JP2006141300A - Method for separately identifying and/or counting bacterium of enterobacteriaceae and bacterium of genus aeromonas by in-situ hybridization method combined with cultivation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for quickly and separately identifying and/or detecting Aeromonas bacterium and bacterium of Enterobacteriaceae in a specimen likely to contain a bacterium of the genus Aeromonas and a bacterium of Enterobacteriaceae. <P>SOLUTION: The invention provides a method for the simultaneous detection of a bacterium of Enterobacteriaceae and a bacterium of the genus Aeromonas by using in-situ hybridization method combined with cultivation. The method enables the detection of both of the bacterium of Enterobacteriaceae and the bacterium of the genus Aeromonas. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention belongs to the technical field of detection and identification of microorganisms. Specifically, the present invention relates to a technique for rapidly detecting, identifying and / or counting bacteria belonging to the genus Eromonas from the coliform group.

  The coliform is an aerobic or facultative anaerobic Aspergillus oryzae that is a gram-negative bacterium that decomposes lactose to form gas. In addition to Escherichia coli, bacteria such as Klebsiella, Citrobacter, Enterobacter, etc. And is collectively referred to as coliforms. It is regarded as important as a stool contamination indicator bacterium, and detection of this fungus group from water and food means that they are directly or indirectly contaminated by stool.

  For the identification and detection of coliforms, the sample is transplanted into brilliant green lactose bile broth medium (BGLB), cultured for 45 to 51 hours, and observed for gas evolution. Chromocult colliform medium by LB-BGLB method or chromogenic enzyme substrate method that confirms coliform group by degradation of salmon-GAL with β-D-galactosidase and confirms that it is E. coli by degradation with X-GLUC with gluconidase The culture is counted using a selective medium such as (Chromocult® Coliform Agar).

  However, the coliform group includes bacteria that are not taxonomically related, and there is a problem that important enteric pathogens such as Salmonella and Shigella are not detected by the coliform group test.

  Therefore, recently, instead of using the above coliform group as an indicator, bacteria belonging to the family Enterobacteriaceae (referred to as Enterobacteriaceae) are used as safety indicators for processed foods. To date, we have examined 117 representative species belonging to the family Enterobacteriaceae, and 74 of them are positive for ONPG and are subject to the so-called coliform test. Thirty species are negative for ONPG and 13 are unknown for ONPG reactivity. Many ONPG-negative species are isolated from stool and contain pathogens such as Salmonella and Shigella. As a probe for detecting these Enterobacteriaceae bacteria, the present inventors have already developed a probe D (Non-patent Document 1). Furthermore, using this probe D, the number of bacteria of the family Enterobacteriaceae has been measured by a culture combined in situ hybridization method (Non-patent Document 2).

  The genus Aeromonas is a resident bacterium in a freshwater environment and causes infections in fish and animals, and is therefore a bacterium that serves as a safety test index for water, food (fish, vegetables, ice, ice cream). In the genus Aeromonas, (1) the bacteria A. salmonicida that causes gut disease in fish (salmon), (2) it causes infection in humans, for example (i) (well water contamination), etc. A. hydrophila, a bacterium that causes infants or the elderly in developing countries, Australia and the United States (Iowa), (ii) sepsis (infection of leukemia / cancer / liver injury patients, gastrointestinal tract) A. hydrophila, A. caviae, A. veronii, A. schubertii, A. media, (iii) bacteria that cause wound infection are known.

  The examination of the genus Aeromonas can be made only by examining the phenotypes of about 10 items after isolating the strain using a non-selective medium. It takes a few weeks to a month to identify Elomonas.

Journal of Applied Microbiology. Vol.93.60-68 Journal of Applied Microbiology. Vol.95.1182-1190

  Bacteria belonging to the genus Aeromonas are Gram-negative, facultative anaerobic, terrestrial (low salt requirement), oxidase-positive, 16 species known so far, some species ferment lactose at 37 ° C Produce gas.

