JP2017169455A - Signal detection method from bacteria in foods using fluorescence in situ hybridization combined with culture - Google Patents

Abstract

[Problem] Livestock products and marine products have relatively low fluorescence noise, but agricultural products, especially green and yellow vegetables, contain pigments, and thus have a problem of fluorescence noise. Therefore, the development of a new fluorescent signal automatic detection method that does not generate noise in a wider variety of foods was an issue.
By means of detecting a fluorescence signal derived from a DNA probe on a membrane filter with a specific color and brightness using a low-magnification optical system using a fluorescence in situ hybridization method combined with culture. Provided is a microorganism testing method for automatically detecting a new fluorescent signal free from noise by detection.
[Selection] Figure 1

Description

  The present invention detects the fluorescence signal of the micro-colony of the target microorganism by distinguishing it from noise based on color, brightness, and size using in-situ hybridization with culture in industrial fields such as food manufacturing and environmental analysis. On how to do.

  Microbiological tests that detect or count specific microorganisms such as E. coli, Salmonella, Listeria, Vibrio parahaemolyticus are essential for sanitary evaluation in food and the environment. In general, these tests are performed by a culture method, and multiple items (enrichment culture, isolation culture, estimation test, confirmation test) must be performed and the number of days required for the test must be 1-8 days until positive confirmation is obtained. It took time and effort.

  The present inventors have developed a culture combined fluorescence in situ hybridization method using a membrane filter for the above-mentioned microorganism inspection in the industrial field (Patent Document 1, Patent No. 4785449).

Since this method can measure per 0.1 g of food, it has the same limit of detection (10 CFU / g) as the pour plate culture method, and it can detect and count live specific bacteria in food quickly (7 hours) Within 24 hours). In addition, a fluorescence image acquired over the entire membrane filter was input to the image processing means and subjected to image processing, so that the number of microorganisms to be detected on the membrane filter could be automatically counted. In many fresh foods, the image processing method is cheaper because the fluorescence image of the microcolony is much better in terms of light quantity and light emission area than the fluorescence image of inclusions and the like that cause noise, and can be easily distinguished. Counting is possible with a general particle counting process that can be realized in real time even with image processing equipment. For example, when TAMRA is labeled on a DNA probe for detection of Enterobacteriaceae and the fluorescence in situ hybridization method combined with culture is used, food with E. coli added ((Bacterial count: approx. 10 ³ CFU / g) pork , Squid, mackerel, chicken cutlet, etc.) were accurately counted.

  In addition, a membrane filter with a high outer frame was invented as a test kit that simplifies the fluorescence in situ hybridization method combined with culture (Patent Document 2, Patent No. 4950433).

  As a standard test method for microorganisms from food, there is NIHSJ-16 (Non-patent Document 1).

Japanese Patent No. 4785449 Japanese Patent No. 4950433

Enterobacteriaceae community count method NIHSJ-16 Standard test method for microorganisms from food (http://www.nihs.go.jp/fhm/mmef/index.html)

Fluorescence in situ hybridization combined with culture has paved the way for application to microbial testing of livestock and marine products in industrial fields such as food manufacturing. As described in Example 2, the method of Patent Document 1 is a food sample (cabbage, lettuce) in which the detection target is Enterobacteriaceae and Escherichia coli as a test bacterium is added at a final concentration of 1.0 × 10 ² or more CFU / g. , Beer orange, tofu, pork, chicken cutlet, squid, etc.) were accurately counted for Enterobacteriaceae.

  By the way, it is known that there are components that emit autofluorescence in various foods. In particular, since agricultural products, especially green and yellow vegetables contain pigments, there is a concern that autofluorescence may affect measurement. . In order to further examine the applicability of the technique of Patent Document 1 to a wide range of foods, the inventors have used the fluorescence in situ hybridization method combined with culture described in Patent Document 1 to automatically count the fluorescence signal of the food autofluorescence signal. The effect on the test was tested. Twelve kinds of foods shown in Table 1 were used as samples. In the test, a simple test kit was prototyped and used. This kit consists of a 55 mm diameter and 15 mm high plastic petri dish with 5 sheets of 50 mm diameter filter paper, and a membrane filter with an outer frame described in Patent Document 2 (diameter 47 mm, pore size 0.8 μm) The following disc-type membrane filter and ring-shaped acrylic with an outer diameter of 47 mm, an inner diameter of 42 mm and a height of 15 mm are bonded together) with the filter face down and covered with a plastic petri dish with a diameter of 60 mm and height of 10 mm And

  25 g of each food sample was placed in a stomacher bag, and further 225 ml of a bacterial test diluent (phosphate buffer) was added and suspended to prepare a 10-fold diluted sample (sample 0.1 g / ml). 1 ml of that was added to a simple test kit. After the diluted solution was filtered from the filter surface onto the filter paper, the membrane filter with an outer frame having the height was immersed in ethanol for 20 minutes to be fixed and dried. Next, the membrane filter with the outer frame with the height is placed in a plastic petri dish with a diameter of 55 mm and a height of 15 mm, and a hybridization buffer (20% formamide, 0.01% sodium dodecyl sulfate [SDS], 0.9 M sodium chloride, 20 mM). 1.5 ml of Tris-HCl [pH 7.4]) was added and the mixture was allowed to stand at 46 ° C. for 20 minutes. Next, a washing solution (0.01% sodium dodecyl sulfate [SDS], 180 mM sodium chloride, 20 mM Tris-HCl [pH 7.4]) was added and left at 46 ° C. for 15 minutes, and then drained (washing). The membrane filter with an outer frame having a height was rinsed with distilled water and dried. As a result, a food sample specimen containing 0.1 g of the food sample and containing no bacterial microcolony (size 20 μm to 150 μm) was prepared. Automatic counting (means for discriminating microcolony from noise in terms of light intensity and light emitting area) described in Patent Document 1 was performed on the entire membrane filter surface of various food sample specimens.

  The fluorescence detection optical conditions are the excitation wavelength 460-500 nm and the fluorescence wavelength 515 nm or more as the first fluorescence detection optical condition, the excitation wavelength 520-560 nm, the fluorescence wavelength 570 nm or more as the second fluorescence detection optical condition, The fluorescence detection optical conditions of 3 were an excitation wavelength of 590-650 nm and a fluorescence wavelength of 660 nm or more. When a fluorescently labeled DNA probe is not used, all the fluorescence counted under the above conditions is noise (a food-derived autofluorescence whose light amount and light emitting area are equivalent to a microcolony). As a result, many foods were found to contain noise. Although the number of noises varies depending on the type of sample and the fluorescence detection conditions, in general, the first fluorescence detection optical condition was very noisy for all 12 types of food samples. Under the second fluorescence detection condition, noise was detected in cabbage, carrots, tofu and mentaka, but not in other foods. Under the third fluorescence detection condition, 100 units / g of noise was detected from lettuce and green beans of green produce (Table 1).

  In order to set the detection limit of the combined fluorescence in situ hybridization method to 10 CFU / g, noise must not be detected in the measurement of 1 ml of a 10-fold diluted food sample. The automatic counting means of the in situ hybridization method with a detection limit of 10 CFU / g described in Patent Document 1 can be applied to yellow-red agricultural processed products, livestock products and marine products in the second fluorescence detection means, and the third fluorescence detection means Then, it was applicable to yellow-red agricultural products, yellow-red agricultural products, white agricultural products, livestock products, processed livestock products, and marine products.

  For application in the industrial field, it is desirable to be able to apply to as wide a variety of food samples as possible. Therefore, the development of a new fluorescent signal automatic detection method that does not generate noise in a wider variety of foods was an issue.

  As shown in Table 1 above, livestock products and marine products have relatively low fluorescence noise, but agricultural products, especially green and yellow vegetables, contain pigments, so it is considered that the problem of fluorescence noise is large.

  The present inventor has developed a new automatic signal detection method for distinguishing between fluorescence signals in culture combined fluorescence in situ hybridization and food-derived fluorescence noise, so that autofluorescence of various foods and fluorescence used for labeling of DNA probes are used. The fluorescence signals of the substances were compared.

