JP2002000257A - Test sheet and method for detecting staphylococcus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect Staphylococcus aureus existing in a food and a beverage, etc., specifically, simply and surely. SOLUTION: The test sheet and the method for detecting a staphylococcus are characterized in that a sheetlike substrate body is impregnated with a detection solution obtained by adding 4-methylumbelliferyl phosphate as a reaction substrate to a culture reagent containing a tellurite and lithium chloride.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a simple and convenient site-oriented test sheet for staphylococcal detection and a method for detecting staphylococci.

[0002]

BACKGROUND OF THE INVENTION Staphylococci are Gram-positive bacteria belonging to the Micrococcus family which are widely distributed in nature and are currently classified into many types (23 bacterial species, 4 subspecies). Among them, S. aureus (S. aureus) mainly invades human and animal skin and mucous membranes and causes various purulent diseases and causes toxin-type bacterial food poisoning. Staphyloc
occus aureus).

[0003] In addition, since these staphylococci are detected in large amounts in healthy human skin, nasal mucosa, feces, soil, sewage, and the like, there are very many opportunities to contaminate foods and the like.

[0004] As a test method for such Staphylococcus aureus, a bacterial laboratory has used an egg yolk reaction (lipase reaction and lecitoviterin reaction) in which yolk is added to various staphylococcal selection media to react with lipase produced by staphylococcus. ) And Staphylococcus epidermidis (Staphylococcus epidermi)
dis) and other bacteria of the Microcassaceae family.

[0005] However, in the test sheet, the yolk could not be technically adsorbed on the test paper and dried, and it was difficult to incorporate the yolk reaction.

[0006] For this reason, attempts have been made to improve the composition of the medium that suppresses the growth of other than staphylococci, but the results are inconsistent with the results of the yolk-added mannitol salt medium (MSEY medium) and are not sufficiently reliable. Was.

[0007] The present invention has been made in view of the above problems, and provides a highly specific method for detecting the presence or absence of pathogenic Staphylococcus aureus in a sample of food or the like and the degree of contamination with a simple operation. An object is to provide a test sheet for detection and a method for detecting staphylococci.

[0008]

Means for Solving the Problems and Embodiments of the Invention As a result of intensive studies to achieve the above object, the present inventor has found that a culture reagent containing tellurite and lithium chloride is used as a reaction substrate as a reaction substrate. -A test sheet for staphylococcus detection obtained by impregnating a sheet-like substrate with a detection solution to which methylumbelliferyl phosphate is added, combining tellurite, lithium chloride and 4-methylumbelliferyl phosphate That they work effectively,
It has been found that the detection of highly specific staphylococci, which is consistent with the results of the yolk-added mannitol salt medium (MSEY medium), can be performed easily and reliably.

That is, the present invention provides the following test sheet for detecting staphylococci and a method for detecting staphylococci. Claim 1: A staphylococci characterized by impregnating a sheet-like substrate with a detection solution obtained by adding 4-methylumbelliferyl phosphate as a reaction substrate to a culture reagent containing tellurite and lithium chloride. Test sheet for detection. Claim 2: 0.4 to 0.75 g / L of potassium tellurite
Reagent 1000 containing 5 and 7 g / L of lithium chloride
pH 6.7 to which 0.05 to 0.5 g of 4-methylumbelliferyl phosphate was added as a reaction substrate to mL.
7. A sheet-like substrate made of water-absorbing paper or cloth impregnated with the detection liquid of 7.1, wherein the specimen is brought into direct contact with the sheet-like substrate to transfer the specimen from the specimen. 2. The test sheet for detecting staphylococci according to 1. Claim 3: The test sheet according to claim 1 or 2 is brought into direct contact with a test object to transfer a sample from the test object,
This test sheet is placed in a petri dish or a transparent bag made of plastic film and cultured at 36 to 44.5 ° C. for 18 to 48 hours. The cultured sheet is irradiated with ultraviolet rays to determine the presence and degree of the fluorescent reaction. A method for detecting staphylococci characterized by the following.