  Among the strains belonging to the genus Aeromonas, A. caviae, A hydrophila, A. media, and other motile Aeromonas strains include bacteria that ferment lactose at 37 ° C to produce gas. included. In addition, A. caviae, A. eucrenophila, A hydrophila, A. media, and A. sobria grow on a chromocult (R) Coliform Agar (hereinafter referred to as CC medium). On the other hand, some bacteria belonging to the genus Aeromonas, such as A. salmonicida subsp. Achromogenes and A. salmonicida subsp. Salmonicida, do not grow on the CC medium and are not sufficiently identified.

  On the other hand, coliform bacteria are counted using a selective medium such as CC medium, but it is known that the count of coliform bacteria lacks accuracy if bacteria belonging to the genus Aeromonas are mixed. ing. In fact, in 1962, Leclerc and Buttiaux judged that 30% of 9,036 cases of drinking water were positive for false coliforms due to contamination with Aeromonas bacteria.

  In addition, in the method using the probe D described above, bacteria belonging to the genus Aeromonas could not be detected.

  Therefore, the present invention distinguishes between Eromonas bacteria and bacteria belonging to Enterobacteriaceae from a sample in which bacteria belonging to the genus Aeromonas and bacteria belonging to the Enterobacteriaceae may coexist and quickly identifies and / or detects. It is an object of the present invention to provide a method for performing the above.

  To date, the inventors have invented a culture combined in situ hybridization method that rapidly counts Enterobacteriaceae known as an alternative indicator of coliforms.

  In addition to this, we have also developed gene probes that rapidly detect Aeromonas bacteria. In order to investigate contamination of E. coli bacteria in samples that are positive for coliforms by the current culture identification method, both enterobacteriaceae bacteria and bacteria belonging to the genus Aeromonas can be detected using the in situ hybridization method combined with culture. Thus, various conditions were examined, and a method for detecting both simultaneously and rapidly was developed and the invention was completed.

  Furthermore, the present inventors also examined the culture and / or preparation conditions of the sample for simultaneous detection of both these bacteria, and formed a microcolony when a sample in which E. coli and A. hydrophila were mixed was used. When the culture conditions were determined, it was revealed that the cells developed into microcolonies that could be detected with a gene probe at 30 ° C for 6-7 hours.

  By using the method of the present invention, both Enterobacteriaceae bacteria and bacteria belonging to the genus Aeromonas can be detected.

1. 1. In-situ hybridization with culture 1-1. Features / advantages of in situ hybridization combined with culture The conventional in situ hybridization using a fluorescently labeled probe has the following problems. (1) Particularly for identification and detection of microorganisms that contaminate foods, a fluorescently labeled probe cannot be detected unless the microorganisms have grown, that is, contamination has not progressed considerably. (2) In food contamination, where the number of viable bacteria is particularly a problem, since the probe hybridizes to dead bacteria, it was impossible to distinguish between live bacteria and dead bacteria.

  On the other hand, in the in-situ hybridization method combined with culture, a well-diluted sample is cultured for about 1 to 10 hours, so that only viable bacteria are proliferated to form a microcolonial shape, thereby allowing a normal fluorescence microscope. Below, colonies can be observed and counted.

1-2. Steps for in situ hybridization combined with culture (1) Collection of sample on membrane filter A sample separated from food, environment, etc. is aspirated on a sample collector such as a membrane filter. As the membrane filter, for example, a nucleopore filter, an isopore filter, and a mirex filter can be used. Preferably, for example, an isopore membrane filter having a pore diameter of 0.2 to 0.4 μm can be used.

(2) Sample culture Formation of microcolony The sample collector such as a membrane filter to which the sample is attached is transferred to a Petri dish containing the medium. The culture is performed at 30-37 ° C. until a micro colony is formed, for example, for 1-10 hours, preferably 5-7 hours, for example, 6 hours.

(3) FISH and bacteria count (a) Cells on a slide glass or filter are fixed with ethanol or the like. The filter is fixed to the glass slide.
(B) Dried in ethanol or oven.
(C) The filter and the slide glass are immersed in the hybridization buffer and incubated at the hybridization temperature.
(D) The oligonucleotide probe is added and the incubation is continued.
(E) The filter or slide glass on which the sample is placed is washed and air-dried.
(F) Observe with a fluorescence microscope. Usually, it can be observed with X10 to X1000.