  Usually, the fluorescence measurement is performed by measuring the fluorescence intensity (brightness) in a specific wavelength range with respect to the excitation wavelength. As a result of the examination, the inventors of the present application have studied the fluorescence of the combined fluorescence in situ hybridization method. In order to distinguish fluorescent noise from signal and food, it is difficult to distinguish it from noise only by the brightness of the limited wavelength range of the fluorescence for observation, but the color and brightness of the observation object are measured, It was found that automated measurement can be performed by evaluating a signal that falls within a certain range as a signal derived from a fluorescent probe.

  More specifically, the inventors of the present invention provide a membrane filter having an effective filtration area equal to or greater than the area of a circle having a diameter of 24 mm in a microorganism testing method such as food using a combined fluorescence in situ hybridization method. Filtered using, cultured so that the microcolonies are 20 μm to 150 μm in size, and propagated the specific microorganisms grown by the fluorescence in situ hybridization method combined with the culture using the low-magnification optical system of the membrane filter with a specific color. Thus, the present invention has been completed by finding that bacteria can be automatically detected with the accuracy required by food hygiene standards by automatic detection with a solid state element with brightness and brightness.

  Here, the signal (signal) and noise discrimination conditions can be generally determined as follows. That is, using a sample with known noise and signal (signal), the hue and brightness of the noise and signal (signal) are actually measured, and the discrimination condition based on the hue and brightness is determined from the optical characteristics of the signal (signal) and noise. be able to. Specifically, the Enterobacteriaceae count is counted from the method of the present invention by fluorescence measurement for each of the sample added with bacteria such as E. coli and the sample not added with bacteria such as E. coli, and at the same time, the count is performed by the standard method (control method). carry out. The sample may or may not include Enterobacteriaceae.

  For each sample, the device measurement conditions (lightness, hue) are examined so that the value of the device count according to the method of the present invention matches the count of the control method. When the count values of the two substantially match, noise and signal can be distinguished in apparatus measurement, and appropriate brightness and hue range can be determined. The validity of the developed method of the present invention can be evaluated by measuring food samples using the method developed in the present invention and a standard control method, and comparing the results of both.

1. First Fluorescence Detection Optical Condition The inventors of the present application first studied on the first fluorescence detection optical condition (excitation wavelength: 460-500 nm, fluorescence wavelength: 515 nm or more). In addition, any fluorescent substance can be used as the first fluorescent substance as long as it exhibits fluorescence under these conditions and the fluorescent luminance (%) is 90% or more of the maximum fluorescent luminance obtained at the optimum fluorescent wavelength. Is, for example, Alexa fluor488, FITC, FluorX, Dylight488, 5-FITC, FAM (5'FAM), DY-495-X5, DY-495, Fluorescein, ATTO495, ECD, DY-485XL, PE, Quantum Red, ParCP , 0regon green488, spectrum green, etc. can be used. Specific examples include Alexa fluor488. Here, an Enterobacteriaceae DNA probe labeled with Alexa fluor488 was used. As food samples, three-color mixed vegetables (green beans, corn, carrots), mentakatsu, and hirame were used. Each food sample was provided with two sections: E. coli added (signal + noise section) and no addition (noise section). A 10-fold diluted solution (0.1 g / ml) was prepared by adding a diluent for a bacterial test to a food sample (signal + noise group or noise group) and mixing. 1 ml of each 10-fold diluted solution and 1 ml of microcolony formation medium were mixed and added to the above-described simple test kit.

  In the case of the signal + noise group, the cells were cultured at 37 ° C. for 6.5 hours to form bacterial microcolony (microcolony formation culture). Thereafter, 2 ml of 100% ethanol was added to the simple test kit and fixed for 10 minutes (immobilization). Next, after removing the membrane filter with a high outer frame and drying it, 2 ml of hybridization solution containing Alexa fluor488-labeled Enterobacteriaceae detection probe is added to the membrane filter with various outer frames. Reacted for 30 minutes at 10 ° C, then added 10 ml of washing solution, allowed to stand at 46 ° C for 15 minutes, then drained and washed, rinsed the high membrane filter with outer frame with distilled water and dried .

  Next, in the case of the noise zone, mix 1 ml of the 10-fold diluted food sample and 1 ml of the 2-fold concentration bacterial culture liquid medium, add it to the simple test kit, do not perform microcolony formation culture, It carried out like signal + noise section. A color photograph was taken on these culture combined fluorescence in situ hybridization specimens, that is, a membrane filter with an outer frame having a height of a signal + noise section and a noise section under the first fluorescence detection optical conditions described above. The hue and brightness of the bright spots of the captured images of each category were measured. The measurement data were compared and the signal and noise discrimination conditions were examined.

  As a result, the hue and brightness characteristic of the signal of the first fluorescent substance were found under the first fluorescence detection optical condition. Specifically, hue range: H64-H98, brightness: V150 or more.

2. Second fluorescence detection optical condition Next, the second fluorescence detection optical condition (excitation wavelength: 520-560 nm, fluorescence wavelength: 570 nm or more) was examined. Any fluorescent substance can be used as the second fluorescent substance as long as the substance exhibits fluorescence under this condition and the fluorescent brightness (%) is 90% or more of the maximum fluorescent brightness obtained at the optimum fluorescent wavelength. For example, PhycoerythrinB, TRITC, Cy3, DY521XL, Alexa fluor555, Dylight547, Rhodamine, DY-548, DY-554, Alexa fluor546, DY-556, Northernlight557, Oyster550, 5-TAMRA, DY-505-X5, DY-547 Oyster556, DY-549, ATTO550, B-PE, DY-560, Spectrum Orange, TAMRA, and the like can be used. Specific examples include TAMRA. Here, Enterobacteriaceae DNA probe labeled with TAMRA was used. As food samples, three-color vegetables (green beans, corn, carrots), mentakatsu, and hirame were used. Each food sample was provided with two sections with addition of E. coli (signal + noise section) and without addition (noise section). Preparation of a culture combined fluorescence in situ hybridization method specimen was performed in the same manner as the first fluorescence detection optical condition described above. Next, a color image of the specimen was taken under the second fluorescence detection optical condition described above. The hue and brightness of the bright spots of the captured images of each category were measured. The measurement data were compared and the signal and noise discrimination conditions were examined.

  As a result, the hue and brightness characteristic of the signal of the second fluorescent substance were found under the second fluorescence detection optical condition. Specifically, hue range: H44-H54, brightness: V150 or more, hue range: H54-H74, brightness: V100 or more.

3. Third fluorescence detection optical condition Next, the third fluorescence detection optical condition (excitation wavelength: 590-650 nm, fluorescence wavelength: 660 nm or more) was examined. Any fluorescent substance can be used as the third fluorescent substance as long as the substance exhibits fluorescence under this condition and the fluorescent luminance (%) is 90% or more of the maximum fluorescent luminance obtained at the optimum fluorescent wavelength. However, for example, Allophycocyanin (APC), Cy5, APC-XL, Alexa Fluor 647, Oyster 645, DY 635, DY-636, DYLight 649, HILytePlus 647, and the like can be used. Specifically, Cy5 can be mentioned. Here, an enterobacteriaceae DNA probe labeled with Cy5 was used. As food samples, three-color vegetables (green beans, corn, carrots), mentakatsu, and hirame were used. Each food sample was provided with two sections with addition of E. coli (signal + noise section) and without addition (noise section). Preparation of a culture combined fluorescence in situ hybridization method specimen was performed in the same manner as the first fluorescence detection optical condition described above. Next, a color image was taken on the specimen under the third fluorescence detection optical condition described above. The hue and brightness of the bright spots of the captured images of each category were measured. The measurement data were compared and the signal and noise discrimination conditions were examined.

  As a result, the hue and brightness characteristic of the signal of the third fluorescent substance were found under the third fluorescence detection optical condition. Specifically, hue range: H340-H360, brightness: V80 or more, hue range: H0-H20, brightness: V80 or more.

  The specific color tone and lightness were clarified in the signal of the specific fluorescent material used in the first, second, or third fluorescence conditions. Using these as signal detection conditions, a method for automatic detection was established and investigated for applicability to a wide variety of foods. As a result, it has become clear that the first, second, and third fluorescence detection conditions can be applied to a wide variety of foods.

  In the following examples, examination was performed using typical fluorescent substances under the first, second, and third fluorescence conditions, but the excitation wavelength and fluorescence were also obtained when other fluorescent dyes were used. It will be apparent to those skilled in the art that hues and brightnesses of similar wavelengths do not change significantly.

