JPH0363571A - Inspection kit for coliform bacilli - Google Patents

Abstract

PURPOSE:To detect most of coliform bacilli by using a carrier coated with the polyclonal antibody of the strain belonging to the enteric bacteria and a compd. which conjugates a labeling enzyme with this antibody as essential constituting elements. CONSTITUTION:An anti-Salmonella antibody soln. and an Enterobacter cloacae antibody soln. are each partly mixed and the mixture is diluted with a phosphoric acid buffer soln. The soln. is dispensed into a 96-hole type microtiter plate and is incubated; thereafter, the antibody soln. is discarded and a sodium carbonate buffer soln. contg. egg white albumin is added to the plate and the plate is freeze-dried. The anti-Salmonella antibody is dissolved in the sodium carbonate buffer soln. to prepart the enzyme-labeled anti-Salmonella antibody soln. An enzyme-labeled ante-Enterobacter antibody is similarly prepd. The most of the coliform bacilli are detected in this way.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a test kit for coliform bacteria, which is a sanitary indicator for foods, drinking water, and the like.

(Prior art) The official definition of coliform bacteria is "all Durham-negative, non-spore-forming short rods that are aerobic and facultatively anaerobic and that decompose lactose and produce gas." ing. Escherichia and Talepsia
bsiella) and other bacteria belong to this category. Food and drinking water containing coliform bacteria must have come into direct or indirect contact with animal feces, urine, etc. during handling, and must be unclean food that may be contaminated with pathogenic bacteria. means. Therefore, testing for coliform bacteria is a very important test in terms of food hygiene.

Traditionally, the official method for measuring coliform bacteria is to use Brilliant Green Lactose Bile Broth Medium (BGLB) or lactose broth medium containing a Durham tube, and then add a food sample to it and wait for 24 or 48 hours for 35 to 35 hours.
Cultivate at 37° C. and observe whether gas accumulation is observed in the Durham tube. If gas accumulation is observed, further confirmatory testing using Endo medium or the like must be performed. If a definitive test is performed, a long period of culture of 48 to 72 hours is required. As a result, pre-shipment inspections and sanitary controls in each process at processed food manufacturing facilities are nearly impossible. Although there is a simple method using paper strips, etc., it is inaccurate and requires about 12 hours of culturing time.

These tests require sterile equipment and well-trained personnel.

(Problems to be Solved by the Invention) Recently, measurement kits applying enzyme-linked immunoassay and DNA probe technology for testing several types of Escherichia coli and Salmonella bacteria have been commercially available. These methods are simpler and require shorter testing time (approximately A) than the above methods. However, these are created for the purpose of specifically detecting individual bacterial species such as E. coli or Salmonella, and are not intended to detect the entire coliform group.

The following conditions are required for the method of testing coliform bacteria. 1) Most coliform bacteria can be detected, and 2) It is easy to operate. Therefore, an object of the present invention is to provide a coliform testing method that satisfies these conditions and an instrument used therefor.

(Means for Solving the Problems) The present invention relates to the Enterobacteriaceae (enterobacteriaceae).
This is a test kit for coliform bacteria whose main components include a carrier coated with a polyclonal antibody against a strain belonging to B. ceae), and a compound in which a labeled enzyme is bound to the antibody. Preferably, Escherichia coli comprises a carrier coated with a polyclonal antibody obtained from boiled bacterial cells of a bacterium belonging to the family Enterobacteriaceae, an enzyme-labeled antibody obtained by binding a labeled enzyme to the antibody, a substrate for the enzyme, and a chromogenic substance. This is a group test kit.

Any bacterial strain belonging to the Enterobacteriaceae family can be used in the present invention as long as it belongs to this family, but antibodies that have cross-reactivity to coliform bacteria and pathogenic Enterobacteriaceae bacteria To obtain Enterobacter cloacani (E.
terobacter cloacae), or Salmonella typhimurium
himurium) is preferred.

Polyclonal antibodies can be obtained from the serum of animals such as rabbits, Guinea Big, rats, and mice by injecting boiled bacterial cells into the animals.