1-3. Preparation of Probe 1-3-1 A probe for detecting Enterobacteriaceae was prepared according to the previously filed Japanese Patent Application Laid-Open No. 2001-136969.
Specifically, (1) Enterobacteriaceae is systematically closest to Pasturellaceae, followed by Vibrionaceae (Vibrioaceae), so enterobacteriaceae, Pasteurellaceae and Vibrioaceae The 16S rRNA nucleotide sequences of the bacterial species belonging to the above were compared, and the nucleotide sequences common to the Enterobacteriaceae family and different from those of the latter two Pasteurellaceae and Vibrioaceae were studied. Therefore, intestinal bacteria 12 genera 29 species (β-galactosylase (+) 9 genera 19 species, β-galactosylase (-) 5 genera 9 species, β-galactosylase (unknown) 1 species) and Pasteurellaceae 16S rDNA base sequence information of 3 genus bacteria belonging to 6 species and 8 species belonging to 3 genera belonging to Vibrioaceae is collected, and multiple alignment of these sequences is performed to obtain a base sequence of about 20 bases common only to Enterobacteriaceae bacteria. searched. (2) By searching, the base sequence common to Enterobacteriaceae bacteria and the base sequence of the same region of 16S rRNA of each of the above-mentioned bacterial species (Enterobacteriaceae, Pasteurellaceae and Vibrioaceae) used for multiple alignment were compared. 5'-GAAGCCACGCCTCAAGGGCACAA-3 '(SEQ ID NO: 1), which has a relatively large mismatch number for 14 species of Pasteurellaceae and Vibrioaceae and a small number for 29 species of Enterobacteriaceae : Called probe B) and 5'-TGCTCTCGCGAGGTCGCTTCTCTT-3 '(SEQ ID NO: 2: called probe D) were selected as enterobacteriaceae bacteria-specific probe candidates. (3) Enterobacteriaceae and non-intestinal bacteria were assayed by the FISH method using the above Enterobacteriaceae bacterial probe candidates labeled with a fluorescent label. As a result, fluorescence was detected from most of the Enterobacteriaceae bacterial samples regardless of whether β-galactosylase was positive or negative, and no fluorescence was observed in FISH without adding a probe. In particular, when probe D is used, 75 out of 77 species belonging to the family Enterobacteriaceae can be detected by in situ hybridization, and the genus Aeromonas, Bacillus, Lactobacillus, Staphylococcus, Fluorescence was not detected from microorganisms belonging to the genus Photobacterium and Vibrio. (The number of Enterobacteriaceae bacteria detected was 29 (85%) for probe 1 and 32 (94%) for probe 2 against 34 strains of test bacteria. No signal was detected as a result of the FISH performed in the same manner as in No. 1 and No. 2. From the above, the Enterobacteriaceae bacteria detection probe 1 and No. 2 can specifically detect the Enterobacteriaceae bacteria. The detection rate was high, and the probe 1 was 85% and the probe 2 was 94%.

1-3-2 Probe for detecting Aeromonas genus The probe was designed in the same manner as the probe for detecting Enterobacteriaceae bacteria.
Specifically, (1) 16S rRNA of bacteria belonging to the species belonging to the genus Aeromonas and related strains, specifically Alcaligenes, Vibrio, Shewanella, Brenneria, Pseudoalteromonas, and fungi belonging to the family Enterobacteriaceae Base sequences collected and common to Aeromonas spp. And mismatched with closely related bacteria, specifically Alcaligenes, Vibrio, Shewanella, Brenneria, Pseudoalteromonas, and Enterobacteriaceae Search for a base sequence of about 20 bases. (2) By comparing the base sequence common to the bacteria of the genus Aeromonas and the base sequence of the same region of the 16S rRNA of each of the above strains used for multiple alignment, a relatively mismatch is found for strains other than the genus Aeromonas Those having a large number and few mismatches to the species of Aeromonas were selected as candidates for probes specific to the genus Aeromonas. This operation can be verified with the on-line probemer (http://probemer.cs.loyola.edu/) and the ribosome database project (http://rdp.cme.msu.edu/index.jsp). is there. The results are shown in Table 1.