According to the present invention, the action of tellurite and lithium chloride suppresses the growth of bacteria other than staphylococci, such as Staphylococcus aureus and Staphylococcus epidermidis, and staphylococci reduces and reduces tellurite. By forming black colonies, staphylococci can be primarily sorted. Then, the reaction substrate 4-methylumbelliferyl phosphate is hydrolyzed by the phosphatase enzyme produced by the pathogenic Staphylococcus aureus, as shown in the following reaction formula, and the fluorescent substance 4-methylumbelliferone is released. ,
By irradiating the released 4-methylumbelliferone with long-wave ultraviolet rays (around 366 nm) and measuring the fluorescence reaction, only the pathogenic Staphylococcus aureus can be specifically detected.

[0011]

Embedded image

In this case, the growth of bacteria other than staphylococci is inhibited by the action of tellurite and lithium chloride, and the phosphatase positive rate of Staphylococcus aureus is 100%. Slightly positive strains can be seen in cocci, and other micrococcus family, Aerococcus bacteria are completely negative,
The combination of 4-methylumbelliferyl phosphate, tellurite and lithium chloride makes it possible to test for highly specific pathogenic Staphylococcus aureus. In addition, the test sheet of the present invention does not require any special equipment, allows the specimen to be adhered on site and cultured, and simply irradiates with ultraviolet light to quickly and easily detect the pathogenic Staphylococcus aureus specifically. In addition to being able to be performed, the detection solution is impregnated in a sheet-like substrate, so that it can be detected at low cost using only a small amount of culture medium, culture reagents and reaction substrates, and is easily burned after inspection. And it can be safely disposed of.

Hereinafter, the present invention will be described in more detail. The test sheet for detecting staphylococci of the present invention is obtained by impregnating a sheet-like substrate with a detection solution obtained by adding 4-methylumbelliferyl phosphate as a reaction substrate to a culture reagent containing tellurite and lithium chloride. Things.

As the culture reagent, a culture reagent containing tellurite and lithium chloride is used. Particularly, 0.4 to 0.75 g / L of potassium tellurite and 5 to 7 g / L of lithium chloride are used. It is preferable to use a culture reagent having the following composition. <Culture reagent composition> Yeast extract 10 to 15 g / L polypeptone 5 to 10 g / L mannitol 5 to 7 g / L Potassium tellurite 0.4 to 0.75 g / L Lithium chloride 5 to 7 g / L Sodium chloride 35 to 80 g / L Casamino acid 5-7g / L Sodium pyruvate 5-7g / L

[0015] The culture reagent of the present invention can suppress the growth and growth of microorganisms other than staphylococci by containing tellurite and lithium chloride. Since black colonies are formed, staphylococci can be primarily sorted, and specificity can be improved.

The 4-methylumbelliferyl phosphate added as a reaction substrate to the culture reagent is
As described above, it is hydrolyzed by the enzyme phosphatase produced by Staphylococcus aureus to produce a fluorescent substance (4-methylumbelliferone). It can be detected. The amount of the reaction substrate 4-methylumbelliferyl phosphate to be added is not particularly limited, but is usually 0.05 to 0.5 g, preferably 0.05 to 0.1 g per 1000 mL of the culture reagent.
g. If the amount is too small, a good fluorescent reaction may not be obtained. On the other hand, the detection accuracy does not change even if there is too much,
On the contrary, it may be disadvantageous in terms of cost.

The test sheet of the present invention is obtained by impregnating a sheet-like substrate with a detection solution obtained by adding the above-mentioned culture reagent to the above-mentioned 4-methylumbelliferyl phosphate, and the detection solution has a pH of 6.9. It is preferable to be about ± 0.2. If the pH is out of the range of 6.9 ± 0.2, the staphylococcus is not suitable for growth and growth, and the fluorescent substance (4-methylumbelliferone) may show only weak fluorescence.
Various additives other than those described above may be added to the detection solution as long as they do not inhibit the growth and growth of staphylococci, and culture reagents and bacterium selection agents other than those described above can be appropriately added.