These antisera may be used as they are, or may be purified using known means. Moreover, two or more types of antisera may be mixed. As a carrier, a plastic test tube, a microtiter plate, a filter paper, a nitrate cellulose membrane, a plastic bead, a glass bead, a nonwoven fabric, or the like is used. As the labeling enzyme, horseradish peroxidase, alkaline phosphatase, β-galactosidase, etc. are used. As a substrate for peroxidase,
Hydrogen peroxide or urea peroxide is used.

The antibody and the labeled enzyme are bound by known means such as the glutaraldehyde method, the periodic acid method, the maleimide method, and the pyridyl disulfide method. ABTS as a chromogen
(2,2'-azinodi(3-ethylbenzothiazoline)
-6'-sulfonic acid), 5-aminosalicylic acid, luminol, or TMB (3,3",5.5'-tetramethylbenzidine). As a substrate for alkaline phosphatase, p-nitrophenyl phosphoric acid is used. acid, 0- or p as a substrate for β-galactosidase
-nitrophenyl-β-D-galactoside, or 4
-Methylumbelliferyl-β-D-galactoside is used.

The method of using the coliform test kit of the present invention will be described in detail below. Dilute food or drinking water samples to an appropriate amount with sterile physiological buffer, add to bouillon medium, and incubate at 37°C.
Incubate for at least 4 hours. This culture solution is added to the antibody-coated carrier and incubated for 30 minutes at room temperature. At that time, Bacillus subtilis was also used as a negative control.
) A suspension of boiled IFO13719 cells is simultaneously added to the carrier and incubated. After that, the unadsorbed substances are
Wash thoroughly several times with buffer, etc. A solution of an antibody bound to a labeled enzyme is added thereto and incubated for 30 minutes at room temperature. Wash several times with buffer, etc., add a substrate or a substrate and a chromogenic substance, and incubate at room temperature. The same thing is done for the negative control at the same time as the sample.

After 30 minutes, dilute sulfuric acid is added to stop the reaction, and absorbance or fluorescence intensity is measured. When the amount of coliform bacteria in the culture solution after incubation is 10 s/ml or more, the absorbance or fluorescence intensity is proportional to the logarithm of the amount of bacteria. The absorbance or fluorescence intensity of the sample is 2.5% higher than that of the negative control.
When the amount is more than double that, the coliform group in the sample can be determined to be positive.

(Effects of the Invention) According to the present invention, it is possible to provide a test kit that is easy to operate, has high sensitivity, can detect almost all coliform bacteria, and can be tested in a short time.

(Example) Next, the present invention will be explained with examples. Note that the present invention is not limited only to the examples.

Example 1 Boiled Salmonella typhimurium IFO12529 cells in physiological buffer pH 7.4 were intravenously injected into the abdomen of rabbits once a week for 3 weeks, then intravenously into the same rabbits once a week for 3 weeks. did. After 8 weeks, blood was collected and antiserum was obtained. Add this antiserum 5-80 to phosphate-buffered saline.
．． Column 1 using 2n11 diluted 10 times with a solution containing 5% and bound to formylcellulofine (manufactured by Chisso 01, trade name) and lipopolysancalide as a carrier.
0 m1 (size: diameter 1 cm, height 13 cm). The column was washed with the same 3M buffer, and then the anti-Salmonella antibody was eluted with glycine-HCl buffer pH 2.5.

Tris hydroxylamine was added to the eluate until the pH reached 8.5, and this was dialyzed against phosphate buffered saline overnight to obtain an anti-Salmonella antibody solution.

An antibody solution of Enterobacter cloacani IFO13535 was also obtained in the same manner.

Mix these antibody solutions one part at a time and add 1 ml to 0.
．． Diluted 500 times with 01M phosphate buffer pH 7.2,
100 μl each was dispensed into a 96-well microtiter plate. After incubating the plate at 37°C for 1 hour, the antibody solution was discarded. Next, egg white albumin 3%
Including 0. Add 300 pl of OIM sodium carbonate buffer p) 17.5 to each well and incubate for 1 hour at 37°C, then discard the contents and wash 3 times with 0.01 M phosphate buffer pH 1,0. I drained the water. The plate was freeze-dried, placed in a plastic bag containing a desiccant such as silica gel, and stored at 4°C.

It remained stable in this state for at least 6 months.