(3) Next, the genus Aeromonas and non-Eronomonas were actually assayed by the FISH method using the probe candidates for the genus Aeromonas. As a result, it is possible to use a probe for detecting Aeromonas as a probe for which fluorescence is detected from most of the bacteria of the genus Aeromonas and no fluorescence is observed in FISH without adding a probe.

2. 2. Establishment of in-situ hybridization method combined with culture in samples coexisting with Enterobacteriaceae and Aeromonas bacteria 2-1. Study on detection of enterobacteriaceae bacteria in combination with in situ hybridisation method Using the probe for identification of enterobacteriaceae bacteria selected in 1-3-1. Identification conditions by the hybridization method were examined.

2-1-1 Examination of culture conditions It is difficult to detect one bacterial cell by observation with a fluorescently labeled probe for 16SrRNA using a normal fluorescence microscope. Therefore, the cells were cultured to increase to 100 cells that can be observed with a 100-magnification fluorescence microscope. For example, when culturing in Tryptic Soya Agar medium at 37 ° C., it can be cultured for about 6 hours to form microcolonies.

2-1-2 Examination of hybridization conditions Cell immobilization: In normal FISH, paraformaldehyde is used for immobilization, but in culture in situ hybridization method, ethanol is used for about 5 minutes. Can be immobilized. When paraformaldehyde is used, washing after immobilization is necessary to remove paraformaldehyde and maximize fluorescence intensity, but ethanol does not.

  The identification of Enterobacteriaceae is examined by changing the hybridization temperature from 45 ° C to 65 ° C. For example, in the case of probe D, the species with the most mismatches in the region corresponding to probe D in the family Enterobacteriaceae is the 4 bases of Serratia rubida. Therefore, 20% of formamide is compared to Serratia rubidae. Under the conditions, it was found that the identification was enhanced by hybridization at 60 ° C. Hybridization may be performed for about 5 minutes when a DNA probe is used.

2-2. Examination of simultaneous detection of genus Eromonas in the detection by in-situ hybridization with combined culture of Enterobacteriaceae bacteria 2-2-1. Selection of probes for simultaneous detection Probes that can simultaneously detect Aeromonas bacteria under hybridization in the in situ hybridization method combined with culture in the Enterobacteriaceae family, in other words, probes for Enterobacteriaceae have specificity. A probe is selected so that the hybridization conditions possessed by the probe for the genus Aeromonas overlap with the hybridization conditions having specificity. Hybridization conditions are also affected by the reaction temperature, salt concentration, and formamide concentration. Specifically, for example, when probe D is used for detection of Enterobacteriaceae bacteria, 20% formamide and a temperature of 60 ° C. are adopted as hybridization conditions. A genus specific probe is selected. Specifically, AER66 (CTACTTTCCCGCTGCCGC: SEQ ID NO: 3) can be used.

  For comparison of hybridization conditions, the hybridization specific specificity condition values are compared, and a set of probes for Enterobacteriaceae and a probe for Aeromonas bacteria that approximate the hybridization specific specificity condition values are selected. You can also.

  Here, the hybridization specific specificity condition value is defined as follows using the hybridization temperature and the formamide concentration (%) when the probe undergoes hybridization. (As a hybridization buffer, one having the same composition as that other than formamide is used. For example, a hybridization solution in which the composition other than formamide is 0.9 M NaCl, 20 mM Tris-HCl, 0.01% SDS (pH 7.4). Can be mentioned.)

[Equation 1]
Hybridization-specific specificity condition value (K) (° C) = hybridization temperature (° C) + 0.7 (° C) X formamide concentration (%)

2-2-2 Examination of culture conditions Enterobacteriaceae bacteria and bacteria belonging to the genus Aeromonas were co-cultured and then identified by FISH.
As co-culture conditions, temperature and time were changed. Then, after the co-culture, the size of the microcolony was measured, and the culture conditions suitable for the culture combined in situ hybridization method were determined.

