JPH08242888A - Examining sheet for detecting escherichia coli determination - Google Patents

Abstract

PURPOSE: To simply and surely detect Esherichia coli occurring in food and beverage specifically. CONSTITUTION: 4-Methylumbeliferyl-β-D-glucuronide is added to bouillon medium containing culture reagents as a reaction substrate to prepare the detection solution and the base test sheet 2 is impregnated with the solution to give this coli bacterium detection sheet. Thus, even in the case that the amount of coli bacteria is small in the other intestinal bacteria, good fluorescent reaction is recognized and the coli bacteria can be surely detected.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an inexpensive test sheet for detecting Escherichia coli which can easily and surely detect Escherichia coli, which is an indicator of fecal contamination of foods and drinks and causes E. coli contamination.

[0002]

2. Description of the Related Art Since coliform bacteria (Coliform organisms) are normally resident bacteria in the intestinal tract of warm-blooded animals, if these bacteria are detected in water or food, Since it is suspected that water and food are directly or indirectly contaminated with human or animal feces, and there is a possibility that various digestive system infectious disease bacteria and food poisoning bacteria are present, the food and drink are naturally hygienic. Considered unfavorable.

Therefore, the coliform bacteria have been conventionally referred to as fecal contamination indicator bacteria or simply contamination indicator bacteria, and this coliform bacteria is generally tested as an indicator of fecal contamination of food and drink.

Recently, for some fresh foods, milk, processed meats, etc., which are relatively highly contaminated with coliform bacteria, Escherichia coli (Escherichia coli) is used rather than the coliform bacteria inspection. Since direct testing is more specific and efficient, it is becoming more common to detect E. coli instead of coliforms. Furthermore, in recent years, fatal accidents due to E. coli contamination of well water and E. coli contamination in sand fields in parks have been reported, and interest in E. coli contamination is rapidly increasing.

However, the test for Escherichia coli is more complicated in detection method than the test for coliforms, and moreover, it takes a long time for final identification and requires abundant knowledge and experience for its identification. It is inferior in sex.

Recently, various test kits for detecting Escherichia coli have been developed and sold. Most of these test kits are test tubes such as test tubes filled with a liquid or solid medium. In terms of operability, disposal method, and price, it was difficult to widely reach end users. In particular, in a test kit using 4-methylumbelliferyl-β-D-glucuronide (MUG) as a reaction substrate, this MUG is expensive, so the test kit is expensive, which is costly from the viewpoint of versatility. Is disadvantageous to Under these circumstances, it is desirable to develop an inexpensive test kit that allows end users to detect E. coli easily and reliably.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an inexpensive Escherichia coli detector which can easily and surely detect Escherichia coli in a short time.

[0008]

In order to achieve the above object, the present invention provides 4-methylumbelliferyl-β-D as a reaction substrate in a broth medium containing a culture reagent.
-A test sheet for detecting Escherichia coli characterized in that a detection solution containing glucuronide is impregnated in a sheet-shaped substrate.

That is, when detecting Escherichia coli using the test sheet of the present invention, attach a sample to the test sheet,
The fungus in the sample is cultured in the detection solution contained in the test sheet. At this time, if E. coli is present in the sample, the reaction substrate 4-methylumbelliferyl-β contained in the detection solution by the enzyme β-D-glucuronidase produced by E. coli.
The glucuronide bond of -D-glucuronide (hereinafter abbreviated as MUG) is hydrolyzed, and the fluorescent substance 4-methylumbelliferyl is released. And U on this test sheet
Escherichia coli is detected by irradiating ultraviolet rays from a V lamp or the like and measuring the fluorescence reaction of the 4-methylumbelliferyl.