On the other hand, 40 mg of the anti-Salmonella antibody mentioned above was added to ETA
Grade horseradish peroxidase 30n+
g of 0.1M sodium carbonate buffer (pH 7.0) 5
- mixed with a solution dissolved in. While slowly stirring with a magnetic stirrer, 50 mg of 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide was gradually added, and the pl (pl) was diluted with 0.IN hydrochloric acid and 0.IN sodium hydroxide to 6.0~ 20 at room temperature while adjusting to 7.0.
Allowed time to react. Transfer the reaction solution to Cellulofine GCL-10
The mixture was passed through a column of 00sf (manufactured by Chisso ■, trade name) to separate and remove unreacted substances. This was used as an enzyme-labeled anti-Salmonella antibody solution.

Similarly, an enzyme-labeled anti-Enterobacter antibody was prepared.

On the other hand, 121FO3301 (Es.
cherichia coli), Salmonella typhimurium IFOL2529, Enterobacter cloacani IFO13535, Yersinia ardovuae J
CM5892 (Yersinia aldovae),
Klebsiella terrigena JCM 1687 (Klebs
iella terri ena), Aeromonas hydroxyl subsp. hydrophila JCM
1027 (Heromonas h dro hila
5ubs eciesh dro hilla), Serratia marcescens JCM1239 (Serra
tia marcescens), Proteus vulgaris IFO3851 (Proteus vulgaris)
1 platinum loop of bacterial cells of s) was incubated in broth medium at 35°C.
, and cultured for 3 hours. This culture solution was soaked in boiling water for 5 minutes, allowed to cool, and then added in 100 pl portions to the previously prepared microtiter plate and incubated at 37°C. After 30 minutes, each well of the plate was washed three times with distilled water containing 0.05% Tween 20.

Next, a mixture of two types of enzyme-labeled antibodies was added to each well and incubated at 37°C. After 30 minutes, each well of the plate was again washed three times with distilled water containing 0.05% Tween 20. After draining well, AB
A mixture of TS (2,2'-azinodi(3-ethylbenzothiapurine)-6'-sulfonic acid) and 0.1M hydrogen peroxide in citrate buffer pH 5.5 was added at 0.21N! In addition 1
The mixture was left standing for 5 minutes to develop color, and the absorbance at 405 nm was measured using an immunoreader. The results are shown in Table 1.

Table 1 *Second-class luminous intensity of 0.2 or more was considered positive. Negative control is 0.0
It is 8.

As shown in Table 1, positive values were shown for Salmonella, Escherichia, Yersinia, Enterobacter, and Klebsiella. Results were negative for Aeromonas, Serratia, and Proteus. This result is favorable because Aeromonas, Serratia, and Proteus do not belong to the coliform group. Strictly speaking, Salmonella and Yersinia do not belong to the coliform group, but this result is favorable because it is desirable to detect them together with coliform bacteria as bacteria that cause food poisoning.

Example 2 After adding 12 pieces of Escherichia coli to 50 g of ground beef, diluting and suspending it with 200 ml of physiological saline. 1 ml of solution diluted 103 times and 10' times with physiological saline
1, 0.1-10.01- were inoculated into BGLB medium containing five Durham fermentation tubes and BGLB medium 10- without Durham fermentation tubes, respectively.

The BGLB medium containing the Durham fermentation tube was cultured at 37°C for 24 or 48 hours, and the presence or absence of gas generation was examined. The results are shown in Table 2 as a comparative example.

Table 2 As shown in the table, Example 2 had better sensitivity than Comparative Example. The time required was 48 hours in the comparative example, but in Example 2
The time was significantly shortened to 7 hours.

After culturing the BGLB medium without fermentation tubes at 37°C for 5 hours, enzyme immunoassay (EL, l
5A) was performed. The results are shown in Table 3.

Claims (2)

[Claims] (1) Enterobacteriaceae
A test kit for coliform bacteria, the main components of which are a carrier coated with a polyclonal antibody against a strain belonging to the group Ae), and a compound in which a labeled enzyme is bound to the antibody. (2) The strain is Salmonella typhimurium (￥Sal
monella typhimurium￥) and Enterobacter cloacae (￥Enterobacter
2. The test kit according to claim 1, wherein the test kit is both R cloacae ¥).