  Based on these facts, the fluorescence signal of the microcolonies of the target microorganisms is classified into noise and noise based on color and brightness under the first, second, or third fluorescence detection conditions using the culture combined in situ hybridization method. A detection method was constructed separately.

  In the fluorescence in situ hybridization combined with culture, it has been clarified that the automatic signal detection method for bacteria described in Patent Document 1 may cause noise depending on the type of food sample and cause an error in measurement. However, the bacterial signal detection method of the present invention can accurately and automatically count the bacteria to be detected from the food, distinguishing it from noise, and can be applied to a wide variety of foods. Also, in many industrial processes where accuracy and speed are required, such as for use in inspections during the manufacturing process and inspections prior to product shipment in industrial fields such as food manufacturing and food processing. It is available and very useful for industry.

Schematic of the measuring device used in the present invention. Measurement result of noise from three-color mixed vegetables under the first fluorescence detection optical conditions. The measurement result of the signal and noise which come out from the three-color mixed vegetable which added colon_bacillus | E._coli in 1st fluorescence detection optical conditions. The measurement result of the noise which comes out of the flash under the first fluorescence detection optical condition. The measurement result of the signal and noise which come out of the hirame which added colon_bacillus | E._coli on the 1st fluorescence detection optical conditions. The measurement result of the noise which comes out of a punch under the first fluorescence detection optical conditions. The measurement result of the signal and noise which come out of the cutlet which added colon_bacillus | E._coli on the 1st fluorescence detection optical conditions. Measurement result of noise from three-color mixed vegetables under the second fluorescence detection optical condition. Signal and noise measurement results from a three-color mixed vegetable supplemented with E. coli under the second fluorescence detection optical condition. The measurement result of the noise which comes out of the flash under the second fluorescence detection optical condition. The measurement result of the signal and noise which come out of the hirame which added colon_bacillus | E._coli on 2nd fluorescence detection optical conditions. The measurement result of the noise which comes out of a punch under the 2nd fluorescence detection optical conditions. The measurement result of the signal and noise which come out of the cutlet which added colon_bacillus | E._coli on the 2nd fluorescence detection optical conditions. Measurement results of noise from three-color mixed vegetables under the third fluorescence detection optical condition. The measurement result of the signal and noise which come out from the three-color mixed vegetable which added colon_bacillus | E._coli on the 3rd fluorescence detection optical conditions. Measurement result of noise coming out of the flash under the third fluorescence detection optical condition. The measurement result of the signal and noise which come out of the hirame which added colon_bacillus | E._coli on 3rd fluorescence detection optical conditions. The measurement result of the noise which comes out of Menchikatsu in the 3rd fluorescence detection optical condition. The measurement result of the signal and noise which come out of the cutlet which added colon_bacillus | E._coli on 3rd fluorescence detection optical conditions.

<Outline of the present invention>
The present invention relates to a microbe colony in a microorganism testing method such as food using a combined fluorescence in situ hybridization method, using a membrane filter having an effective filtration area equal to or greater than the area of a circle having a diameter of 24 mm. Culturing to a size of 20 μm to 150 μm, and the above-mentioned test is performed by using a low-magnification optical system of a membrane filter for a specific color of a specific microorganism grown by a culture combined fluorescence in situ hybridization method. And a method for detecting bacteria characterized by means of automatic detection by a solid state device with brightness.

(1) Culture combined fluorescence in situ hybridization method In the present invention, the culture combined fluorescence in situ hybridization method uses a fluorescent probe for detecting microorganisms after culturing a sample containing microorganisms in a medium to form microcolony. Contains detecting. As the fluorescent probe, those targeting rRNA can be used, and those described in Japanese Patent No. 4785449 can also be used.

  Apparatus using fluorescence in situ hybridization combined with culture provided with means for automatically detecting the color and brightness of a fluorescent signal under a specific color, for example, the first, second, or third fluorescence detection optical conditions Can be used. Specifically, for example, it can be performed as follows.

(B) Filtration of sample A membrane filter is incorporated in the suction filtration filter unit, and the sample solution is filtered.
When using the membrane filter with an outer frame described in Patent Document 2, the sample solution is filtered by placing it on the filter base for suction filtration. Alternatively, 1 ml of the sample solution and 1 ml of the microcolony formation medium are mixed, and a simple test kit (configuration: 5 sheets of filter paper with a diameter of 55 mm and a height of 15 mm in a petri dish with a diameter of 55 mm and a height of 15 mm is described on Patent Document 2. An outer frame membrane filter with a height of 47 mm (diameter 47 mm, pore type 0.8 μm or smaller disk-type membrane filter and 47 mm outer diameter 42 mm inner diameter 42 mm height 15 mm height acrylic laminated together) is placed with the filter face down This may be added to a plastic petri dish lid having a diameter of 60 mm and a height of 10 mm) for filtration.

(B) Culture The membrane filter on the base is placed on a solid medium (gel medium or fibrous pad containing liquid medium), and the filter is brought into contact with the other side (back side) and the medium. Culture is performed under predetermined conditions, and microcolony is formed on the surface on the front side of the filter. For example, by placing a membrane filter with a high outer frame on an agar medium and culturing for about 5 to 6 hours at the optimum temperature of the bacteria to be detected, a microcolony is formed on the front side of the membrane filter. be able to. Or you may culture the simple test kit which provided the above-mentioned sample solution as it is.

(C) Fixation Immerse the membrane filter in an appropriate fixative (such as ethanol) and then dry it. When using a filter with a high outer frame, for example, the membrane filter can be fixed by placing it on a filter paper containing ethanol.

(D) Hybridization For example, the filter can be transferred to a dedicated container containing a hybridization buffer and a fluorescently labeled probe for detecting a specific bacterium for hybridization. When using a filter with a high outer frame, a fluorescent labeling probe for detecting a specific bacterium and a hybridization buffer are added to the filter and reacted at a predetermined temperature.

(E) Probe labeled fluorescent substance In fluorescence in situ hybridization combined with culture using the first fluorescence detection optical conditions (excitation wavelength: 460-500 nm, fluorescence wavelength: 515 nm or more), fluorescence is emitted under these conditions, and the fluorescence brightness A substance exhibiting fluorescence whose (%) is 90% or more of the maximum fluorescence luminance obtained at the optimum fluorescence wavelength is used as a first fluorescent substance, and this is used as a labeled fluorescent substance of a DNA probe for detecting target bacteria. For example, Alexa fluor488 or FITC can be used.

  In fluorescence in situ hybridization combined with culture using the second fluorescence detection optical condition (excitation wavelength: 520-560 nm, fluorescence wavelength: 570 nm or more), fluorescence is displayed under this condition, and the fluorescence intensity (%) is obtained at the optimum fluorescence wavelength. A substance exhibiting fluorescence that is 90% or more of the maximum fluorescence brightness is used as the second fluorescent substance, and this is used as the labeled fluorescent substance of the DNA probe for detecting the target bacteria. For example, TAMRA can be used.

  In the fluorescence in situ hybridization combined with culture using the third fluorescence detection optical condition (excitation wavelength: 590-650 nm, fluorescence wavelength: 660 nm or more), fluorescence is exhibited under this condition, and the fluorescence brightness (%) is obtained at the optimum fluorescence wavelength. A substance exhibiting fluorescence that is 90% or more of the maximum fluorescence luminance is used as a third fluorescent substance, and this is used as a labeled fluorescent substance for the DNA probe for detecting the target bacteria. For example, Cy5 can be used.

(F) Cleaning For example, the membrane filter can be transferred to a dedicated container using tweezers, and the filter can be immersed in a cleaning solution for cleaning. When a membrane filter with a high outer frame is used, a washing solution can be added on the filter and reacted at a predetermined temperature.

(G) Preparation of specimen For example, a membrane filter is placed on a slide glass, an encapsulant is added thereon, and then a cover glass is put on to form a specimen. However, when a membrane filter with a high outer frame is used, the work is not necessary.

(H) Fluorescence signal counting Fluorescence signal counting The first, second, and third fluorescence detection optical conditions are provided, and the fluorescence derived from the DNA probe on the membrane filter is obtained with a low magnification optical system (1 to 6 times magnification) attached to the fluorescence detection unit. A specimen can be provided in an apparatus that can execute an automatic signal detection method having means for detecting a signal with a specific color and brightness, and a fluorescence signal can be measured.