In this case, the enzyme β-D-glucuronidase of Escherichia coli is widely distributed in animals, higher plants, microorganisms, etc. ) Genus, Enterobacter genus and other genus Proteus (Proteus genus, Salmonella genus, etc.), which belong to the group of enterobacteria, do not have this enzyme. Therefore, it is possible to perform highly specific tests. Moreover, as described above, the sample can be adhered to the test sheet, cultured, and the Escherichia coli can be easily detected in a short time only by irradiating the ultraviolet rays, and the sheet-shaped substrate is impregnated with the detection liquid. Therefore, the test can be carried out inexpensively with a very small amount of the medium, the culture reagent and the reaction substrate, and after the test, it can be simply and safely discarded by incineration.

The present invention will be described in more detail below. The test sheet for detecting Escherichia coli of the present invention is obtained by impregnating a sheet-like substrate with a detection solution prepared by adding a culture reagent and MUG to a broth medium as described above. However, in this case, as the broth medium, a broth having a basic composition that has been generally used for culturing bacteria can be used, and specifically, a broth having the following composition can be exemplified. Peptone 20 g / L Lactose 5 g / L Bile salt 1.5 g / L Monopotassium hydrogen phosphate 4.0 g / L Dipotassium hydrogen phosphate 1.5 g / L Sodium chloride 5.0 g / L

The 4-methylumbelliferyl-β-D-glucuronide (MUG) added to the broth as a reaction substrate is bound to the glucuronide by the enzyme β-D-glucuronidase produced by Escherichia coli as described above. Is hydrolyzed to produce a fluorescent substance (4-methylumbelliferyl), and Escherichia coli is detected from the fluorescent reaction of the fluorescent substance by ultraviolet irradiation. The amount of the reaction substrate MUG added is not particularly limited, but is usually 0.1% per 1000 ml of the broth medium.
It is about 05 to 0.1 g, and if it is less than 0.05 g, a good fluorescence reaction may not be obtained, while if it exceeds 0.1 g, the detection accuracy does not change and MUG is an expensive reagent. It is a cost disadvantage.

Further, the above-mentioned culture reagent is added in order to effectively culture Escherichia coli in a short time, and any reagent may be used as long as it does not affect the reaction of the reaction substrate MUG, and it is usually used for the culture of Escherichia coli. What is possible can be used conveniently. Specifically, L-asparagine, casamino acid,
Examples thereof include yeast extract, glucose, sodium citrate and the like. Among these, it is preferable to use L-asparagine, casamino acid and yeast extract in combination. The amount of the culture reagent added can be a usual amount, specifically about 0.5 to 3 g per 1000 ml of the above broth, and in particular, the above L-asparagine,
When using a combination of casamino acid and yeast extract, about 1 g of L-asparagine, about 0.5 g of casamino acid,
It is preferable to use about 1 g of yeast extract.

The test sheet of the present invention is obtained by impregnating the above broth medium with a detection solution containing the above MUG and a culture reagent, and the detection solution is preferably about pH 6.5, and pH 6.5. In the following, the above fluorescent substance (4-methylumbelliferyl) is not suitable for the growth and growth of Escherichia coli.
May show only weak fluorescence. In addition, additives other than those described above may be added to the detection solution as long as they do not inhibit the growth of Escherichia coli, and culture reagents and bacterium selection agents other than the above may be appropriately added.

Next, as the sheet-shaped substrate impregnated with the above-mentioned detection liquid, there is no particular limitation on the material, shape, etc., and paper or cloth having water absorption property can be used, but water absorption such as filter paper is particularly preferable. A small strip formed from a paper having good properties is preferably used. Specifically, the sheet-like substrate shown in FIG. 1 is exemplified. That is, FIG. 1 shows an example of the test sheet of the present invention. This test sheet 1 is a sheet-like substrate 2 formed in a strip shape using a paper having good water absorption such as a filter paper.
Is impregnated with the above detection liquid. In this case, 3 in the figure
Is a cut line formed on one end side of the sheet-like substrate 2, and the cut line 3 serves as a boundary, and one side is configured as a knob part 4 and the other side is configured as a test part 5, whereby the test sheet 1 is inoculated with a sample. However, since the fingers do not directly touch other portions of the test sheet 1 except for picking up the knob portion 4 with a finger and lifting, the test portion 5 is not contaminated, and the test portion 5 is not touched. It is possible to judge that all the reactions appearing in 1) are derived from the sample, and therefore the test results can be judged without error.

