice-producing bacterial strains. As a result, a wide range of measures can be taken in infection prevention and treatment.

 Pacteriophages having high specificity for specific pathogenic bacteria, especially pathogenic Escherichia coli, as described above, can be used alone, but phage with different properties are used. The use of a cocktail in which two or more types are mixed makes it possible to simultaneously cope with other types of pathogenic cells, which is extremely convenient and advantageous.

 The present invention also provides such a novel method for screening pateriophages. In addition, the screening method has an effect that a novel phage having extremely high specificity for pathogenic bacteria can be reliably and easily screened.

 The present invention provides a novel bactericidal material containing such a novel bacterium phage, and the effect achieved by providing the phage as described above can be practically used. Will be able to In other words, by including such phage in this novel bio-sterilized material, for example, food products such as fresh foods, places where such food products are stored, prepared, processed, equipment, etc., and those food products, etc. It can be used for handling humans, etc., and can reliably prevent infection with pathogenic bacteria including pathogenic colon bacilli such as enterohemorrhagic colon 、. Even if infection is expected, the pathogenic bacteria can be sterilized easily, in a short time, by disinfecting the infected article or place or the suspected article or place. There is a big effect that it can be done.

By adding the preservative or stabilizing agent according to the present invention to this novel biocidal material, the novel biocidal material can be stably stored for a long period of time while maintaining the action of the phage. It is. In addition, by causing the novel biocidal IS material to contain a plurality of novel bacteriophages according to the present invention, the biocidal material can simultaneously cope with multiple types of pathogenic bacteria. There is a big effect that.

 By providing a method for producing such a novel bacteriophage according to the present invention, the phage can be produced in a large amount and at low cost. In particular, the novel bacteriophage according to the present invention can be produced. This is extremely advantageous in commercializing a new bio-sterilizing material containing.

 Further, in this production method, the addition of gold ions such as calcium ions to the medium in which the phages are cultured has the effect of promoting the growth of phages requiring such metal ions and hastening the increase of the phages. .

 In addition, the present invention provides a stabilizing agent or a preservative that enables stable storage of the novel Bataterio phage, whereby the phage can be stored stably for a long period of time. Thus, the novel bacterio phage according to the present invention, which is prepared by adding the phage stabilizing solution, also has a great effect that it can be stably stored for a long time.

 Further, the present invention provides a method for detecting a pathogenic bacterium and a kit for detecting the pathogenic bacterium, whereby a pathogenic bacterium serving as a host can be easily and quickly obtained using the phage according to the present invention. It is extremely useful because it can be detected in a collision. By using this detection method and the reagent kit for detection, even if urgent measures such as suspected infection by pathogenic bacteria are required, the suspected pathogenic bacteria can be collected at the site where the sample was collected. The use of only the detection reagent kit to determine whether or not the sample is present means that the assay can be reliably performed in a short period of time without using any other measurement equipment for detection. There are advantages. Furthermore, this method for detecting pathogenic bacteria can be performed by increasing the amount of at least one pathogenic bacterium to be detected, even if the amount of the specimen is very small. Since phagocytic bacteria act as a host, it is an extremely sensitive method that can detect the bacteria.

In addition, the reagent kit for detecting pathogenic bacteria using this method for detecting pathogenic bacteria is extremely limited to a normal container such as a petri dish containing an agar medium containing phage. The kit can be used with a simple configuration.For detection, the kit needs to be released in an environment where bacteria grow and spread for a short period of time, that is, only for the time required for phage to phagocytize the fine sprout. It is also a great advantage that it is not necessary at all.

Sequence listing

SEQ ID NO: 1-1

Array length: 628

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Of the array; Genomic DNA

The origin of the array

Array

 TCATCATCCC ATACTTCTTC AGTACTATAT TCAGCACTGT GAAGTTGGTG 50 TTCAGGAATG AAATCAGGAA TTTGCATTTC ACGTTCTTTT TCTTGGATTA 100 GTTCTTCACG GGTTTTAATG ACCGTACCAC CTGAACCAAT CTTATCACCA 150 CGAGCATTTA GGTTTGCATT ACCTAGTGCT ACTTGGTGCT GGTTTTGATA 2 0 0 CTTTAGCATA TCCATATCAA TTGTTGTTCC ACGATAAGTT GTGTGTTTTG 25 0 ACATATAAAA ATCCTTTTTC TATGTGAGTG AA AACGCC TCAAACAACA 3 00 TTATATCACA TGTTTTTCTA CCATTTCAAA AATTCATTTA CATCTAGTTT 350 ATACTTCAAA GAATCAACCA TATGCAAATC AATCAGGAAC AAACAATACG 400 AAGCCACTGA ACTACCACGC CCAAGACCCC AAAAAATATT GTGCTCTTCC 450 ATATAATCAA CTAACCAGAT CATACATCTT AAAAGCTTGG CTGTTTTGGC 500 GGATGAGAGA AGATTTTCAG CCTGATACAG ATTAAATCAG AACGCAGAAG 550 CGGTCTGCCGA GATCAGTCGATC TGCCTCGATC TGCCTGGATC