(2) Setting of various conditions in a signal automatic detection method having means for detecting a fluorescent signal derived from a DNA probe on a membrane filter with a specific color and brightness In the present invention, a fluorescent signal derived from a DNA probe on a membrane filter is specified The conditions of the automatic signal detection method having means for detecting by color and brightness are adjusted as follows.

(A) Membrane filter Specifically, it is only necessary to be able to inspect a sample of food such as 0.1 g at a time. For this purpose, for example, as a membrane filter, the effective filtration area is equivalent to the area of a circle with a diameter of 24 mm or A membrane filter having an area larger than that, for example, a membrane filter having a diameter of 47 mm can be used.

  The material of the membrane filter may be any commercially available material, and for example, polycarbonate or cellulose acetate can be used. The average pore size is preferably 0.4 μm to 0.8 μm so that bacteria can be collected. The bottom surface of the membrane filter may be any shape that can be placed on the suction filtration filter base, can be in close contact with the fixed medium during fixed culture, and can be placed on the operation console of the microscope.

  If necessary, an outer frame having a height (hereinafter referred to as an outer frame) can be provided on the outer surface of the membrane filter.

  By attaching the outer frame to the membrane filter, a certain amount of liquid can be accommodated on the membrane, and the solution for hybridization and washing can be held on the membrane filter. Further, the outer frame allows the membrane filter to be held without sagging. For example, the outer frame can be directly bonded to the membrane filter without any gaps, but a holding frame is provided around the membrane filter to hold the membrane filter so that it does not sag. You can also. The material of the outer frame may be a material that can withstand a certain tension and a certain tension for stretching the filter without slack, such as plastic, metal, and cellulose, and that does not allow liquid to permeate. The height of the outer frame is the height at which the objective lens does not contact the outer frame when the objective lens (1 to 10 times) is used to focus on the filter surface with the outer frame. If it exists, it can be set to 3 mm-30 mm, for example. The shape of the outer frame can be arbitrarily formed, for example, in a cylindrical shape, a square, or the like, but is preferably a cylindrical outer frame. The columnar outer frame can be formed, for example, in a funnel shape or a funnel shape so that the area of the opening is larger than the area of the membrane filter. As the membrane filter with a high outer frame, a commercially available product in which an outer wall is provided on the membrane filter, for example, a permeable support transwell can be used.

(B) DNA probe
Various fluorescent probes can be used as the DNA probe. Specific examples include oligonucleotides to fluorescently labeled rRNA. Enterobacteriaceae (TGCTCTCGCGAGGTCGCTTCTCTT), Cl. Perfringens (AATGATGATGCCATCTTTCAACA), etc. can be used (patent document 1).

(C) Low-magnification fluorescence detection optical system in automatic measurement In the present invention, a low-magnification fluorescence detection optical system can be used in order to proliferate a specific bacterium for detection in a sample by a combined fluorescence in situ hybridization method. Microorganism fluorescence signals can be detected by a low-magnification fluorescence detection optical system with a magnification of 1 to 6 times. However, in automatic measurement using a membrane filter with an outer frame with a diameter of 47 mm, the magnification is 2 times. In the low-magnification fluorescence detection system of 6 times, since one field of view is a part of the whole surface of the membrane filter of the sample, a two-dimensional scan measurement of the sample is required and a moving sample stage is required. In a low-magnification fluorescence detection system with a magnification of 1 ×, one field of view covers the entire surface of the sample, and the entire surface of the sample is measured with only one field of view measurement.

(D) Fluorescence detection optical conditions in automatic measurement The automatic measurement device has the first fluorescence detection optical condition, the second fluorescence detection optical condition, and the third fluorescence detection optical condition described above. Can be switched and measured.

(3) Automated measuring device used in the present invention In the present invention, a fluorescence image of the entire membrane filter can be obtained with a small number of observations by using a high-resolution low-magnification optical system with a magnification of 1 to 6 times, and a color imaging device It is possible to automatically distinguish noise components such as signal fluorescence and autofluorescence based on hue and brightness.
Hereinafter, a specific mode of the apparatus used in the present invention will be described with reference to FIG.

(B) Sample stage The use of a high-resolution, low-magnification optical system makes it unnecessary to move the sample stage in a 1x optical system. The sample stage is installed in a completely dark room in the apparatus and is in a state where there is no incident light other than excitation light. In addition, when a 2 to 6 times optical system is used, a moving stage is adopted so that the entire membrane filter can be imaged with a small number of imaging times.

(B) Excitation light source Light from a halogen lamp light source is passed through an excitation wavelength filter via a fiber as excitation light, and the sample on the sample stage is irradiated with uniform excitation light through a light guide.

(C) Optical filter changer (excitation filter, fluorescent absorption filter)
The excitation light (white) from the excitation light source is automatically switched to the excitation light filter according to the selected fluorescent label on the display unit. In addition, the absorption filter for fluorescence in the previous stage of the fluorescence detection unit is automatically switched to the corresponding absorption filter.

  As the first fluorescence detection condition, a band-pass filter 460-500 nm can be used as the excitation light filter, and a long-pass filter 515 nm or more can be used as the fluorescent signal absorption filter.

  As the second fluorescence detection condition, a band-pass filter 520-560 nm can be used as an excitation light filter, and a long-pass filter 570 nm or more can be used as an absorption filter for a fluorescence signal.

  As the third fluorescence detection condition, a bandpass filter 590-650 nm can be used as the excitation light filter, and a bandpass filter 660 nm or more can be used as the fluorescent signal absorption filter.

(D) Fluorescence detector (camera)
As a digital color camera, for example, a CMOS device with a 35 mm full size (35.9 x 24.0 mm, total number of images 7360 x 4912) is used, and a high-resolution digital color camera is used for the fluorescence detection unit. What can detect a colony can be used. The camera can use a low magnification optical system: 1 to 6 times magnification. This is because a low-magnification optical system can be used due to the formation of microcolony and the high-resolution fluorescence detection section. Setting the optical system magnification to 1 makes it possible to measure the entire filter device with a single observation, contributing to a significant reduction in observation time.

(E) Image processing unit Image processing discrimination based on hue and brightness is performed on the fluorescent color image captured by the fluorescence detection unit, and the discrimination result is displayed on the monitor. It also issues control commands to the control board.

(F) System control board Upon receiving a command from the image processing unit, it controls the light source (ON / OFF, light amount adjustment) and the filter switching device. Also, error signals from various devices are received and transmitted to the image processing unit.

(G) Monitor The processing result of the image processing unit is displayed, and the operation status of the device is also displayed.

(H) Device characteristics The first fluorescence detection condition, the second fluorescence detection condition, or the third fluorescence detection condition can be selected from the monitor screen and automatically switched. Shooting conditions (light source intensity, ISO, exposure, shutter speed) can be selected and input from the monitor screen. The measurement range is the entire filter device.

(4) Automated measurement of a specific sample The measurement sample prepared by the fluorescence in situ hybridization method combined with the culture in (1) above is a fluorescence signal derived from the DNA probe on the membrane filter in (2) with a specific color and brightness. It can adjust as various conditions in the signal automatic detection method which has a means to detect, and can perform as follows using the measuring apparatus of (3) description.

  Specifically, the sample is photographed with a color camera using a culture combined fluorescence in situ hybridization method with respect to a membrane filter with a frame having a height under the first fluorescence detection optical conditions described above. First, (i) determine whether the conditions of the hue and lightness are met for the pixels of the entire photographed image, and exclude pixels that do not meet the conditions of hue and lightness by, for example, masking. I can do it. Next, (ii) image processing for searching for and detecting an aggregate (bright spot) of pixels that are colonies from the applied pixels is performed. In image processing, discriminating shapes such as a circle, an ellipse, a triangle, and a rectangle from a captured image is generalized. However, since a colony is basically a circle or an ellipse, A pixel aggregate having a circular or elliptical shape and a size, that is, a diameter or a major axis and a minor axis of 20 to 150 μm can be automatically detected.