The means for impregnating the sheet-shaped substrate 2 with the detection liquid is not particularly limited, but a method of immersing the sheet-shaped substrate 2 in the detection liquid and impregnating the sheet-shaped substrate 2 with the detection liquid is preferably adopted. It In this case, the amount of the detection liquid impregnated into the sheet-shaped substrate is not particularly limited, and the E. coli detection test sheet of the present invention can be obtained by drying the sheet-shaped substrate impregnated with the detection liquid.

The test sheet for detection of the present invention comprises various foods and drinks such as fresh foods (vegetables, fruits, tofu, etc.), processed livestock products (meat, ham, cheese, etc.), pooled water, tap water, It can be widely used for well water, etc. and is not limited to indicators of fecal pollution of food and drink, but it is also a simple public health test such as water quality survey, pollution of sandboxes in parks, hygiene management in restaurants and homes, etc. It can be effectively used as a seat.

In the method of using the test sheet for detecting Escherichia coli of the present invention, a sample is first attached to the test sheet of the present invention. In this case, when the test substance is water-soluble, it can be used as a sample as it is, and when the test substance is a mucous liquid or a solid state, it can be appropriately diluted and mixed with physiological saline and used as the sample. The amount of the sample adhered to the test sheet is not particularly limited, but usually about 1 ml is suitable. When the test substance is a solid substance such as various foods, it is possible to directly contact the test sheet and attach the sample.

Next, the test sheet with the attached sample is subjected to culture. The culture condition is preferably a temperature of 36 to 44.5 ° C, and particularly preferably 35 to 37 ° C because it is a general culture condition. Although the culture time is not particularly limited, it is generally 18 to
It will be about 24 hours. As a specific culturing method, for example, the test sheet to which the sample is attached is placed in a transparent bag made of a petri dish or a plastic film in order to avoid germs and drying, and stored for 24 hours in an incubator at 35 to 37 ° C. It can be done by doing.

After culturing, the test sheet is taken out and irradiated with ultraviolet rays using a UV lamp or the like, and the presence or absence and degree of the fluorescent reaction are measured. In this case, in the present test sheet, when the number of E. coli is large, a fluorescent reaction covering the entire sheet is obtained, and when the number of bacteria is small, a fluorescent reaction occurs like spots on the sheet. Therefore, in this test sheet, the degree of fluorescence reaction that occurs on the test sheet with the naked eye (the number of spots, 1 /
It is possible to inspect approximately the amount of bacteria by determining the fluorescence reaction in 2 and the fluorescence reaction in the entire test sheet. When the test sheet with spots is stored, it is suitable to store it in a refrigerator, and the frozen spots are attenuated in about 10 days. Also, the wavelength of the ultraviolet light that irradiates the test sheet is 3
About 65 nm is preferable.

As a method for storing the test sheet for detecting Escherichia coli of the present invention, it is desirable to store it in a cool and dark place (refrigerator, etc.) in view of the stability of the reagent. However, even after 7 months of storage at room temperature, spots are observed, and storage at room temperature is possible for a certain period of time.

Since the used test sheet may be contaminated with other bacteria even if the fluorescent reaction does not occur, it must be disposed of by incineration in order to prevent infection and the like. In this case, since the test paper sheet of the present invention is a sheet made of paper or cloth, it can be easily incinerated and discarded, and the disposal is simpler and more economical than the conventional detection kit method using a test tube or the like. Is.

[0023]

EXAMPLES The present invention will now be specifically described with reference to examples, but the present invention is not limited to the following examples.