Array length: 7 8 0

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology Linear

Sequence type Genomic DNA

Sequence origin Phage

Array TTCAGTTAAA CAATATTGTG AGACTACGTG CTGCTGGTAT TGAAGATGCA 50

CGTTTAGAAT AACTATCCAT AAGGNCGCAT TACGCGTCTT TTTCTATGCG 100 AGAATAAAAT GACAAAATTA GATGAGTTCC TATCAAACGT ATCAGTACTA 15 0

GACACCGAAA CAACTGGTGT CGAAAGTGAA GATGATATTA TTGAATTTAG 200

TATTTCATAT CCTCACGATG CACATGAGAA TATTGATACT ATTGATAACT 2 50

ACACTTTGCG TTATAAACCA CTAAAAGATA TACCACCAGA AGCAAGTGCT 30 0

GTGCATTTTA TCAGTACTGA AGATGTAGCA AACTGCATTG GTTATAAAGA 3 5 0

TGACTTAGAA AACATTGACG CACTAATGGG GTGTCGTAAT TATTTTATTG 40 0

GACACAACGT TCAATTTGAC CGCCGAATGA TGGTAGATAA CGAATATAAA 4 50

TATCGTAACT CAGTTTCGCA GTACTTGCTC GATGAAGATA AATGGATTTG 50 0

TACCCTTCGT TTAGCTAAGA AGATGTTTGC AGAAGACACT GAATTTAAAA 550

ACTTAACTCT AAGTTATTTG TGGTATAAAT TTGGTTGCTA TCGTGATGTA 600

CATCGTGCAG TCAATG CTCA CGCAGCAAAA GATGACGTGT TTATGTGTTA 650

TCAAGTTCTA ATCAAATTGA TTGAAGTTGC GATTGAACGT GGACATATTG 7 0 0

ACCCTAATGG TGACATTGGT GAACAAATCG TAACATTCTG TAATACACCA 7 50

ATGCGTTATA AATTCATGCC ATTTGGTAAA 780 SEQ ID NO: 1-3

Array length 5 1 8

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology Linear

Sequence type Genomic DNA

Origin of sequence

Array

 AAAGATAGTG GATGTATTAT TCTCGAAAAT GGTTCTGACG TTGTTACTGA 50 TGACTTCAGT GTAGCGTTTG ATGTTTCACC TGATGGTTCG CTATCAATGC 10 0 CACGCTTAGG GACTGGTGAC ATGACTGTGT GGGTAGGTTT CACTATGGGG 15 0

AATGTACCAG GCACGGTGTA TGTAGATGAT GCTGAGTTGA AAGAAAGCTT 2 0 0

GGCTGTTTTG GCGGATGAGA GAAGATTTTC AGCCTGATAC AGATTAAATC 2 5 0

AGAACGCAGA AGCGGTCTGA TAAAACAGAA TTTGCCTGGC GGCAGTAGCG 3 0 0

CGGTGGTCCC ACCTGACCCC ATGCCGAACT CAGAAGTGAA AGNCCGTAGC 3 50

GCCGATGGTA GTGTGGGGTC TCCCCATGCG AGAGTAGGGA ACTGCCAGGC 40 0

ATCAAATAAA ACGAAAGGCT GAGTCGAAAG ACTGAGCCTT TCGTTTTATC 45 0 TGTTGTTTGT CGGTGAACGC TCTCCTGAGT AGGACAAATC CGCCGGGAGC 50 0 GGATTTGAAC GTTGCGAA 518 SEQ ID NO: 1-4

Array length: 5 9 9

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Sequence type Genomic DNA

Origin of the sequence Fern

Array

AAGCTTCAAC AAGTTCTTTA AATGGAACCA TTGGAGCTTC TTTCTTATCT 50 TTATCAGTCA GGCTTGACAG CATCTGTGCA AATGTTGGAA CAAAAATTT 100 ACCCAACTTC ATAGACATCT TAATACCATC TCTTGCCCCA AGCAGAACGA 150 TATTTACTTT CTTACCATTA ATTACTCTAG ATTCTGTTTT CATTGTGATT 200 CCTTAATACT TTAAAAGAAA CAAAAAAGGG GAAGACCTTT TAAAGTCTCC 250 CCCTTATAGG ATTTATTAAA CACTTGACGC TGGAATTGTA GAAGTGTAGT 3 00 CTAACTTCTC ACAACCAAAA ATCCAAGTTT TAGAGTTCTG GTCACGACCA 3 50 AGTTCAATCT GTGGTAATTC CTGCAACCAA GCATTAA AC CAGTTGCCAG 400 AACAGAGCCT GATGGGTCGT AGATTACGAA GTAGAAGAGA TATCTTCTTC 450 AAGTTCCATA TTGTCTTGTT TAGCTTGAAT TGCAGAAANN A CTTGTTAN 500 GAGANAGAGN CTGGATTANN TCAATCTCAA TAAGACCTGG CNTTGCTCNA 550 TTTCTTTGCA NGGAACCTGG CCACCTTNAC CTACAACTGG GNGCACAAT 59 9 SEQ ID NO: ⁹ - 1