  Here, when the measurement is performed under the first fluorescence detection optical condition, for example, the photographing conditions are a halogen light source output 10%, a camera setting exposure time 2.0 seconds, an exposure F3.5, and an ISO sensitivity 12800. Next, the hue range of the pixel aggregate (bright spot) of the photographed image is H64-H98, the brightness is V150 or more, the shape is circular or elliptical, and the size is the diameter or major axis and minor axis 20 μm to 150 μm. Can be the target of automatic detection. For measurement under the second fluorescence detection optical condition, for example, the photographing conditions are a halogen light source output of 10%, a camera setting exposure time of 0.6 seconds, an exposure of F3.5, and an ISO sensitivity of 25600. Next, the bright spot of the photographed image has a hue range of H44-H54, lightness of V150 or higher, or a hue range of H54-H74, lightness of V100 or higher, circular or elliptical shape, diameter or major axis and short axis. A pixel aggregate having a diameter of 20 μm to 150 μm can be an object of automatic detection. When measurement is performed under the third fluorescence detection optical condition, for example, the photographing condition is a halogen light source output 10%, camera setting exposure time 1.0 second, exposure F3.5, and ISO sensitivity 1250. Next, the hue range of the pixel aggregate (bright spot) of the photographed image is H340-H360, the brightness is V80 or more, or the hue range is H0-H20, the brightness is V80 or more, and it has a circular or elliptical shape. In addition, a pixel aggregate having a diameter or a major axis and a minor axis of 20 μm to 150 μm can be an object of automatic detection. Note that even if the intensity of the light source is changed, the hue is not greatly affected. It is also possible to make a similar determination by shortening the exposure time of the camera in accordance with the intensity of the light source.

  The present invention is not limited to the actual hue / brightness ranges described above, and by adjusting the hue and lightness conditions, it is possible to discriminate between food-derived fluorescent noise and fluorescent probe-derived signals. It can cope with food noise.

(5) Combination of fluorescence detection optical conditions In addition, in the automated measurement of the sample of (4) above, measurement under the first fluorescence detection optical condition using a DNA probe using the first fluorescent substance, second fluorescence Three types of fluorescence consisting of measurement under the second fluorescence detection optical condition using a probe using a substance and measurement under a third fluorescence detection optical condition using a DNA probe using a third fluorescence substance It is also possible to measure more accurately by combining two or more fluorescence detection optical conditions selected from the detection optical conditions.

[Example 1]
Development of a new automatic fluorescence signal detection method Autofluorescence of various foods and fluorescence used for labeling of DNA probes to develop a new automatic counting method to distinguish fluorescence signals from food-borne fluorescence in situ hybridization methods and food-derived noise The fluorescence signals of the substances were compared as follows.

  First, investigation was performed under the first fluorescence detection optical conditions (excitation wavelength: 460-500 nm, fluorescence wavelength: 515 nm or more). In addition, Alexa fluor488 was used as a first fluorescent substance that exhibited fluorescence under these conditions and whose fluorescence brightness (%) was 90% or more of the maximum fluorescence brightness obtained at the optimum fluorescence wavelength. A DNA probe ((TGCTCTCGCGAGGTCGCTTCTCTT) Patent Document 1 Patent No. 4785449) for detecting Enterobacteriaceae bacteria labeled with Alexa fluor488 was used. In addition, a simple inspection kit was prototyped and used. In this kit configuration, 5 sheets of filter paper with a diameter of 50 mm are placed in a plastic petri dish with a diameter of 55 mm and a height of 15 mm, and a membrane filter with an outer frame described in Patent Document 2 (diameter: 47 mm, pore size: 0.4 Place a disk-type membrane filter of μm or less and ring-shaped acrylic with an outer diameter of 47 mm, an inner diameter of 42 mm and a height of 15 mm, and place the plastic face lid (diameter 60 mm, height 10 mm) on the filter surface. Cover the outer frame of the membrane filter with outer frame and the outer edge of the plastic petri dish.

  As food samples, inspiration, three-color mixed vegetables (green beans, carrots, corn), and chili cutlet were used.

  Each food sample was provided with two sections: a sample added with E. coli (signal + noise section) and a sample not added (noise section).

  25 g of a food sample (signal + noise group or noise group) was collected in a stomacher bag, mixed with 225 ml of a diluent for bacterial testing, and mixed to prepare a 10-fold diluted solution (0.1 g / ml). 1 ml of food sample diluent and general bacterial microcolony formation medium (casein peptone 34 g, soy peptone 6 g, yeast extract 12 g, glucose 5 g, sodium chloride 10 g, sodium pyruvate 2.2 g, potassium dihydrogen phosphate 2.7 g, dipotassium hydrogen phosphate Sterilize 5 g, disodium hydrogenphosphate 19.2 g, bile salt No. 3 1.12 g, and purified water 1 L (121 ° C, 15 minutes). Mix 1 ml and add it to the above-mentioned simple test kit. .

  In the case of the signal + noise group, the cells were then cultured at 37 ° C. for 6.5 hours to form bacterial microcolony (microcolony formation culture). Thereafter, 2 ml of 100% ethanol was added to the simple test kit and fixed for 10 minutes (immobilization). Next, the membrane filter with a high outer frame is taken out, dried, transferred to a plastic petri dish with a diameter of 55 mm and a height of 15 mm, and 5 μl of a probe for detecting 10 μM Enterobacteriaceae bacteria labeled with various fluorescent substances. Hybridization buffer solution (20% formamide, 0.01% sodium dodecyl sulfate [SDS], 0.9 M sodium chloride, 20 mM Tris-HCl [pH 7.4]) 1.5 ml was added and reacted at 46 ° C. for 30 minutes (hybridization). Drained. Next, add 10 ml of washing solution (0.01% sodium dodecyl sulfate [SDS], 180 mM sodium chloride, 20 mM Tris-HCl [pH 7.4]), leave it at 46 ° C. for 15 minutes and drain (wash), and then the height The membrane filter with outer frame was rinsed with distilled water and dried.

  In the case of the noise group, mix 1 ml of the 10-fold diluted food sample and 1 ml of the microcolony formation medium, add it to the simple test kit, do not perform the microcolony formation culture, and the subsequent operations are the same as the signal + noise group Went to.

  These culture combined fluorescence in situ hybridization method specimens, that is, the signal + noise section, the membrane filter with an outer frame having a noise section height, were taken with a color camera under the first fluorescence detection optical conditions described above. . The equipment is a digital camera (CMOS element size 35mm full size (35.9 x 24.0mm, pixel number 7360 x 4912)), halogen light, filter switching device (excitation light side: small rotary filter switching device (6 sheets switching), Fluorescent side: Thin-type rotary filter switching device (8-sheet switching) etc. is used as a material, and an automated measuring device is prototyped and used as described above. Halogen light source output is 10%, Alexa fluor488 filter (excitation: bandpass filter) 460-500 nm, absorption: long pass filter 515 nm or more). The camera settings were: exposure time: 2.0 seconds F: 3.5 ISO12800. Projected images of each section are displayed on the monitor screen, and the signal pixel aggregates and noise pixel aggregates are visually distinguished for the aggregates (pixel groups) of bright spots of various colors, brightness, and size. . Next, hue / lightness was measured at 10 points or more at random on an aggregate of pixels (corresponding to a size of 20 to 150 μm), which was noise identified visually. Similarly, hue / lightness was measured at 10 points on a pixel aggregate (equivalent to a size of 20 to 150 μm) as a signal.

(result)
The measurement results under the first fluorescence detection optical condition are shown in Tables 2 to 10 and FIGS. 2 to 7. Three-color mixed vegetable results (noise group (Table 2, Fig. 2), signal + noise group (Table 3, Table 4, Fig. 3), inspiration results (noise group (Table 5, Fig. 4), signal + noise group) (Table 6, Table 7, and Fig. 5)), Menchikatsu results (noise zone (Table 8, Fig. 6), signal + noise zone (Table 9, Table 10, Fig. 7)), the first fluorescence detection optical conditions In order to distinguish the first fluorescent substance, the hue range was different because the appearance of the fluorescent label contained in the colony differs depending on whether it is strong or weak. As a result, a bright spot having a hue range of H64-H98 and a brightness of V150 or higher was set as a discrimination condition for discriminating from a signal, and this was set as an automatic measurement condition.

  Therefore, among the measurement results of the three-color mixed vegetable signal + noise section, Table 3 shows the signal and Table 4 shows the noise, and these are shown in FIG. Next, in the signal / noise section of the inflection, Table 6 is a signal and Table 7 is a noise. Further, in the signal + noise section of Menchikatsu, Table 9 is a signal, and Table 10 is a noise, which are collectively shown in FIG.