The sheet-like substrate impregnated with the detection liquid is not particularly limited in its material, shape and the like, and may be paper or cloth having water absorption. Those formed in a strip shape using paper having good properties are 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 filter paper.
Is impregnated with the above-mentioned detection solution. In this case, 3
Is a score line formed on one end side of the sheet-like substrate 2, one side is configured as a knob portion 4 and the other side is defined as a test portion 5 with the score line 3 as a boundary, whereby the test sheet 1 is inoculated with a specimen. Other than picking up the knob 4 with a finger and lifting it up, the other parts of the test sheet 1, especially the test section 5, are not directly touched by a finger, so that the test section 5 is not contaminated and the test section 5 is not contaminated. Can be determined to be derived from the sample, and the test results can be determined without error.

The means for impregnating the sheet-like substrate 2 with the detection liquid is not particularly limited, but a method of immersing the sheet-like substrate 2 in the detection liquid and impregnating the sheet-like substrate 2 with the detection liquid is preferably employed. You. In this case, there is no particular limitation on the amount of the detection liquid impregnated in the sheet-like substrate, and the test sheet for staphylococcal detection of the present invention can be obtained by drying the sheet-like substrate impregnated with the detection liquid.

The test sheet for detection of the present invention can be used for various foods and drinks, for example, fresh foods (vegetables, fruits, tofu, etc.), processed livestock products (meat, ham, cheese, etc.), furthermore, pool water, tap water, It can be widely used for well water, etc., and is not limited to indicators of food poisoning, fecal contamination of food and drink, water quality surveys, contamination of sandboxes in parks, and simple hygiene management at food factories, restaurants and homes. It can also be effectively used as a test sheet for public health inspection.

In the method of using the test sheet for detecting staphylococci of the present invention, first, a specimen is attached to the test sheet of the present invention.
In this case, when the test substance is water-soluble, the test substance can be used as it is, and when the test substance is in a viscous or solid state, the test substance can be appropriately diluted and mixed with physiological saline to prepare the test substance. The amount of the sample attached to the test sheet is not particularly limited, but usually about 1 mL is suitable. In addition,
When the test object is a solid such as various foods, the test sheet can be directly contacted to adhere the sample.

Next, the test sheet to which the sample is attached is subjected to culture. The culture conditions are preferably at a temperature of 36 to 44.5 ° C, and particularly preferably at a temperature of 35 to 38 ° C, which is a general culture condition. The culturing time is not particularly limited, but is generally 18 to
48 hours, preferably 24-48 hours. As a specific culture method, for example, a test sheet to which a sample is adhered is placed in a petri dish or a transparent bag made of plastic film or the like in order to avoid germs and drying, and placed in an incubator at 35 to 38 ° C. for 24 to 48 hours. It can be done by preserving it.

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 the degree of the fluorescent reaction are measured. In this case, in the present test sheet, when the number of staphylococci is large, a fluorescent reaction that covers the entire sheet is obtained, and when the number of staphylococci is small, a fluorescent reaction occurs in a spot shape on the sheet. Therefore, in the test sheet of the present invention, the degree of the fluorescent reaction generated on the test sheet by the naked eye (the number of spots, a fluorescent reaction on half of the test sheet, a fluorescent reaction on the entire test sheet, etc.)
By determining, the approximate amount of bacteria can also be examined. When the test sheet with spots is stored, refrigerated storage is suitable.
Decreases in 0 days. Further, the wavelength of the ultraviolet light applied to the test sheet is preferably about 366 nm.

In the method for storing the test sheet for detecting staphylococci of the present invention, it is desirable to store the test sheet in a cool and dark place (such as a refrigerator) from the viewpoint of the stability of the reagent and the like. However, spots are observed even after 7 months of storage at room temperature, and storage for a certain period of time is possible even at room temperature.

It is to be noted that the used test sheet may be contaminated with other bacteria even if no fluorescent reaction occurs. Therefore, it must be disposed of by incineration in order to prevent infection and the like. In this case, the test sheet of the present invention is
Since it is a sheet made of paper or cloth, it can be easily incinerated and disposed, and disposal is simpler and more economical than the conventional detection kit method using a test tube or the like.

[0026]

EXAMPLES The present invention will be described below in detail with reference to Examples and Comparative Examples, but the present invention is not limited to the following Examples.