Array length 5 8 5

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Sequence type Genomic DNA

Origin of the sequence Fern Array

 GGATCCGATT GATTAAGACA CCTGTACCAA TTTTTGGATT TCCTTCTATA 50 GAAGAGTTCA AAGTTTATCT TGACAAAAAC TTCTACAATG AGCAGCCTG 10 0 TACTTTGCTG AAGAGTGACT TATCAGAGCT TCTTGATATG GTTATCAAGG 150 CAACTTCTGA GAAGGAACCT GAGCAGAAAG CTGAGAAGAA GACTAGTAAG 2 0 0 AAGTCCGATA AGAAGACTGA AAAGCCTGAG TAGTAACTTG AGGGGTTNCT 2 5 0 TGATAGCCCC TTTATAGAAC TTAGAAGGTA GCGAAATGAA AGAGCTTATA 3 00 GGTNAAGAGC TTGACATTGT TGATGCNAAG ACACAGAGAT ATATATCTAC 3 5 0 TGTTAAATTT CTTGGAATGA ATGATGCGAG TNATGCTTAC CCTCTCNACT 400 GCNTANTNCT AGACAAATTT GAGGTTTGTG GTNTTGATTT TAATGATGAT 450 AACTTTATAA GCTTTGATAA GGACGGTTTC TGGCGTGGTN AGANTCATCC 50 0 TC AGCAAAT GAATTTGATA 9CCGATCAATCAGTCATGATCATGATCATAGATAGATCGATCATGATCATGATCGATCATGATCGATCATGATCGATCATGATCGATCATGATCATGATCGATCATGATCGATCATGATCGATCATGATCC

Array Length 5 7 8

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Genomic DNA

The origin of the array

Array

 GGATCCGATC AAGNGATGGT ATTAAGATGT CTATGAAGTT GGGTAAAATT 50 GTTGTTCCAA CATTTGCACA GATGCTGTCA AGCCTGACTG ATAAAGATAA 10 0 GAAAGAAGCT CCAATGGTTC CATTTAAAGA ACTTGTTGAA GCTTGTTTTG 15 0

ACAGAATTGA AGAAATCAAC CTTGAAGAAA TGGCTACCCT GTTATTTCAA 2 0 0

GGGGCAACTG TTGATGACTT CCCACTTAAT ATTGATACAT ACTTCCAAGC 2 5 0

AAACTACGGT GAATTTATTG ATTACTTAGC ATTTGCGCTG GAGGCAAACT 3 0 0

TCGGAAGTTT TTTCGAAGCA AGCATTTTCA AAAGCCTAAC TTCTCAGTAA 3 50

ACATGGGTNA CACTCTACAG ACACCACTGA CTGATGCTGC TGTNNAGGCN 40 0

ACCTATGAAG AAGCNGACGA NATGAAATTT GTGCTTGCTA TTTATGGTAT 4 50

GGAANGGTGT NAAGAAACAC TTGACCAACT CTTTGCTATG ACATTCTCTG 50 0

ATTTATTATC NTTGAGACAA TTTCTTGAGA TTCAGA GTC GTATNAAGAG 55 0

GAAATTGCTT ACNACGAACT TNNAANAA 57 8 SEQ ID NO: 2-3

Array length 5 9 8

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Sequence type Genomic DNA

Origin of sequence

Array

 GGATCCGATT TCGTTACAGA CTTCATGTAC AGAACATCTG CATTGTATTA 50 CTATGCAAGA GCTTGG ATA AAGACCTTGA CAACAGTCAG CAAAAGCTAA 100 TCAAAAGTGC TGGTGAATTT CTGGGAACAG TCGTGACAAT TGGTGGTGCA 150 GTTGCTGTAG TATCAAAATC AGTCAAGCTC CTAAGTGGTT TGGTCGGTGG 200 TGGCATCTTT GGTAAAATCT TACAAAGACT TGGTGTTAGT GCAGCAGGTA 250 CAGCAGCAGC AGGAGAAGCA GCCGCAGCGG CAGGTGGAGT TACAGCAACG 3 00 AGAATGGCAC TTGGTACTGT TGGCTCTGCA TTGATGCTAA NAAGTNCTAC 350 AGACCCNAAT GCTGCTAAAA ACTACAGTGA AGTTACACTT CCNAAACCAT 400 TTGAAAATGC TGTTGCNAAT ATTACAAACC CNAAAAGACC AATGTTCTTT 450 GATGAAAATG GTCAACTCCA GTTTGCACAG TATACTCNAG ACATTGAAGG 500 TTACAGAAAG TTAATTGACA ATGGCCTATC TAATTGGGAG AT ATCATGG 550 ATAAACTATC NACATCTATT GATAATTAGCAGA 2

Array length 6 0 0

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Sequence type Genomic DNA

Origin of sequence

Array

GGATCCGAAG ACGAAGCTAA GACTGGCACT GTAATCAACG GTGAAGAAAT 50 TCACGTAGTT GTTGACCGTG TATTCTTCAG CAAGCTGACT AAACACCCTA 100 AGATTCGTGA TGCCTATCTT GCACAGCAGA CCCCACTGGC TTGGCAACAG 1 50 ATTACTGGTT CTCTGAGAAC TGGTGGTACT GACGGCGTTC AGGCTCACAT 2 0 0 GAACACTTTC TACTACGGTG GTGTTAAGTT TGTCCAGTAC AACGGTAAGT 2 50 TCAAAGACAA GCGTGGTAAG GTTCATACTC TGGTGAGCAT TGATGGTGCT 3 0 0 GGTGCANAAG TTGGTGTTTG ACACGCTTTC CCTAACGTTT CTATGCTGGG 3 50 TGAAGCAAAC AACATCTTCN AAGTGGCTTA TGGCCCATGC CCTAAGATGG 40 0 GTTACGCNAA TNCCTTGGTC NNGGAACTGT TTGTTTTCNA ATACCAAAAA 45 0 GACCGTGATG AANGTATTGA CTTCGAANCT CACTCTTACA TGCTGCCATA 50 0 CTGTNCTCGT CCTCAGTTGC TGGTANACGT TCGTTCTGAC GCTNAAGACG 55 0 AATAATATTC TTAA GAAGG TTATGAAATG TGTTATNCAG GCGACCCACC 60 0 SEQ ID NO: 2-5

