JPH10211000A - Detection of bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for expediting the detection of amplified DNA fragments after a sampling stage and PCR. SOLUTION: The E. coli O157 strains in a sample of food, drinks, etc., are collected by adding water to this sample, subjecting the sample to a homogenization treatment and mixing the sample with the magnetic particles coated with an anti-O157 lipopolysaccharide antibody. The sample subjected to the repetition of the operation is put into a PCR reaction vessel. The magnetic particles are attracted from the outer side of the reaction vessel to recover the magnetic particles. The sample is then suspended in sterilizing water and is washed and thereafter, the sample is again put into the PCR reaction vessel, into which a reaction liquid (PCR reaction liquid) for amplifying a gene contg. FITC-dUTP is put to effect the PCR reaction. If the E. coli O157 strain contg. a Verotoxin gene is included in the sample, the FITC-dUTP is taken into the DNA chain, by which the degree of polarization of fluorescence increases and, therefore, the presence of the E. coli O157 strain contg. the Verotoxin gene may be detected from the value of the degree of the polarization of the fluorescence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for detecting bacteria such as E. coli producing verotoxin. The method of the present invention is used in the fields of safety and hygiene management of foods, beverages and the like.

[0002]

2. Description of the Related Art For example, enterohemorrhagic Escherichia coli (enterohemo
rrhagic Escherichia coli (hereinafter, EHEC) or Verocytotoxin-producing Escher
ichia coli (hereinafter referred to as VTEC) is recognized not only as a food poisoning symptom typified by hemorrhagic colitis, but also as a causative organism of hemolytic uremic syndrome in children. The importance of the detection of this bacterium is increasing.

[0003] In an EHEC (or VTEC) test, the test material is feces, food, or water (drinking water, river water, etc.) collected from the patient's surrounding environment. When detecting and identifying EHEC (or VTEC) from these samples, direct isolation culture, primary confirmation culture test,
It is necessary to perform an operation leading to an agglutination test using an antiserum after a secondary confirmation culture test.

[0004]

However, the time required for these culturing steps is 18 to 24 hours, respectively, and the total required time is 3 to 4 days, which is a very long time. As the serotype of EHEC (VTEC), O157: H7 is currently representative, but a diagnostic antiserum required for serotype identification is not yet commercially available and must be prepared in-house. Furthermore, in EHEC (VTEC), the serotype does not always match the pathogenicity, so that identification by the serotype alone often makes it difficult to determine the causative bacterium. Therefore, the current E
The HEC (VTEC) test lacks speed and simplicity and is not effective.

In recent years, to solve these problems, EHEC
Oligonucleotides that selectively hybridize with the Vero toxin gene produced by (VTEC) are prepared, and the oligonucleotides are used as primers for the gene amplification method (P
CR method) to selectively detect only verotoxin-producing bacteria. However, the method using the PCR method has a problem in terms of rapid detection because the confirmation of an amplified DNA fragment after PCR is performed by agarose electrophoresis.

[0006] Therefore, an object of the present invention is to solve the above-mentioned problems and to provide a method for detecting bacteria more quickly.

[0007]

In order to solve the above-mentioned problems, the present invention provides (a) a step of collecting bacteria to be detected by particles coated with an antibody against the bacteria; and (b) a step of collecting the bacteria. Adding the obtained bacteria to a reaction solution for gene amplification containing a fluorescently labeled nucleotide derivative, amplifying the gene while incorporating the fluorescent dye, and (c) detecting the fluorescence polarization of the gene-amplified nucleic acid fragment. Detecting the presence of the specific gene in the bacterium.

Here, the bacteria to be detected are, for example, EHE
Examples include, but are not limited to, C or VTEC, and include all bacteria required for testing foods, beverages, and the like. As the particle coated with an antibody against bacteria, for example, when the detection target is VTEC, an antibody against the bacterial surface lipopolysaccharide of strain O157 can be used. The production of this antibody was reported by Chart, H. et al. (Char
t, H. et al, Journal of Infection, Volume 24, 257-61, 199
2), the same antibody coated on magnetic particles
(Norway, Oslo).

[0009] The collected bacteria are suspended and washed, and then the gene is amplified. Gene amplification is based on Poly
merase Chain Reaction method (hereinafter abbreviated as PCR method;
Science 230, 1350 (1985)). This method uses certain nucleotide sequence regions (eg, E
When detecting the HEC or VTEC verotoxin gene), an oligonucleotide that recognizes and hybridizes one of the two ends of the region with the + strand and the other with the − strand is prepared. The sample nucleic acid in a stranded state is caused to function as a primer for a template-dependent nucleotide polymerization reaction, and the generated double-stranded nucleic acid is again separated into a single strand, and the same reaction is caused again. By repeating this series of operations, the copy number of the region between the two primers increases until it can be detected. The temperature of the heat denaturation is 90 to 95 ° C., and the temperature of the annealing operation for hybridizing the primer is 37 to 95 ° C.
The polymerization is carried out at 65 ° C. and the polymerization reaction at 50 ° C. to 75 ° C., and the PCR is carried out for 20 to 42 cycles in which the cycle is one cycle.

[0010] Oligonucleotides used as primers are selected from the viewpoints of selectivity, detection sensitivity and reproducibility.
It is a nucleotide fragment having a length of 10 bases or more, preferably 15 bases or more, and may be either chemically synthesized or natural. Further, the primer may not be particularly labeled for detection. EHE defined by primer
The amplification region in the nucleotide sequence of the C (VTEC) verotoxin gene may be 50 bases to 2,000 bases, preferably 100 bases to 1,000 bases.
As an example of the primer, when detecting the VT1 and VT2 genes of EHEC or VTEC, see JP-A-7-8.
No. 280 can be used.