  Next, the second fluorescence detection optical conditions (excitation wavelength 520-560 nm, fluorescence wavelength 570 nm or more) were studied. Under these conditions, TAMRA was used with a substance exhibiting fluorescence and having a fluorescence brightness (%) of 90% or more of the maximum fluorescence brightness obtained at the optimum fluorescence wavelength as the second fluorescent substance. A DNA probe ((TGCTCTCGCGAGGTCGCTTCTCTT) Patent Document 1 Patent No. 4785449) for detecting Enterobacteriaceae bacteria labeled with TAMRA was used. As food samples, three-color vegetables (green beans, corn, carrots), mentakatsu, and hirame were used. Each food sample was provided with two sections with addition of E. coli (signal + noise section) and without addition (noise section). Preparation of a culture combined fluorescence in situ hybridization method specimen was performed in the same manner as the first fluorescence detection optical condition described above. Next, a color image of the specimen was taken under the second fluorescence detection optical condition described above. The equipment is a digital camera (CMOS element size is 35mm full size (35.9 x 24.0mm, total number of images 7360 x 4912)), halogen light, filter switching device (excitation light side: small rotary filter switching device (6 sheets switching), Fluorescent side: Thin-type rotary filter switching device (8-sheet switching) etc. is used as a material, and an automated measuring device is prototyped and used as described above. Halogen light source output is 10%, TAMRA filter (excitation: bandpass filter 520 -560 nm, absorption: long pass filter 570 nm or more). The camera settings were: exposure time: 0.6 seconds F: 3.5 ISO25600.

  The image was projected on a monitor screen, and a signal pixel aggregate and a noise pixel aggregate were visually distinguished for a collection (pixel collection) of bright spots of various colors, brightness, and size. Next, the pixel group (equivalent to a size of 20-150μm), which is the noise identified by visual observation, is randomly assigned to 10 points, and the pixel group, which is a signal (equivalent to a size of 20-150μm), is randomly assigned to the hue / The brightness was measured. The measurement data were compared and the signal and noise discrimination conditions were examined.

  The measurement results of the second fluorescence detection optical condition are shown in Tables 11 to 19 and FIGS. Three-color mixed vegetable results (noise group (Table 11, Fig. 8), signal + noise group (Table 12, Table 13, Fig. 9)), inspiration results (noise group (Table 14, Fig. 10), signal + noise 2nd fluorescence detection optics from ward (Table 15, Table 16, Figure 11)), Machikatsu results (Noise (Table 17, Figure 12), Signal + Noise (Table 18, Table 19, Figure 13)) The conditions for discriminating the second fluorescent material were examined. investigated. The range of the hue was given to the hue because the appearance differs depending on whether the fluorescent label contained in the colony is strong or weak. As a result, the hue range is H44-H54 and the brightness is in the range of V150 or higher, and the hue range is H54-H74, and the bright spot in the range of the brightness is V100 or higher is set as the discrimination condition. Automatic measurement conditions were used.

  In the measurements from Table 11 to Table 19, therefore, among the measurement results of the three-color mixed vegetable signal + noise section, Table 12 is a signal and Table 13 is a noise, and these are shown in FIG. Next, in the signal / noise section of the inflection, Table 15 is a signal and Table 16 is a noise. Further, in the signal + noise section of Menchikatsu, Table 18 is a signal, and Table 19 is a noise.

  Next, examination was performed under the third fluorescence detection optical condition (excitation wavelength: 590-650 nm, fluorescence wavelength: 660 nm or more). Under this condition, a substance that exhibits fluorescence and whose fluorescence brightness (%) is 90% or more of the maximum fluorescence brightness obtained at the optimum fluorescence wavelength was used as a third fluorescent substance, and Cy5 was used. A DNA probe for detecting Enterobacteriaceae bacteria labeled with Cy5 ((TGCTCTCGCGAGGTCGCTTCTCTT) Patent Document 1 Patent No. 4785449) was used. As food samples, three-color vegetables (green beans, corn, carrots), mentakatsu, and hirame were used. Each food sample was provided with two sections with addition of E. coli (signal + noise section) and without addition (noise section). Preparation of a culture combined fluorescence in situ hybridization method specimen was performed in the same manner as the first fluorescence detection optical condition described above. Next, a color image was taken on the specimen under the third fluorescence detection optical condition described above. The equipment is a digital camera (CMOS element size is 35mm full size (35.9 x 24.0mm, total number of images 7360 x 4912)), halogen light, filter switching device (excitation light side: small rotary filter switching device (6 sheets switching), Fluorescent side: Thin-type rotary filter switching device (8-sheet switching) etc. is used as a material, and an automated measuring device is prototyped and used as described above, the halogen light source output is 10%, and a Cy5 filter (excitation: bandpass filter 590 -650 nm, absorption: 660 nm or more long pass filter). The camera setting was exposure time: 1.0 sec. F: 3.5 ISO1250.

  The image was projected on a monitor screen, and a signal pixel aggregate and a noise pixel aggregate were visually distinguished for a collection (pixel collection) of bright spots of various colors, brightness, and size. Next, the hue of the pixel aggregate (equivalent to a size of 20-150μm), which is the noise identified visually, is randomly assigned to 10 points, and the pixel group (signal equivalent to the size of 20-150μm), which is a signal, is randomly assigned to 10 or more -The brightness was measured. The measurement data were compared and the signal and noise discrimination conditions were examined.

  The measurement results under the third fluorescence detection optical condition are shown in Tables 20 to 28 and FIGS. 14 to 19. Three-color mixed vegetables (noise group (Table 20, Figure 14), signal + noise group (Table 21, Table 22, Figure 15)), inflection (noise group (Table 23, Figure 16), signal + noise group (Table 24) , Table 25, Fig. 17)), Machikatsu (Noise section (Table 26, Figure 18), Signal + Noise section (Table 27, Table 28, Figure 19)), the third fluorescence under the third fluorescence detection optical conditions. Substance discrimination conditions were investigated. The brightness range is H340-H360, the brightness is V80 or more, and the hue range is H0-H20, and the brightness point is in the range of V80 or more. Condition.

  Therefore, among the measurement results of the three-color mixed vegetable signal + noise section, Table 21 shows the signal and Table 22 shows the noise, and these are shown in FIG. Next, in the signal / noise section of the inflection, Table 24 is a signal and Table 25 is a noise. Further, in the signal + noise section of Menchikatsu, Table 27 is a signal, and Table 28 is a noise.

[Example 2]
Fluorescence noise measurement of food by the fluorescence signal measurement method of the present invention The fluorescence measurement method described in Patent Document 1 showed fluorescence noise for the food samples described in Table 1. The same food sample was measured by the method of the present invention as follows.

  Thirteen kinds of foods shown in Table 1 were used as samples. In the test, a simple test kit was prototyped and used. This kit consists of a 55 mm diameter plastic petri dish with a height of 15 mm and 5 filter papers with a diameter of 50 mm, and a membrane filter with an outer frame as described in Patent Document 2 (however, 47 mm in diameter, pore size) A disc-type membrane filter of 0.8 μm or less and a ring-shaped acrylic having an outer diameter of 47 mm, an inner diameter of 42 mm, and a height of 15 mm were attached together with the filter face down.

  25 g of each food sample was placed in a stomacher bag, and further 225 ml of a phosphate buffer for bacterial testing was added and suspended to prepare a 10-fold diluted sample (sample 0.1 g / ml). The 1 ml was used for a simple test kit. After the diluted solution has been filtered, the membrane filter with a high outer frame is immersed in ethanol for 20 minutes, fixed and dried, and then a hybridization buffer (20% formamide, 0.01% sodium dodecyl sulfate [SDS], 0.9M chloride) 1.5 ml of sodium, 20 mM Tris-HCl [pH 7.4] was added and placed at 46 ° C. for 20 minutes. Next, a washing solution (0.01% sodium dodecyl sulfate [SDS], 180 mM sodium chloride, 20 mM Tris-HCl [pH 7.4]) was added and left at 46 ° C. for 15 minutes, and then drained and washed. The membrane filter with an outer frame having a height was rinsed with distilled water and dried. As a result, a food sample specimen containing 0.1 g of food sample and not containing bacterial microcolony (circular colony, diameter 20 μm to 150 μm) was prepared. Next, by measuring the fluorescence of the membrane filter with a high outer frame with two or more specific colors and brightness, it is measured under the conditions for automatic detection of the fluorescence signal of the first, second and third fluorescent substances. did. The fluorescence counted here becomes noise (a food-derived autofluorescence whose light amount and light emitting area are equivalent to those of a microcolony). As an apparatus to be used, the same automated measurement apparatus used in Example 1 was used, and a low-magnification fluorescence detection system having a magnification of 1 was used. The fluorescence signal detection conditions are as follows.