Example 1 4-methylumbelliferyl-β-D-glucuronide (MUG) 0.05 g and L-asparagine 1.0 per 1000 ml of broth medium.
g, 0.5 g of casamino acid and 1.0 g of yeast extract are added to prepare a detection solution, and a filter paper cut into strips is immersed in the detection solution to put the above detection solution on the filter paper (sheet-like substrate). After impregnation, this was dried to obtain a test sheet for detecting Escherichia coli.

Next, each of the following test strains (1) to (15)
After culturing at 37 ° C for 18 hours in ordinary broth,
The resulting culture stock solution was diluted with physiological saline 10 times.⁹Dilute to twice, 1
0^Five-10⁹Up to 1 ml of the bacterial solution up to
After culturing at 36 ° C and 44.5 ° C, U
Fluorescent reaction by irradiating ultraviolet rays (wavelength 365nm) with V lamp
Was measured. The amount of inoculum is calculated according to the official method.
Was. The results are shown below. Test strain Using a total of 15 strains of the following 6 strains of Escherichia coli and 9 strains of other enterobacteria
Was. (1) Escherichia c
oli, Sun Chemical Co., Ltd. stock): hereinafter abbreviated as Ec-1
You (2) Escherichia c
oli, stool-derived strain): hereinafter abbreviated as Ec-2. (3) Escherichia coli
oli, stool-derived strain): hereinafter abbreviated as Ec-3. (4) Escherichia c
oli, strain derived from feces): hereinafter abbreviated as Ec-4. (5) Escherichia c
oli, food-derived strain): hereinafter abbreviated as Ec-5. (6) Escherichia coli
oli, food-derived strain): hereinafter abbreviated as Ec-6. (7) Enterobacter / SPP (Enterobac)
ter SPP, food-derived strain): hereinafter abbreviated as En-1. (8) Enterobacter / SPP (Enterobac)
ter SPP, food-derived strain): hereinafter abbreviated as En-2. (9) Citrobaca Freundy
cter frundii, food-derived strain): Cf
Abbreviated. (10) Klebsiera Pneumonia
Lla pneumoniae, food-derived strain): K below
Abbreviated as p. (11) Klebsiel Oxytoca
la oxytoca, food-derived strain): hereinafter abbreviated as Ko
You (12) Serratia marcescens
marcescens, food-derived strain): hereinafter abbreviated as Sm
You (13) Proteus Mirabilis
irabilis, food-derived strain): hereinafter abbreviated as Pm. (14) Salmonella Newport
(la newport, strain derived from feces): hereinafter abbreviated as Sn
You (15) Salmonella typhimurium
lla typhimurium, fecal-derived strain):
Abbreviated as St.

Judgment result of fluorescence reaction on the test sheet of various bacterial solutions Bacterial species Inoculated amount 36 ° C culture 44.5 ° C culture Ec-1> 300 ++++++++ 144 +++++++++ (27) ++ (21) 2+ (9) + (2) 0+ (1) -Ec-2> 300 +++++++ 59 +++++ 5+ (12) + (8) 0+ (1) + (1) 0−−Ec-3> 300 ++++ +++ 212 +++++++ 32 (++) (32) ++ (19) 0+ (1) + (1) 0−−Ec-4> 300 +++++++++ 168 ++++++ (25) +++ (17) 2+ (8) + (4) 0−−Ec-5> 300 ++++++++ 288 +++++++ 23 +++ (29) ++ (20) 4+ (10) + (6) 0−−Ec-6> 300 +++++++ 284 ++++++ 17 +++ (21) ++ (19) 2+ (7) + (4) 0−−En−1> 300−−43−−2−−0−−0−− En-2> 300 −−> 300 − − 69 − − 11 − − 0 − − Cf> 300 − − 93 − − 10 − − 1 − − 0 − − Kp> 300 − − 288 − − 32 − − 4 − − 0 − − Ko> 300 − −192−−18−−3−−0−−Sm> 300−−172−−16−−11−−0−− Pm>300−−164−−15−−2−−0−−Sn> 300 −−224−−30−−2−− 0 −−− St> 300−−196−−22−−1 −−− 0−− Judgment standard +++: Fluorescence reaction over the entire test sheet ++: Half of the test sheet Fluorescent reaction + If the spot on the test sheet is counted -: If the spot on the test sheet is not detected (): number of spots

From the above results, for all 6 E. coli strains,
A fluorescent reaction could be detected even when the amount of inoculated bacteria was less than 1 to 10, and no fluorescent reaction was detected for the other 9 strains of enterobacteria.