Array length 6 0 3

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology Linear

Sequence type Genomic DNA

Sequence origin Phage

Array

GGATCCGATG ATAAGATTGC AGAACTTGGT CGTTTTGATG ACTTCAAAAT 50 CTTCGTAGGT ACTCGTTTCG AGACAGATGC CTTCAAACAT TTAGAAGCAG 10 0 CATTACTAGA CCCTGCAACA GCAGGTTTTG CGGCTAAGTG GTTACCACGA 15 0 GTCAAACCAC GTCATAAGCA GTTTGTAAAA CGTTTCTGCA AGTTTGCCAA 2 0 0 CTTGAGTGAG AAAGAGTACC GCACACTGTT GTCTGCACTA TCTGATACAG 2 50 TTGAGCAAAA AATCTCTGCT AATGAGTTTG GTAAGATTGA CTACAGTAAG 3 0 0 ATTCCTTCAC TTGCTGCTGC ACGTTACCAA AAACTGTTTA ACCGTNAAGA 3 50 TGGAGAGCGT TACAAAGCTT ACATCGAGTC CTTATCAGAA GGTGAGACTA 4 0 0 AGATTAACGC TGGTGCTGTT TACCCATACG ATGTGATTAA ATCTGTCAAG 4 50 NTGGTAATG CNGATGTTGC TAATGAGCAG TGGAAAGCAC TACCAATGTG 0TCTNGTGATCGATCTGTCATCTGTCGATCTGTCGATCTGTCGATCTGTCTC SEQ ID NO: 2-6

Array length: 6 0 1

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Sequence type Genomic DNA

Origin of the sequence

Array

 GGATCCGATT TGAAAAAGCT TTAAAGCTTG TTATGCCGCA TTTAGAATCT 50 GGAAAGCTGA CTTTAGAGAT GCTTAATAGG GTTATTCGGA GAGCTTATGA 100 AAATTAAAGA AGTGGTTCAA AAAGCAATGC TTGACAACTC AACTAAAGA 150 GAAATGTACA AAGAAATTTG TGATAAGTTG AATTGTTCAA GACATGCTGC 200 TAAGGTTCTT GTCTCGTGTT TTATCTGGGA ATGCTCAGAA GCTTATATGC 2 50 AAGCTTATAT GCAACATGCA GTGTTTGAAA GTTCTAACTT ACTAGGTGAT 3 00 GTAGAAGCTA GTGAGAAGCT AAAAGAACCT GAAGTGAAAA CAGTCCCTAA 3 50 AGTTGGTAAT ACATACCCTC TTAAAGACCT CNAGACTGGA AAGATTATTG 400 CAAAAGGTGT AGTAGAGTCT GTCTACCATG ATGGTAAATA CTTACTTAAA 450 ATATTTGAGT NTGACAGCCA CTATACGCAC TTATGTGGGA TTACATTCTT 500 AGTAACNGAA GAAGACCTTA TTAGGAACAA TAGCAACAAG TTTGCAGTAC 550 CAGCTTACCA AGTTTAGGA ATAGTTAGGA ATAGTTAGGAAATTTGAGATGATATAGATA

Array length: 6 0 7

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Sequence type Genomic DNA

Origin of sequence

Array GGATCCGATA ACTTCCACCA GATGTTACCC AAGCTCTTGA GTCAGCATTT 50 ATGAGGATTT TGGTAAAGTC CACTAAGCAG AATAATTTGC TTATCTCGG 100 AAATGATATT CACAGTATTG TTGAAGGTGC TTTGGCAGAG GTCAATCACG 150 AGATTTATGA GTCTTACTCA ACATACAGAA ATTACCGTAA AGAGGTTGCT 200 CAAAATTGGG ACGAACTCTA CCAGAAGACT AAAGATACAC TCTTCTTAGG 250 TGACCGTGAA AATGCTAACT TTGACAGCAG TTTAATTTCT ACGAAAGGTT 300 CAATTATCCG TGGTTACCTG ACTAAAGAAA TCTTTAAACA GTATCATCTA 350 ACACCAGA G AACTTGAAAC CATTGAGAAA GGCTTTATCT ATATCCNCGA 40 0 TTTGAGAGAC CTGATTTTTG GTGGTATTAA CTGTTGCCTG TTTGACATTG 450 GTAAAGTACT AAAAGGTGGC TTTGAAATGT CCGGCNTTGA NTNCTGTGAA 50 0 CCTAAATCTG TTCTGTCAGC GTTGCAGGTT ATTGGGTGAC GTAATACTTT 550 CAGCAACTGC NCAGCATTTGGATGAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TTAG