Example 1 A detection solution having the following composition was prepared, and a filter paper cut into strips was immersed in the detection solution to impregnate the filter paper (sheet-like substrate) with the detection solution. This was dried to obtain a staphylococcal test sheet. <Detection liquid composition> Yeast extract 10.0 g / L polypeptone 5.0 g / L mannitol 5.0 g / L potassium tellurite 0.4 g / L lithium chloride 5.0 g / L sodium chloride 75.0 g / L casamino acid 5.0 g / L sodium pyruvate 5.0 g / L 4-methylumbelliferyl phosphate 0.1 g / L pH: 6.9

Next, the detection sensitivity of the obtained test sheet was
The measurement was performed using the following test strains (1) to (5). In addition,
All of the test strains used were stocks of Sun Chemical Co., Ltd. (1) Staphylococcus aureus (Staphy
lococcus aureus; hereinafter abbreviated as Sa. (2) Staphylococcus epidermidis (Sta)
phylococcus epidermidis; hereinafter abbreviated as Se. (3) Staphylococcus Ssp (Staphylo)
coccus Ssp; hereinafter abbreviated as Ssp. (4) Escherichia coli
oli; hereinafter, abbreviated as EC. (5) Klebsia
lla aerogenes; hereinafter abbreviated as Ka. )

Measurement of Detection Sensitivity The test strains (1) to (5) were cultured in tryptic soy broth at 35 ° C. for 24 hours. Less than 1-10,
Serial dilution was performed so that the number was 10 or more and less than 100, 100 or more and less than 1000, and 1000 or more.

The test sheet was immersed in each of the diluted bacterial liquids and cultured in a thermostat at 35 ° C. for 24 hours and 48 hours, taken out at each cultivation time, and the black color initially formed on the test sheet with the naked eye Spots were measured according to the following criteria. Table 1 shows the results. <Criteria> +++: 100 or more black spots ++: 30 or more and less than 100 black spots +: 1 or more and less than 30 black spots-: 0 black spots

[0031]

[Table 1]

From the results shown in Table 1, when the number of inoculated bacteria was very small, no black spots were observed in 24 hours of culture, but Sa became positive after 48 hours of culture. In addition, when the inoculated bacterial amount is up to 100 cells / mL, it can be detected even after culturing for 24 hours, but the number of spots increases after culturing for 48 hours, and black spots become clear. In contrast, the amount of inoculated bacteria was 100 or more and less than 1000 cells / mL or 1
When the number of cells exceeds 000 cells / mL, the species of Se and Ssp become 2
Even when cultured for 4 hours, black spots were formed, and when cultured for 48 hours, the number of bacterial cells was slightly small, but black spots were clearly observed.

Fluorescence Reaction Test In addition, the test sheet tested in the same manner was irradiated with ultraviolet rays (wavelength: 366 nm) by a UV lamp, and the fluorescence positive spot was measured according to the following standard. Table 2 shows the results. <Judgment criteria> +++: 100 or more fluorescent positive spots ++: 30 or more fluorescent positive spots and less than 100 spots +: 1 or more fluorescent positive spots and less than 30 spots-: 0 fluorescent positive spots

[0034]

[Table 2]

From the results shown in Table 2, Se and Ssp which formed black spots with the naked eye in Table 1 did not emit fluorescence, and of course, other bacterial species did not emit fluorescence. On the other hand, it was confirmed that Sa emits fluorescence and can specifically detect only Staphylococcus aureus.

[Example 2] Foods 8 foods such as cooked rice, 5 meats, 3 fried eggs,
Using a total of 30 specimens of 6 salads and 8 prepared dishes, 10 g of each specimen was aseptically weighed in a vinyl bag for stomacher, and 90 mL of sterile physiological saline was added thereto. Emulsions were prepared, and these were used as sample stock solutions.

Next, about 1 mL of each sample stock solution was added to a test sheet.
The cells were adsorbed and cultured in a thermostat at 35 ° C. for 24 hours and 48 hours. The cells were taken out at each culturing time, and the black colonies and fluorescent colonies formed on the test sheet at first were visually observed. Table 3 shows the results. In addition, each sample stock solution was diluted with sterile physiological saline for 1 hour.
Samples diluted 0-, 100-, and 1000-fold were cultured in the same manner, and black colonies and fluorescent colonies were measured. Table 4 shows the results.