[Embodiment 2] Escherichia coli (E. coli) bacteria amount of 1 to less than 10, 10 to less than 100, 100 to 10 ³
The bacterial solution in three stages was adjusted so that the bacterial amount was less than approximately.
In addition, coliform bacteria other than E. coli (Citrobacter,
Enterobacter and Klebsiella are mixed respectively,
The bacterial solution was adjusted in three stages so that the bacterial amount was 10 ⁴ to less than 10 ⁵ , 10 ⁵ to less than 10 ⁶ , and 10 ⁶ or more.

Next, 9 kinds of mixed bacterial liquids were prepared by combining the bacterial liquids of both 3 stages. 1 ml of each mixed bacterial solution was adsorbed on the same test sheet as in Example 1 and cultured in the same manner to measure the fluorescence reaction. The inoculum size was calculated according to the official method, and the determination was made in the same manner as in Example 1. The results are shown in Table 1.

[0030]

[Table 1]

From the results shown in Table 1, it was confirmed that the fluorescence reaction of Escherichia coli was not affected by the amount of other coexisting coliform bacteria and that the test sheet of the present invention could specifically detect Escherichia coli.

Example 3 10 g of each of the following foods (12 samples) and 90 ml of physiological saline were mixed to prepare 12 kinds of 10-fold emulsion. Next, 1 ml of each emulsion was adsorbed on a test sheet similar to that in Example 1, and after culturing in the same manner as in Example 1, the fluorescence reaction was measured. In addition, a small amount of Escherichia coli was added to the same sample, the culture was performed in the same manner, and the fluorescence reaction was measured. The number of coliform bacteria in the food was calculated by the official method, and the determination was made in the same manner as in Example 1. Table 2 shows the results. Foods used Ground pork 5 samples Cut raw vegetables 3 samples Tofu 1 sample Tamari water 2 samples Cut sales ham 1 sample

[0033]

[Table 2]

From the results in Table 2, when actually inspected using food, coliform bacteria were recognized by the official method from foods other than tofu and cut ham, of which 4 out of 5 samples of ground pork in the test sheet of the present invention Fluorescent reaction was observed in one of the three samples in the samples and cut vegetables, and in two of the two samples in the pooled water, and Escherichia coli was detected.

E. Upon inoculation of E. coli, a fluorescence reaction was also observed in a sample in which no fluorescence reaction was observed before inoculation, and it was confirmed that the test sheet of the present invention specifically reacts only with E. coli.

[0036]

As described above, according to the test sheet for detecting Escherichia coli of the present invention, Escherichia coli present in foods and drinks can be specifically, easily and reliably detected. In this case, it is possible to detect Escherichia coli satisfactorily and specifically even in the presence of a mixture of bacteria other than Escherichia coli belonging to the coliform group and even when the amount of E. coli is small. Furthermore, it is cheaper than a conventional test kit using a test tube or the like, and is easily and economically disposed of.

[Brief description of drawings]

FIG. 1 is a plan view showing an example of a test paper used in the present invention.

[Explanation of symbols]

 1 Test paper 2 Sheet body 3 Cut line 4 Knob part 5 Test part

Claims (2)

[Claims] 1. A method for detecting Escherichia coli characterized in that a sheet-shaped substrate is impregnated with a detection solution prepared by adding 4-methylumbelliferyl-β-D-glucuronide as a reaction substrate to a broth medium containing a culture reagent. Test sheet. 2. The test sheet for detecting Escherichia coli according to claim 1, wherein L-asparagine, casamino acid and yeast extract are used as culture reagents.