3. A method and kit for simultaneously identifying and / or measuring a sample that may contain a genus Aeromonas and a bacterium belonging to the family Enterobacteriaceae.
(Sample preparation)
In the case of a food sample, a test sample is prepared by homogenizing the sample with a blender with a 0.8% NaCl solution.

(Preparation of probe)
The Enterobacteriaceae-specific probe and the Aeromonas genus-specific probe are labeled with a first fluorescent label or a second fluorescent label, which are fluorescent labels having different fluorescence wavelengths. For example, the probe D can be labeled with an enterobacteriaceae-specific probe, and the AER66 can be labeled with a TAMRA or FITC as an Aeromonas bacterium-specific probe, respectively.

(FISH pre-culture)
The homogenized sample is vacuum-sucked onto a sample collector such as a membrane filter with a pore size of 0.2-0.4 μm that does not allow bacteria to pass through a specific reaction, preferably a membrane filter with a pore size of 0.4 μm (Isopore Millipore). Is done. Filters are transferred to Petri dishes containing appropriate media and incubated at 25-37 ° C. for 2-8 hours. As the culture medium, a culture medium in which Enterobacteriaceae bacteria and Aeromonas bacteria can be similarly grown can be employed. Specific examples include bouillon medium, tryptic soya agar (Difco) medium (trypsin-treated soybean casein agar medium), normal nutrient agar medium, brain heart infusion medium, and the like.

(FISH)
Fix the bacteria on the sample collector such as the cultured filter. Preferably, it is fixed with ethanol at room temperature. The filter is then placed on a permeable filter fixing support. When measurement is performed with a fluorescence microscope, a glass slide can be used as the fixed support. However, when measurement is performed with another fluorescence measurement device, a support that can be attached to the fluorescence measurement device and can be measured is used. Any fixed support can be used if present. The filter fixed to the support is dried. Preferably, it is dried with a dryer at 80 ° C. for 10 minutes.

  A hybridization buffer is applied to a fixed support such as a dried slide glass. Examples of the hybridization buffer include a hybridization solution consisting of 0.9 M NaCl, 20% formamide, 20 mM Tris-HCl, 0.01% SDS (pH 7.4). After preincubation for 1-5 minutes in the hybridization buffer, an Enterobacteriaceae specific probe labeled with the first fluorescent label and an Aeromonas bacterium specific probe labeled with the second fluorescent label are added. Hybridization with the probe can be performed for 5 minutes to 1 hour. After hybridization, the fixed support on which the filter is placed is washed with a buffer. As the washing buffer solution, for example, a washing solution containing 20 mM Tris-HCl, 180 mM NaCl, 0.01% SDS (pH 7.4) can be used. After washing with buffer, wash with distilled water.

  Hybridization and washing can be carried out at 60 ° C. for 5 minutes to 1 hour and washing at 60 ° C. for 20 minutes when the labeled probe D and labeled AER66 are used.

  After washing, drying, dipping, and using a device for observing or measuring fluorescence such as a fluorescence microscope using fluorescence using two excitation wavelengths, microcolony can be observed and counted.

(kit)
A reagent or the like for distinguishing and identifying and / or counting Enterobacteriaceae bacteria and Aeromonas genus bacteria by a combined culture in situ hybridization method can be used as a kit. Specifically, a kit comprising a first fluorescently labeled Enterobacteriaceae bacteria-specific probe and a second fluorescently labeled probe specific to bacteria belonging to the genus Aeromonas, more specifically, the first fluorescently labeled Examples thereof include a kit comprising a probe comprising a DNA represented by fluorescently labeled SEQ ID NO: 2 and a probe comprising a second fluorescently labeled DNA represented by SEQ ID NO: 3. These kits may further contain any one of a reaction solution (hybridization buffer), a washing solution (buffer solution), a membrane filter, a medium, or a combination thereof. As the reaction solution, the washing solution, and the medium, those described above can be used.

  Examples of the present invention will be described below, but the present invention is not limited to the following examples.