[Comparative Example 1] As a comparison, 0.1 mL of the sample stock solution was placed on a plate of yolk-added mannitol salt medium (MSEY medium).
Was dropped, applied uniformly to the surface of the medium with a sterile conical rod, cultured in a thermostat at 35 ° C. for 24 hours and 48 hours, taken out at each culture time, and the number of colonies showing an egg yolk reaction was measured. Table 3 shows the results. In addition, each sample stock solution was diluted 10-fold, 100-fold, and 1000-fold with sterile physiological saline, and similarly cultured, and the number of colonies showing an egg yolk reaction was measured. Table 4 shows the results.

[0039]

[Table 3]

[0040]

[Table 4]

[Example 3, Comparative Example 2] Human-derived sample As a human-derived sample, two fingers of a subject were wiped with a sterile cotton swab, and the sample was shaken well in 20 mL of sterile physiological saline. Wipe with 2 samples undiluted in 20 mL of sterile saline, gargle for about 10 seconds with 30 mL of sterile saline, and use 14 samples undiluted in a stomacher plastic bag for a total of 18 samples Was.

Examples 2 and Comparative Example 1
Culture was performed in the same manner as described above, and the detection rate was measured. The results are shown in Tables 5 and 6.

[0043]

[Table 5]

[0044]

[Table 6]

From the results shown in Tables 3 to 6, it can be seen that the test sheet of the present invention can be tested with high specificity which agrees very well with the results of the yolk reaction in MSEY medium, and that the number of staphylococci is 1
It can be detected if there are 0 or more, 2
Of course, accurate results can be obtained by culturing for 48 hours rather than culturing for 4 hours.

[0046]

As described above, according to the test sheet for detecting staphylococci of the present invention, the pathogenic Staphylococcus aureus present in foods and drinks is specifically and simply and reliably isolated from other cocci. Can be detected. Also,
It is cheaper than conventional test kits using test tubes and the like, and disposal is simple and economical. In addition, it can be easily inspected even in a facility without a laboratory or laboratory facilities, and can constantly inspect food raw materials, employees' hands and cooking utensils, and conduct on-site health management and food quality management for stakeholders. It can be easily incorporated.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a test sheet for detecting staphylococci of the present invention.

[Explanation of Signs] 1 Test sheet 2 Sheet-like substrate 3 Cut line 4 Knob part 5 Test part

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/14 C12Q 1/14 // (C12M 1/34 (C12M 1/34 B C12R 1: 445) C12R 1: 445) (C12N 1/20 (C12N 1/20 A C12R 1: 445) C12R 1: 445) (C12Q 1/04 (C12Q 1/04 C12R 1: 445) C12R 1: 445) F-term (reference) 4B029 AA07 AA09 BB02 CC02 CC07 FA03 FA04 FA07 HA02 4B063 QA11 QA18 QQ06 QQ16 QR50 QR66 QR69 QR84 QS12 QS24 QS28 QS36 QS39 QX02 4B065 AA53X BA23 BB02 BB13 BD50 CA46

Claims (3)

[Claims] 1. A grape obtained by impregnating a sheet-like substrate with a detection solution obtained by adding 4-methylumbelliferyl phosphate as a reaction substrate to a culture reagent containing tellurite and lithium chloride. Test sheet for detection of cocci. 2. 0.4 to 0.75 g of potassium tellurite
Reagent containing 10 / L and 5 to 7 g / L of lithium chloride
To 0.05 mL of a reaction substrate, 4-methylumbelliferyl phosphate was added in an amount of 0.05 to 0.5 g.
The detection solution of 7 to 7.1 is impregnated into a sheet-like substrate made of water-absorbing paper or cloth, and the test object is brought into direct contact with the sheet-like substrate to enable the transfer of the sample from the test object. The test sheet for detecting staphylococci according to claim 1. 3. The test sheet according to claim 1 or 2 is brought into direct contact with a test object to transfer a sample from the test object, and the test sheet is placed in a petri dish or a transparent bag made of a plastic film. 18-48 at ~ 44.5 ° C
A method for detecting staphylococci, comprising culturing for a period of time and irradiating the cultured sheet with ultraviolet light to determine the presence and degree of a fluorescent reaction.