Fluorescent signal automatic detection condition for the first fluorescent substance Halogen light source output 10%, Alexa fluor488 filter (excitation: bandpass filter 460-500 nm, absorption: longpass filter 515 nm or more). The camera setting is exposure time: 2.0 seconds F: 3.5 ISO12800. Hue range: H64-H98, brightness: V150 or higher. In this embodiment, because of the resolution of the camera and the imaged area, the size per pixel is about 10 μm, so a pixel aggregate of 2 pixels vertically and horizontally and 15 pixels or less is 20- Since it corresponds to 150 μm, a pixel aggregate of 2 pixels vertically and horizontally and 15 pixels or less, with a hue range of H64-H98 and lightness of V150 or more was automatically detected as a signal.

Fluorescent signal automatic detection condition for the second fluorescent substance Halogen light source output 10%, TAMRA filter (excitation: bandpass filter 520-560nm, absorption: longpass filter 570nm or more). The camera setting is exposure time: 0.6 seconds F: 3.5 ISO25600. Hue range: H44-H54, brightness: V150 or higher, or hue range: H54-H74, brightness: V100 or higher. In this embodiment, because of the resolution of the camera and the imaged area, the size per pixel is about 10 μm, so a pixel aggregate of 2 pixels vertically and horizontally and 15 pixels or less is 20- Since it corresponds to 150μm, it is a pixel aggregate of 2 pixels vertically and horizontally and 15 pixels or less. Hue range: H44-H54, Brightness: V150 or higher, or Hue range: H54-H74, Brightness: Pixel aggregate of 100 or more Was automatically detected as a signal.

Fluorescent signal automatic detection conditions for the third fluorescent substance Halogen light source output 10%, Cy5 filter (excitation: bandpass filter 590-650nm, absorption: longpass filter 660nm or more). The camera setting is exposure time: 1.0 sec. F: 3.5 ISO1250. Hue range: H340-H360, brightness: V80 or higher, or hue range: H0-H20, brightness: V80 or higher. In this embodiment, from the resolution of the camera and the imaged area, the size per pixel is about 10 μm, so the vertical and horizontal pixels are 2 pixels or more and 15 pixels or less. The vertical and horizontal pixels are 2 pixels or more and 15 pixels or less. Since the pixel assembly corresponds to a size of 20 to 150 μm, it is a pixel assembly of 2 pixels in the vertical and horizontal directions and 15 pixels or less in hue range: H340-H360, brightness: V80 or more, or hue range: H0- H20, lightness: V80 or higher A pixel aggregate as a signal was automatically detected as a signal.

  As a result, no noise was generated at the bacterial detection limit of 10 CFU / g under any of the first, second and third fluorescence detection optical conditions (Table 29).

[Example 3]
Evaluation of Applicability to Counting Specific Bacteria in Foods by Signal Detection Method of Fluorescence In Situ Hybridization Combined with Culture The applicability of the present invention to the counting of specific bacteria in foods was examined.
The specific bacterium was designated Enterobacteriaceae. The same food sample was counted by the combined fluorescence in situ hybridization method and the Enterobacteriaceae count control method, and the values of both methods were compared as follows.

material
Test strain
Escherichia coli ATCC 11775 was used as an enterobacteriaceae bacterium.

VRBG Agar Peptone 7g, Bile Salt No.3 1.5g, Salt 5.0g , Crystal Bio Red 0.002g, Yeast Extract 3.0g, Glucose 10g, Neutral Red 0.03g, Agar 15g, Purified Water 1L Used after cooling.

A food sample was used, three-color mixed vegetables (green beans, carrots, corn), and chili cutlet. Each food sample was divided into two sections: E. coli added (bacteria added) and no addition (non-added).

Microcolony formation medium casein peptone 34g, soy peptone 6g, yeast extract 12g, glucose 5g, sodium chloride 10g, sodium pyruvate 2.2g, potassium dihydrogen phosphate 2.7g, dipotassium hydrogen phosphate 5g, disodium hydrogen phosphate 19.2g Then, 1.12 g of bile salt No. 3 and 1 L of purified water were pasteurized (80-100 ° C., 30 minutes) and cooled to room temperature.

A plastic dish of height 15mm with test kit diameter 55 mm (put a bottom down), the height of the placed 5 sheets of filter paper (No.2 quantitative filter paper Advantec) of diameter 50 mm, on which, Patent Document 2 A membrane filter with an outer frame (a disc-type membrane filter with a diameter of 47 mm and a pore size of 0.8 μm or less and a ring-shaped acrylic with an outer diameter of 47 mm, an inner diameter of 42 mm, and a height of 15 mm bonded together) is placed with the filter face down. To do. Furthermore, a plastic petri dish lid (diameter 60 mm, height 10 mm) is placed on the outer frame of the membrane filter with an outer frame and the outer edge of the plastic petri dish.

DNA probe for detecting Enterobacteriaceae bacteria (Patent Document 1 Patent No. 4785449)
As a DNA probe for detecting Enterobacteriaceae bacteria, a DNA probe having the following base sequence labeled with a fluorescent substance is used (TGCTCTCGCGAGGTCGCTTCTCTT).

If the means for detecting the fluorescence signal of the fluorescent substance membrane filter of the DNA probe with a specific color and brightness is the first fluorescence detection optical condition (excitation wavelength: 460-500 nm, fluorescence wavelength: 515 nm or more), DNA The first fluorescent substance for labeling the probe is called Alexa Fluor 488.

  When the means for detecting the fluorescence signal of the membrane filter with a specific color and brightness is the second fluorescence detection optical condition (excitation wavelength: 520-560 nm, fluorescence wavelength: 570 nm or more), the second probe for labeling the DNA probe The fluorescent substance is TAMRA.

  If the means for detecting the fluorescence signal of the membrane filter with a specific color and brightness is under the third fluorescence detection optical condition (excitation wavelength: 590-650 nm, fluorescence wavelength: 660 nm or more), the third fluorescence that labels the DNA probe Let the material be Cy5.

Hybridization buffer
20% formamide, 0.01% sodium dodecyl sulfate [SDS], 0.9 M sodium chloride, 20 mM Tris-HCl [pH 7.4]

Washing solution (0.01% sodium dodecyl sulfate [SDS], 180 mM sodium chloride, 20 mM Tris-HCl [pH 7.4]) 10 ml

As an apparatus to be used for automatic detection of fluorescent signals, the same automated measuring apparatus used in Example 1 was used, and a low-magnification fluorescence detection system having a magnification of 1 was used.

  In the means for detecting the fluorescent signal of the membrane filter with a specific color and brightness, the fluorescent signal detection conditions of the first, second and third fluorescent substances are as follows.

Conditions for automatic detection of microcolony fluorescence signal derived from the first fluorescent substance Halogen light source output 10%, Alexa fluor488 filter (excitation: bandpass filter 460-500 nm, absorption: longpass filter 515 nm or more). The camera setting is exposure time: 2.0 seconds F: 3.5 ISO12800. Hue range: H64-H98, brightness: V150 or higher.

  In this embodiment, because of the resolution of the camera and the imaged area, the size per pixel is about 10 μm, so a pixel aggregate of 2 pixels vertically and horizontally and 15 pixels or less is 20- Since it corresponds to 150 μm, a pixel aggregate of 2 pixels in the vertical and horizontal directions and 15 pixels or less in the hue range: H64-H98, brightness: V150 or more was automatically detected as a signal.

Automatic detection of microcolony fluorescence signal derived from the second fluorescent substance Halogen light source output 10%, TAMRA filter (excitation: bandpass filter 520-560nm, absorption: longpass filter 570nm or more). The camera setting is exposure time: 0.6 seconds F: 3.5 ISO25600. Hue range: H44-H54, brightness: V150 or higher, or hue range: H54-H74, brightness: V100 or higher. Similarly to the above, a pixel aggregate of 2 pixels vertically and horizontally and 15 pixels or less corresponds to a size of 20 to 150 μm. Therefore, a pixel aggregate of 2 pixels vertically and horizontally and 15 pixels or less has a hue range: A pixel aggregate having H44-H54, lightness: V150 or higher, or hue range: H54-H74, lightness: V100 or higher was automatically detected as a signal.
.