Selection and verification of Aeromonas genus-specific probes that can be used in combination with Enterobacteriaceae bacteria-specific probe D. Enterobacteriaceae bacteria probe D is reacted at 60 ° C using a reaction solution with a formamide concentration of 20%. Highly specific results are obtained. A highly specific Aeromonas probe was examined under these reaction conditions.

The specificity of the Aeromonas probe was evaluated as follows.
1) Bacteria closely related to Aeromonas hydrophila and Aeromonas (Table 2-4) were pre-cultured overnight (30 ° C and 37 ° C) in bouillon medium and Tryptic Soya Broth (Difco), respectively, and collected by centrifugation. Then, 300 μl of 4% paraformaldehyde-phosphate buffer (PBS) was added to the cells and fixed at 4 ° C. for 1-3 hours.
2) 3 microliters of the fixed bacterial cells were placed on a slide glass, air-dried, and then dehydrated by sequentially immersing in 50, 80 and 100% ethanol for 3 minutes.
3) Add 8 microliters of hybridization solution (0.9 M NaCl, 20 mM Tris-HCl, 0.01% SDS, pH 7.4) preheated to 60 ° C onto fixed bacterial cells on a slide glass. After prehybridization at 60 ° C. for 5 minutes, 1 microliter of 5 pmol / microliter probe was added and hybridization was performed at 60 ° C. for 60 minutes.
4) After hybridization, the slide glass is immersed in a wash solution (20 mM Tris-HCl, 40-220 mM NaCl, 0.01% SDS, pH 7.4) preheated at 60 ° C. for 20 minutes, and then the wash solution remains in sterile distilled water. Washed away.
5) After air-drying the slide glass, the bacterial cells on the glass were observed with a fluorescence microscope, and the specificity of both probes was determined from the fluorescence state of the bacterial cells.
The results are shown in Table 2-4.

  As shown in Table 2, the AER-mrf probe showed high specificity when reacted at 60 ° C. using a reaction solution having a formamide concentration of 27.5% or more and 45% or less.

  As shown in Table 3, the AER-mrr probe was reacted at 60 ° C. using a reaction solution with a formamide concentration of 27.5% or more and 45% or less, or with a reaction solution with a formamide concentration of 45%. By reacting at 0 ° C., it showed high specificity except for P.phosphoreum.

As shown in Table 4, the AER66 probe showed high specificity when reacted at 60 ° C. using a reaction solution having a formamide concentration of 20% or more and 35% or less.
From the above, it was found that AER66 can be selected as an Aeromonas genus-specific probe that can be used in combination with the Enterobacteriaceae bacteria-specific probe D. In Table 2-4, the temperature is described as Temp and the formamide concentration% is described as FA (%).

Verification of the specific reactivity of enterobacteria-specific probe D and Aeromonas-specific probe AER66 against E. coli and A. hydrophila fixed cell mixed samples
1) A sample in which E. coli and A. hydrophila fixed cells were mixed was placed on a slide glass, air-dried, and then dehydrated by sequentially immersing in 50, 80 and 100% ethanol for 3 minutes.
2) Add 8 microliters of hybridization solution (0.9 M NaCl, 20 mM Tris-HCl, 0.01% SDS, pH 7.4) preheated to 60 ° C onto fixed bacterial cells on a slide glass. After prehybridization at 60 ° C. for 5 minutes, 1 microliter of a probe solution in which probe D and AER66 were mixed at 5 pmol / microliter was added, and hybridization was performed at 60 ° C. for 60 minutes.
3) After hybridization, the slide glass is immersed in a wash solution (20 mM Tris-HCl, 40-220 mM NaCl, 0.01% SDS, pH 7.4) preheated at 60 ° C. for 20 minutes, and then the wash solution remains in sterile distilled water. Washed away.
4) After air-drying the slide glass, the bacterial cells on the glass were observed with a fluorescence microscope, and the specificity of both probes was determined from the fluorescence state of the bacterial cells.