Array length 5 8 5

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Sequence solitary Genomic DNA

Origin of the sequence

Array

GGATCCGATC TACTTTGAAG GTGATGTACA CATTTCTAAA AACTTGTATG 50 TAACAGAAGA AGTACATGGT TCAGATTT A TCAGTGACAC AACTGGTGTG 100 AGCTTTAATG AACACACGCA CC ATT ATT AC TGGACAGACC CTGCTGGTGA 150 GGCTGATACT ACAGAGGCAC AATAATGAAA ACAGACTTTG CATTAAATCT 20 0 AGGTGGTGAC TATGTTGCCA CTTTGGGTTC AGATTCANTG TATGTGGCTC 250 ATGGTGATTT AAAGATTACT GGTAACCNAA TTAGAATTAT CCCNNAAGAT 3 00 AATAAAGCTA CTCACGTTGC TCAAAGACTC CATATTAGAT GCCTTCTAAG 3 50 GGCTGGTGAA GTCTTCTTTA ATACATCTGC TGGGTTCCCN TATTTACAAC 400 TTGCCNAATT TAAACNGAAA ACTTCTATCT TTGACAATTA TATGAAAGCT 450 TACCTTGTTG AAACAAGANA TGTGTCTCACACCTATAACT ATTCTTCTTC 50 0 NATGGACAAC GCTCAAAGAA AAGTGACT ATCAGTCAGTCACTAGCACT SEQ ID NO: 2-9

Array length: 6 0 7

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Sequence type Genomic DNA

Origin of sequence

Array

 GGATCCGATT TAAGGTCTAC ACACATCCTT ATCGTTGACG GTAAGATTGC 50 AAAAGACCGT ACTGGAGTAC TTAAAGGTGA ACGTATTGAT ATTCTGGAG 10 0 TGCTATGAAG AAAATGTACA GTCTCTGGGG AAGGGTTGGT AAAGGTTTTG 150 ACTGGACACT TCTTCGTTCA AACGTTAAAC GTAGTGAATT ACCAGAACTC 2 00 ATTACCCACT ATTTAAAAAC ATACAGAGAG GTNGACTATC GTGAACAATN 2 50 AGGTTAAGAN TTGTGTGAAA GCAATGGTTG CACTTGGAGT AATTTTTCTA 3 00 TCTGGCTGCA ACCCCTCTTA CGAGGACAAA AACGCTTCTT ATAGCCTCCC 3 50 ACCAGAGATG CAAGATTGCA AAGTCTATAA GTTACATGGT GATGCCATAA 400 GCAGAGATAT TGTTGTTGTC AGATGTCCAA ACTCTCAAAC AACAACATCT 4 50 TATAGTTATG GGAAAAATGG TCAATCACAC ACTACGGTTA TTGAGTGAGG 500 TTTTCACGAT GGAAGTCCTN GTAAACTATA TCTATTGTTA TGATGTTGTCGA CC TAGATGTTGTCGATC TAGATGTTGTCGA CC TAGATGTTGTC CGAT TAG

Array Length 5 6 3

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology Linear

Genomic DNA

Origin of sequence

Array GGATCCGATT TTTAATGGTG TACAGACAGT TGTTACGGAT AATGAAGGTC 50 TGATTGTTAA AAACTCTACT CAGAATAGAC CATTATATAT TCGTGGTGTG 100 GACACTACTA ATGTATCAAG ATGGTGGATT GGTGTTGGTG GTGTTGATAC 15 0 GAATGATGTT ACCCTAAATA ACAGTTATTC TGGAACCCAA TTGGTTCTCG 2 0 0 GGAATACAAC ATCATACATT AACAAAACAT TGACTATTGC TGGACAAGTT 250 CAACCTTCAG ATTTCTCTAA CTTAGATGCT AGATACTTTA CGCAAAGTGC 300 TAGTGATAGT AGATACCTAA GAATCAGAAG CACTA CTTC AATGTGGGAA 3 50 ACACTGATAA GTGGGCTAAA ATTGCCACTG TTGTGATGCC ACAATCAGCA 400 TCCACTGCTG TTATTGAAGT ATTTGGTGGG TCAGGTTTTA ATATTAATAC 450 ACCAAACCAA GCANGTAAAT GTGAAATTGT TCTGCGAACT TCAAATAACA 500 ATCCAAAGGG CTTAAATGTT GTTGCTTGGA GAACATCANA NAACACCATT 550 ATCANGGATA TTG sequence 563

Array length 5 2 8

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Genomic DNA

Sequence origin Phage

Array

AAGCTTGACA ATCCCTGAAG AGGATATCAG AGACAGTATT GTACAAGGTA 50 TTAATGCCTA CGGAAGAACT CTTAAGGTTG GTAGTGACGT TATCCCTAAC 100 AGAATCTACG GATATATCTA TGACGTAATT AAGGTATTG AGATTAACGA 150 GGTTAAAGTA GCCTTATCAA ACAGCCAATC AGTTCCACCT AGTGACGGAC 200 AATATACTAC AGCAAGAATT ACTGTTGACG GTGACCAATA TACTGTTTGG 250 GAAAGCAGCC AGTATACCAT CACTAAGGAG TAACAATGTT TGAAAAGATT 300 GATAGTG1TT ATTATAAAAC ACTTGATGAA AGAACTGTAA CACAGTTTAA 350 AGATAAGITT ATCTATACAA GCATCTTGAA AGCAATCACT GATGAGTTAC 400 AAACATTAGA GGATGTTTGC TGGCAGATGC ACACAGAGAG AAATATCAGG 450 ACGTCAGTAG GCCAACAACT TGATAATATT GGCTCACTGA TAAAGTTCC 500 TAAGACCT1T AGGTGCAGAT GATGAAAC 528 SEQ ID NO: 3 -2