Automatic detection of micro colony fluorescence signal derived from third fluorescent substance Halogen light source output 10%, Cy5 filter (excitation: bandpass filter 590-650nm, absorption: longpass filter 660nm or more). The camera setting is exposure time: 1.0 sec. F: 3.5 ISO1250. Hue range: H340-H360, brightness: V80 or higher, or hue range: H0-H20, brightness: V80 or higher. In the same way as above, a pixel aggregate of 2 pixels vertically and horizontally and 15 pixels or less and 2 pixels vertically and horizontally and 15 pixels or less corresponds to a size of 20-150 μm, so it is 2 pixels vertically and horizontally and 15 pixels. In the following pixel group, a pixel group having a hue range: H340-H360, lightness: V80 or higher, or hue range: H0-H20, lightness: V80 or higher was automatically detected as a signal.

Fluorescence in situ hybridization method combined with culture 25 g of food sample was collected in a stomacher bag, mixed with 225 ml of dilution for bacterial test, and mixed to prepare a 10-fold dilution (0.1 g / ml). 1 ml of the food sample dilution was mixed with 1 ml of the microcolony formation medium and added to the simple test kit.

  In the case of the signal + noise group, the cells were then cultured at 37 ° C. for 6.5 hours to form bacterial microcolony (microcolony formation culture). Thereafter, 2 ml of 100% ethanol was added to the simple test kit and fixed for 10 minutes (immobilization). Next, the membrane filter with a high outer frame is taken out and dried, and then enterobacteriaceae detection probe solution labeled with various fluorescent substances is added and reacted at 46 ° C. for 30 minutes, and then the washing solution is added. The membrane filter was washed for 15 minutes at 46 ° C, and the membrane filter with a high outer frame was rinsed with distilled water and dried.

  In the case of the noise section, 1 ml of the 10-fold diluted food sample was mixed with 1 ml of the microcolony forming medium and added to the simple test kit. Next, the microcolony formation culture was not performed, and the subsequent operation was performed in the same manner as the signal + noise section. Next, in the means for detecting the fluorescence signal of the membrane filter with a specific color and brightness, the fluorescence signals of the first, second and third fluorescent substances were measured under the detection conditions.

Enterobacteriaceae count control method The standard method (colon count method) of Enterobacteriaceae described in Non-Patent Document 1 (Enterobacteriaceae community settlement method NIHSJ-16) described above was performed as follows.

  1 ml of a diluted food sample prepared by the fluorescence in situ hybridization method combined with culture was added to a sterile petri dish, poured into dissolved VRBG agar, and solidified. After culturing at 35 ° C. for 24 hours, typical colonies of Enterobacteriaceae were counted.

Results Results of Enterobacteriaceae count of food (Table 30), Fluorescence in situ hybridization combined with culture in all food samples (bacteria added group) under any of the first, second and third fluorescence detection optical conditions The value of the method was within 1/10 to 10 times the value of the Enterobacteriaceae count control method, and was considered to be within the tolerance of measurement error. For food samples (non-added section), the fluorescence in situ hybridization method combined with culture and the control method did not detect Enterobacteriaceae in any of the first, second and third fluorescence detection optical conditions (<10 CFU / g), a reasonable result.

  Therefore, the combined fluorescence in situ hybridization method (UFU / g) with a detection limit of 10 CFU / g by means of detecting two or more specific colors and brightness of fluorescent signals derived from DNA probes is the first, It was shown that accurate counting was possible under the second and third fluorescence detection optical conditions, and it could be used for a wide variety of food inspections.

  In this example, the above three types of samples were used as representative foods, but foods are roughly classified into three categories: agricultural products, livestock products, and marine products, and these are configured individually or in combination. Elements related to noise in a wide range of foods are included in these three types, and if applicable to these, they can be applied to other foods. In particular, the three kinds of vegetables used are vegetable samples having three kinds of colors (green, yellow, orange to red), which are representative of green-yellow vegetables. If the noise of these green-yellow vegetables can be discriminated, the present invention is extensive. Although it is clear that it can be applied to food, in this example, noise of livestock products and fishery products was also confirmed just in case, and it was confirmed that accurate counting was possible as shown in Table 30 below.

  Thus, it is shown that the combined fluorescence in situ hybridization method (UFU / g) using a means for detecting a DNA probe-derived fluorescent signal with two or more specific colors and brightness can be used for a wide variety of food inspections. As a result, automatic microbiological testing devices that can be used in industrial fields such as the food manufacturing industry and the food processing industry can be provided at a very low cost, which can greatly contribute to the safe and reliable supply of food.

Claims (6)

A microbiological test method for foods, which is examined by fluorescence in situ hybridization combined with culture so that the detectable limit value of a specific microorganism is at least 10 equivalents per 10 g of food sample (10 CFU / g) or less,
According to the fluorescence in situ hybridization method used in combination with culture, (1) a liquid containing 0.1 g or more of food sample is filtered using a membrane filter having an effective filtration area equal to or larger than the area of a circle having a diameter of 24 mm. And (2) culturing the microcolony to a size of 20 μm to 150 μm,
The test detects a specific microorganism grown by the fluorescence in situ hybridization method combined with culture by means of detecting a fluorescence signal derived from a DNA probe on a membrane filter with a specific color and brightness using a low magnification optical system. A method for testing microorganisms for foods.   The method for testing microorganisms for food according to claim 1, wherein the specific color is a hue and brightness is brightness.   The food microbiological examination method according to claim 1, wherein the low-magnification optical system has an optical magnification of 1 to 6 times.   Means for detecting a fluorescence signal derived from a DNA probe on the membrane filter with a specific color and brightness shows fluorescence under conditions of an excitation wavelength of 460-500 nm, a fluorescence wavelength of 515 nm or more, which is the first fluorescence detection optical condition, A substance whose fluorescence intensity (%) is 90% or more of the maximum fluorescence intensity obtained at the optimum fluorescence wavelength is used as a first fluorescent substance, and a specific microorganism labeled with the first fluorescent substance (hereinafter, referred to as “the first fluorescent substance”) (Also referred to as target bacteria) Fluorescence in situ hybridization combined with culture using a DNA probe for detection, color photographed under the first fluorescence detection optical conditions, and characteristics of the fluorescence specific color and brightness of the first fluorescent substance in the photographed image 4. The method for testing microorganisms for food according to claim 1, 2 or 3, wherein a signal from a detection target bacterium and a noise from food are discriminated based on the detection, and the target bacterium is detected.   The means for detecting the fluorescence signal derived from the DNA probe on the membrane filter with a specific color and brightness shows fluorescence under the second fluorescence detection optical conditions of excitation wavelength 520-560 nm, fluorescence wavelength 570 nm or more, A DNA probe for detection of target bacteria labeled with the second fluorescent substance on a food sample with a substance having a fluorescent luminance (%) of 90% or more of the maximum fluorescent luminance obtained at the optimum fluorescent wavelength as a second fluorescent substance Fluorescence in situ hybridization combined with culture was performed, color images were taken under the second fluorescence detection optical conditions, and the signal from the detection target bacteria based on the fluorescence-specific color and brightness characteristics of the second fluorescent substance in the photographed image The food microbe inspection method according to claim 1, wherein the target bacteria are detected by discriminating noise from the food.   The means for detecting the fluorescence signal derived from the DNA probe on the membrane filter with a specific color and brightness shows fluorescence under conditions of excitation wavelength 590-650 nm, fluorescence wavelength 660 nm or more as the third fluorescence detection optical conditions, A DNA probe for detection of target bacteria labeled with the third fluorescent substance on a food sample, with a substance having a fluorescent luminance (%) of 90% or more of the maximum fluorescent luminance obtained at the optimum fluorescence wavelength as a third fluorescent substance Fluorescence in situ hybridization combined with culture was performed, color images were taken under the third fluorescence detection optical conditions, and the signal from the detection target bacteria based on the fluorescence-specific color and brightness characteristics of the third fluorescent substance in the photographed image The food microbe inspection method according to claim 1, wherein the target bacteria are detected by discriminating noise from the food.

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