  Further, a sample in which E. coli and A. hydrophila were mixed was collected on a 0.4 μm pore size nucleopore filter, and the filter was cultured on Tryptic Soy agar at 30 ° C. for 6 hours to form a microcolon. The microcolonies were mixed with the Aeromonas-specific probe AER66 and the enterobacteria-specific probe D, and a FISH reaction was performed at 60 ° C. using a hybridization solution containing 20% formamide.

  By using a reaction solution with a formamide concentration of 20% and reacting at 60 ° C, A. hydrophila reacts with AER66 probe and Escherichia coli with probe D with high specificity, and the fluorescence color of each probe is clearly distinguished. did it. Escherichia coli emitted red fluorescence, and A. hydrophila emitted green fluorescence. The results are shown in Figure 1.

Establishment of optimal culture conditions for detecting microcolonization of E. coli and Aeromonas by in situ hybridization combined with culture
1) E. coli and A. hydrophila were tested and collected on a nucleopore filter having a pore size of 0.4 μm.
2) The filter was cultured on Tryptic Soya agar (Difco) at 25 ° C., 30 ° C. or 37 ° C. for 2, 4, 6 and 8 hours, respectively, to form a microcolon of each bacterium.
3) After culturing, colonies were fixed with ethanol, and FISH reaction was performed at 60 ° C. using a hybridization solution containing 20% formamide, each containing the Aeromonas-specific probe AER66 or the enterobacteria-specific probe D.
4) Thereafter, the formation of microcolony was examined by measuring and measuring the diameter of a small colony with a fluorescence microscope. The result is shown in figure 2.

  As the culture conditions for forming microcolonies, it became apparent that microcolonies that could be detected with a gene probe developed at 30 ° C. for 6-7 hours. Under these conditions, the diameters of E. coli and A. hydrophila microcolonies were formed to the same size (50-100 μm).

  The present invention can be used for inspection of food poisoning bacteria and / or hygiene inspection of environmental samples.

Simultaneous detection of E. coli and Aeromonas * E. Coli and A. hydrophila are incubated at 30 ° C for 6 hours, then reacted with TAMRA-labeled probeD and FITC-labeled AER66 at 60 ° C in a formamide 20% reaction solution, washed, and then fluorescent Observed with a microscope. Optimal temperature and time for the formation of small colonies that can be detected by the FISH method combined with culture

Claims (8)

  Enterobacteriaceae and Aeromonas bacteria are cultured to form microcolonies, specific for the first fluorescently labeled Enterobacteriaceae bacteria-specific probe and for the second fluorescently labeled bacteria belonging to the genus Aeromonas A method for simultaneously detecting and / or counting Enterobacteriaceae bacteria and bacteria belonging to the genus Aeromonas by an in situ hybridization method using a simple probe.   2. The method according to claim 1, wherein the enterobacteriaceae bacteria-specific probe and the probe specific to bacteria belonging to the genus Aeromonas are probes having overlapping hybridization conditions.   The above-mentioned Enterobacteriaceae bacteria-specific probe and the probe specific to bacteria belonging to the genus Aeromonas, respectively, are the first fluorescently labeled DNA represented by SEQ ID NO: 2 or its complementary DNA and the second 3. The method according to claim 2, which is a probe comprising a fluorescently labeled DNA represented by SEQ ID NO: 3 or a complementary DNA thereof.   Counting the microcolonies labeled with the first fluorescent label and the microcolony labeled with the second fluorescent label, respectively, to simultaneously count Enterobacteriaceae bacteria and bacteria belonging to the genus Aeromonas, respectively. 3. Any one of the methods.   2. The method according to claim 1, wherein the first fluorescent label and the second fluorescent label emit fluorescence having different wavelengths.   2. The method according to claim 1, wherein the first fluorescent label and the second fluorescent label are TAMRA or FITC.   The method according to claim 1, wherein the culture is performed in a medium in which Enterobacteriaceae bacteria and Aeromonas bacteria grow in the same manner for 4 to 8 hours.   Simultaneous detection and detection of Enterobacteriaceae and Aeromonas bacteria, including a first fluorescently labeled Enterobacteriaceae specific probe and a second fluorescently labeled probe belonging to the genus Aeromonas Or a kit for counting.

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