Array length 5 4 3

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Sequence type Genomic DNA

Origin of sequence

Array

 GAAGATGATA AAGCTACTCA GGTTGCTCAA AGACTCCATA TTAGATGCCT 50 TCTAAGGGCT GGTGAAGTCT TCTTTAATAC ATCTGCTGGG TTCCCATATT 100 TACAACTTGC CAAATTTAAA CAGAAAACTT CTATCTTTGA CAATTATATG 150 AAAGCTTACC TTGTTGAAAC AAGAGATGTG TCTAACATCT ATAACTATTC 200 TTCTTCAA G GACAACGCTC AAAGAAAAGT GACTGTTAAT TTTGATGCAA 250 CTACTACAAC AGATATTTTA ACAGACATTA CGCAAGAGGT TAATATCTAA 300 TGGCAGGATT AACTACAACA GGATTACAAA CTCTAAGATA TCAGGAAATT 350 TTTGATAATA TCAAATCAAG ACTTCTTAGA GATATCTCAC CAAACCTTGA 400 CGTTTCTGAA GATAGCCAAT TAGGTCTCTT CCTAGCTTCA ATTGCAAGGT 450 CTTTAGCAGA TACACATGAG ATTCTNTCAG AAATCTATGA CGGCGGGGAC 500 AATTGACAAG GCTGAAANGA TTTAACCTTG ATGATATCAC AGC 543 SEQ ID NO: 3 -3

Array Length 5 5 7

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Sequence type Genomic DNA

Sequence origin Phage

Array

CAATGAAGGA TGCTCGTGCA ATTTGCAATG AGTTGAATGC AAAGATTGGT 50 AAGAAATGCA AAGACTGGAA GCCAGTAAAA ACAGTTAAAA CTGTGAAGTA 100 CTATAACAGT CATGTTGTTA ACCACTTTGA GCTTGAGTCA AGTCGCTACA 150 CAGGATTGAT TGATGCACCA TATACACCAA TTGAGTTTGA AGTTTCTCGT 200 ATGACTCAGG TAGCAGTTGT TAAAGACTAC TTGAAATCAG TTGGTTGGAT 250 TCCAGATGAC TGGAACTACA AGAAAGATTC AGACGGTCGC CCTGTCAAAG 300 TTTGTCGTTT CANAGACAAC AAAAAGATGA TTACAAAGCA TCCTAAGTGG 350 CAGGAGATGG TTGAACGGTG TGGGTTGAGT TATGTTGAAC ACGAAAGTGT 400 CCAGTACATC GAACATAACT GGTCTGTGAA GAAGT CACG GATTTGCTTG 450 AACCTTGCTT AATCCGTACT TCCCAAAACT TACTGAATCT TCTTATGATA 500 CGATTGAANG TGAGCTTGGA CAGAAGAT G CTAAATACTA TACTTTGATG 550 CCCGACG 557

SEQ ID NO: 3 -4

Array length: 5 2 8

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology: linear

 S class n: Genomic DNA

Sequence Origin: Fern

Array

 AAGCTTGGAA TGAGTATGT GAAGCATTTG GTCACGCAGA TGGACTGAAG 50 AGAATTACCA AGTACCCTAA GACAAAGTAT CGTCAGCAGG TACGTAACGG 100 TGAGATGCAG ACATATGAAA TCAAGCCATT TGGTAAGCCA ACTACAAAGA 150 TTTTTAACAT TGAAAAGAGA AATTGCTATA CACCAACCAA CTCTGTAACT 200 GGTGAAGAGT ACAAGGAGGG TTTTGTAGCA ATGAAGGATG CTCGTGCAAT 250 TTGCAATGAG TTGAATGCAA AGATTGGTAA GAAATGCAAA. GACTGGGAGC 300 CAGTNAAAAC AGT AAACTG TGGAAGTACT ATTACAAGCA AGGTGNTTAC 350 CACTTITGAG CTTTGAGTCA AGTCGCTACA CAAGGATTGA TTGATGCANC 400 ATATACACCA ATTGAGTTTG AAGTTTCNCC GTATGACTCA NGTAGCAATT 450 GTTAAAGACT ACTTGAAATC AGTTGGGTGG ATTCCAGATG ACTGGAACTA 500 CAAGAAAGAT TCAGACGGTC GCCCTGTC 528 SEQ ID NO: 3-5