The reaction solution for gene amplification includes the above-mentioned primer, heat-resistant DNA polymerase, dNTP solution (N =
A, G, C, T) and a reaction buffer. Heat-resistant DNA
Regarding the origin of the polymerase enzyme, it may be derived from any species as long as the activity is maintained at a temperature of 90 to 95 ° C.
The reaction buffer is generally Tris-HCl, MgCl2, KCl,
It is composed of Tween 20, etc. Further, in the present invention, a nucleotide derivative that is fluorescently labeled is added to the reaction solution for gene amplification. As a fluorescent dye to be labeled, for example, FIT
C, NBD, TRITC, Texas Red, and the like can be used, but are not limited thereto. For example, Fluoresc
ein-12-dUTP by Boehringer Mannheim (Mannheim, German
y) Available from the company. By adding a fluorescently labeled nucleotide derivative, the amplified nucleic acid fragment will elongate while incorporating the nucleotide derivative.

[0012] The nucleotide derivative incorporated in the amplified nucleic acid fragment is detected by suppressing the rotational freedom of the fluorescent molecule and increasing the degree of fluorescence polarization. To measure the fluorescence polarization degree, for example, two polarizing filters are attached to a normal fluorescence spectrophotometer, and p = (I11−I2) / (I11) depending on the fluorescence intensity in the direction parallel and perpendicular to the polarization plane of the excitation light.
+ I2) (I11 and I2 indicate the fluorescence intensity in the parallel and vertical directions, respectively).

The excitation light source differs depending on the fluorescent dye used. For example, when the fluorescent dye is FITC, an argon laser can be used, but the present invention is not limited to this. In addition, a known optical system can be used as the optical system.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The steps of the present invention will be described with reference to the drawings. FIG. 1 is a diagram showing the process of detecting the E. coli O157 strain according to the present invention. First, magnetic particles coated with an anti-O157 lipopolysaccharide antibody are subjected to a homogenization treatment by adding water to a sample of food, beverage or the like (in the figure). (In the figure). Escherichia coli strain O157 is bound to the magnetic particles,
By repeating this operation, Escherichia coli O15
Seven strains are collected. In the case of a sample in a solution state, the sample is directly mixed with magnetic particles coated with an anti-O157 lipopolysaccharide antibody without homogenizing. The sample obtained by repeating the above operation is placed in a PCR reaction vessel (in the figure). In this state, the magnetic particles are attracted from the outside of the PCR reaction vessel, and the magnetic particles are collected. In addition, even when the volume of the sample solution is large, the target E. coli strain in the sample can be easily concentrated and collected by repeating the process of mixing the sample and the magnetic particles and collecting the sample with a magnet. After collecting the magnetic particles from the PCR reaction container, the remaining liquid in the container is discarded, and the container is emptied. The collected magnetic particles are suspended in sterilized water, washed, and then put again in a PCR reaction vessel.

[0015] A reaction solution for gene amplification (P
(CR reaction solution) and suspend the magnetic particles (in the figure). The PCR reaction solution was a reaction buffer (20 mM Tris-HCl
(pH 8.3), 1.5mM MgCl2, 25mM KCl, 0.05% Tween 20,20
0 μM dNTP (N = A, G, C, T) 20 units Taq-DNA polymerase), 50 μM FITC-dUTP, primers for verotoxin gene (for example, JP-A-7-8280). The reaction conditions are, for example, heat denaturation: 94 ° C., 1 minute, annealing: 55 ° C., 1 minute, polymerization reaction: 72 ° C., 1 minute,
The process from heat denaturation to annealing and through to the polymerization reaction is defined as one cycle, and this cycle is performed for 20 to 42 cycles. .
These operations were performed using a DNA thermal cycler (Perkin E
lmer Cetus) under the above reaction conditions.

When the E. coli O157 strain containing the verotoxin gene is not present, FITC-
Since dUTP is not incorporated into the DNA chain, the degree of freedom of rotation of the fluorescent molecule is large (in the figure). If the sample contains the E. coli O157 strain containing the verotoxin gene, FITC-
When dUTP is incorporated into the DNA chain, the degree of freedom of rotation of the fluorescent molecule is suppressed, and the degree of fluorescence polarization is increased (in the figure). If the degree of fluorescence polarization is detected by, for example, a fluorescence spectrophotometer equipped with two polarizing filters, it can be detected from the value of the degree of fluorescence polarization whether or not the E. coli O157 strain containing the verotoxin gene is present in the sample.

In the above description, the presence of the Escherichia coli O157 strain containing the verotoxin gene was detected. However, the present invention is not limited to this, and can be applied to any bacteria by selecting antibodies and primers.

[0018]

According to the present invention, bacteria to be detected are collected by particles coated with an antibody against the bacteria.
The sample concentration effect is increased, and the detection sensitivity is improved. Further, since the amplified nucleic acid fragment is detected by the fluorescence polarization method, the analysis time can be reduced as compared with the conventional agarose gel electrophoresis analysis method.

[Brief description of the drawings]

FIG. 1 is a diagram showing a step of detecting E. coli O157 strain according to the present invention.

Claims (1)

[Claims] 1. A step of collecting bacteria to be detected by particles coated with an antibody against the bacteria, and adding the bacteria obtained in the step to a reaction solution for gene amplification containing a fluorescently labeled nucleotide derivative, A bacterium detection method comprising the steps of: amplifying a gene while incorporating a fluorescent dye; and detecting the presence of a specific gene in the bacterium by detecting the fluorescence polarization of the amplified nucleic acid fragment.