Array length: 5 2 8

Sequence type: nucleic acid Number of chains: 2 chains

 Topology linear

Sequence type Genomic DNA

Sequence origin Phage

Array

 AAGCTTTCTT CGTCATATAA TGAGTAAGAT ACTTTAACAA ATGCGTATTT 50 TGGTGTTGGT CTGCTAAAGT AAATATTATG TGCTAAACCA CCTAAATCAT 100 GGGCTGTACC AAAAATTGAG CCGTAAGCTC TAATACCAGC AGGT TTGTA 150 TCCCAGATTG CTTGAGCAAC ATTATCATTT TGACCACCGA CTACGACAAT 200 CTTAAAAGAT TTTGGTGGAA GACCTTCTGA GTTCGTCTCT TCAGTATCA 250 TTTCAACACC TGAAGCATCT GAGACACCTT GAA CCTTTT AACAGCAGCT 300 ACAAT GCAT CCAAAGTCCC TA ACCAAGT AACTGGTAAA GNGTCNAAAT 350 ATCNCTGNCN AAGTTCTGNG TCAGTTTCCT CGGTTCTAAC AATTGNTAAG 400 TCA ACCTGN TATAGATGCT GTCAAGACCA TCAACAAGTT GTCTCAATCT 450 CAATAAGAGT CCCAAGCTAA AGCAGGAATA GCAACAACCT CTTCAGCCAC 500 TACATCTGAG ATAAGTTGTA ATCTTTGT 528

SEQ ID NO: 4-1

Array length: 5 1 7

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology: linear

Sequence name: Genomic DNA

Sequence origin: phage

Array

AAGCT CGTT CCAACTACCT CTCAAGACTT TTACAAGTCC AGCTTCAGAA 50 GCAGCAGAAA ACCCCGCAAA TCGGGTAACT TTGTCTTTAN TTGTTGGTTT 100 AGCTCTTGCT CGATAACCTT TCTCGGCAAG TTTCCTGATG AGGGATGTTG 150 CGTAGGATTT ACCAGCAGCG CCTGGGTCTT GAGGGATAAA AATACCAGTT 200 CGC1TACCGT CACTTTCAGC AGTCAAATTA ATTTGTGTTT CGACTCCAGA 250 GGGTCTATCT CTAAATCTTA CTACATCAAT GATATAATAA GCAACCGTCT 300 TT TTAAGAT TTACCATCTT AACAACTGCT GGCCAATCCG GGTTAAGGNT 350 AAACCCAAGA TGGGAAAAGT TGCTGGCTAA AGTCCCATGC TCTGACATCG 400

AATACATCTT CTGGGAGTGA ATCAACAATT TCACACCATT GNCTT GCCA 450

AATAAGTTTG AACCTTCTGC ACGAAGCCTT CCAAG TACC GAAACGAAGT 500

CTTGCAACGT 1TACAGG 517 SEQ ID NO: 4-2

Array length 3 5 3

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Sequence type Genomic DNA

Sequence origin Phage

Array

 TGTTAAAGT TGTGAATGCAA AATCACCACG GCTACAAGGT ACGGTATTTA 50 GTTTCATCT CAAGTGACTGA AAACCTTGAG TCAGTTCAAT TTGTAACTGC 100 TCCATTACA TAATTGTGGTA ATGCTCTTTA GGAATACCAT AAGATGATGC 150 TTTCTCAGC ATGATAGCGTO NAAACTTCTT AGCATACGGT ACAA TNCCT 200 TATCAATCT CTGCTAAAGTN AAACCACCNA ATTGCTGTGC ANTTGCTGAA 250 ANTNCTACG TCNCCAATAAC C GCAACGCT GACAGTACAG ATTTAGGTTC 300 ACAGTACTC CATGCCGGACA TTTCAAANCC NCCNTTTA T ACITTACCNA 350 TGT 353

SEQ ID NO: 4-3

Array Length: 4 1 6

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Sequence type Genomic DNA

Origin of sequence

Array GGTGCTCCGT ATGATGCAGG TTCAATTGCT TGGGGTAACG CACAGCTAAC 50 TGGCGTAGCT GCTTCTCTAC AGCCATCTAA TCAGAGACCT CTGACAAGTA 100 TTCAGAANTC AGCTTTA AT GCACGTCACT GTAACTTTAT TGACCT GAT 150 GGTGGTGTTC CAGTGGTTCG TAGANGGATT ACTTC GGTG GGGAATGGAT 200 TNATATCATC CGTGGTGTTG ACTGGTTAAA ATCNGACCTG AAAACTTCTC 250 TGAGAGACTT GCTAATTAAC CAGAAAGGTG GTAAGGATTA CTTA GATGA 300 TACTGGTAIT ACCCGTATTC CCAAGTCATT GAAACCTCTC TGCCCANCAG 350 CAGTC ACAG AAACTTCCTG TCATCT ANA CACTTAATGT TCCTAAATCC 400 TCTCAANTTG CTTTGG 416

SEQ ID NO: 4 -4

Array length: 5 1 7

Sequence type: nucleic acid

Number of chains: 2 chains

 Topology: linear

Sequence type: Genomic DNA

Sequence origin: phage

Array

 AAGCTTACCA ATCTTTCTAA TCTTAGCCCA TCTTTTAACG TGACCTTCAT 50 CACCATCAAT CGGGTTGTCA TATGTCTCCA TCCATCTGAA TCCAAAAGGT 100 AAGTGTCCTT TCTTCTCACT GTAAAATGGA ACTACAAAAG GTGCTAAGAT 150 AACTGCTAAG ATTGCTGCCA ATGTTTCTAG CAAAGCTAAG AAAATCCATG 200 AAGCATACTT CAAGTATCTC ATTTAAATAG CC CTTTAAA TTGGCGCAGG 250 ATAAGGGATT CGAACCCCTA TTAACAGCTT CGTAGACTGT TGCTCTATCC 300 ATTTGAACTA ATCCTGCTTA CCTACAAGAA CACCATACAG TTTTTCACAA 350 GAGTTGTTTT TACGCCTGTT TTTGAATGAC GCTATAAACT TTTTTGCAAT 400 TG TTATTA CTCATAGTGT TCTCCTAAAA TTGGTGTTCC AAGACGGNTT 450 CGAACCGTCA CTAGTACAAG GGTTGAGCTT GCATCCTCTG CCAATTGGGA 500 TACTGGGACA TGG ACT 517 SEQ ID NO: 4-5

Array length: 5 2 8 Sequence type: nucleic acid

Number of chains: 2 chains

 Topology linear

Sequence type Genomic DNA

Sequence origin Phage

Array

 AAGCTTTGAC CCAGAAAGAC TGAATAAGTG GGCATCATGG GCAGATAAGC 50 GTGGAATTAT CTGGTCAGAA GTCACTATGG AAGCCATGAA ACGTGTCTAT 100 GAGGGTTGCA CTACAAAAGA GATGCACCAA GCCATGAITG ATGTTTGTGT 150 TGATAAGCAA ACTCAAGAGT ACTCAGATAT GGCTGGACGG CTACTTCTGG 200 GAATTATCTA CAAAGAAGCC TTTGGAGGTT TACTAAGGTT CCTACGCTGG 250 TTACCTTCGN TAAAAATATG GAGAGAGCAA GACTTTGGGA GAAGATGGAC 300 TATTCACAAG AAAGAGCTTG AATATCTGCA AGGGTACATT GTGCACTCA 350 AAAGATATCT CTTACGGTTA TGCAGTCTTG AAACAGTTCA AGAGACAAGT 400 ATGGTATCCG TGATATTAAA ACGGGAAGAC TTTTTGAGTC ACCACAATTT 450 ATGTTTATGG GTATGGCTAT GAAAGCCTTT GAGAAGCAAC CAAAGCACCG 500 TAGACTGCAA GATGTTATCA AGCTGTAC 528

Claims

Claims IIH

1 Novel pacteriophage characterized by having specificity only for specific pathogenic cells.

2. The bacteriophage according to claim 1, wherein the pathogenic bacterium is enterohemorrhagic Escherichia coli.

3 ^ In the pacteriophage of the battle in the range 1 项 or 2 of the request, the pacteriophage is SEQ ID NO: 1-1 to 1-4, SEQ ID NO: 2-1 to 2-10, SEQ ID NO: 3-1 to It is characterized by having a DNA containing a fragment having a base sequence represented by 3-5 or SEQ ID NOs: 4-1 to 4-5, respectively.

4 Infect the pathogenic bacterium with a pathogenic bacterium using the restriction enzyme 尜 -minus and the modifying enzyme positively and amplify the bacteriophage. A method for screening for paterio phage, comprising:

5. A novel bio-sterilizing material comprising the bacteriophagy according to any one of claims BB to circa 3.

6. The novel bio-sterilizing material according to claim 5, wherein a stabilizing agent or a preservative for stabilizing bacteriophages is contained.

7. The novel biocidal material according to claim 5 or 6, wherein the pateriophages are singly or two or more pateriophages having different properties. It is characterized by consisting of a cocktail of di.

8 A new bio-sterilizing material containing the pacteriophage according to any one of claims 1 to 3 or the pacteriophage according to any one of claims 5 to 6 is used to kill pathogenic bacteria. A method of using a bacteriophage to kill pathogenic bacteria, the method comprising using the bacteriophage.

9.The use of the bacteriophage for sterilization according to claim 8, wherein the bacteriophage or the sterilized biomaterial is infected with or suspected of being infected by the pathogenic bacterium. The invention is characterized by being used for sterilizing the pathogenic bacteria or preventing the pathogenic cells from being infected.

10.The method according to claim 8 or 9, wherein the bacterium is a bacterium, wherein the bacterium comprises one or more cocktails of bacteriophages having different properties. Features.

11 After the pathogenic cells have been cultured in the medium and the number of cells has reached a predetermined number, the pateriophage is added to the medium, and the pateriophages are infected with the pathogenic bacteria. A method for producing a culture solution of Pacteriophage, characterized in that a culture solution of the bacteriophage is obtained by removing bacterium.

12. The method for producing a culture solution for a tactical phage phage described in ffl 11 of the claims, wherein the medium contains gold ions such as calcium ions.

13 A bacteriophage stabilizer characterized in that the pH is adjusted from 6.5 to 7.5 using a food material such as an amino acid. 14. The method of claim 13, wherein the amino acid is glycine, arginine or lysine.

 15 A method for detecting a protozoan bacterium, comprising using the tactical phage phage described in any one of the claims 1 to 3 for the detection of a pathogenic bacterium.

 16. A reagent or kit for detecting a pathogenic cell, characterized by using the method for detecting a pathogenic bacterium according to claim 15 埂.