JPH0436198A - Method for examining food poisoning bacterium - Google Patents

Abstract

PURPOSE:To quickly, simply, safely and inexpensively obtain a bacterial cell ingredient and simply identifying and examining the title bacterium by passing a suspension through a plural filters having different diameters to catch the bacterial cell ingredient and subjecting the bacterial cell ingredient to polymerase chain reaction. CONSTITUTION:A suspension containing a food poisoning bacterium is successively passed through the first and second filters having different diameters to remove an insoluble solid content and then a bacterial ingredient is caught by the third filter different in diameter from the above-mentioned filters and a solution containing the caught bacterial ingredient is subjected to polymerase chain reaction and DNA of desired bacterium is amplified to carry out examination of desired food poisoning bacterium.

Description

[Detailed description of the invention] (a) Industrial application field Is the present invention a disease caused by bacteria? Food poisoning bacteria are collected from suspensions (e.g. feces) of human or animal specimens that caused the disease, and the DNA
A method for extracting and testing oil.

(b) Conventional technology 1) In the field of diagnosis and research, 7) food poisoning in biological specimens was used to identify type 1 food poisoning and to analyze its contents on agar plates using various drug-blind methods. Cultivation is carried out from the beginning to the end.

Solid content (food 'J'
Is it possible to make a suspension of a sample using the conventional method of dissolving the food poisoning measures contained in the sample (such as rQ shallow beak or aquaculture) to remove the bacterial cells? After culturing in a medium for several days, the resulting colonies are collected and centrifuged to collect bacterial cells. And this f'L'z 7-lotibus KL:D! Bacteria can be lysed using an enzyme, a surfactant such as sodium sibdodecyl sulfate, or an alkali such as sodium hydroxide, ultrasonic waves, or a physical crushing method such as a mill. Is the bacterial component contained in the pieces that are crushed? obtain.

In addition, in conventional bacterial identification methods, for example, the sample is spread on a medium such as agar containing a certain antibiotic, and the resulting bacterial growth is investigated. Separate and further separate and culture the separated bacterial groups by spreading them onto a glass containing a different antibiotic λ and repeating the separation culture method, or by fixing them on the antibody specific to the bacterial cells in Latex Nuppies. The latex a collection method uses 0 and t, which is added during wiping and the antibody on the bead surface recognizes the bacterial cells, causing the bacteria to aggregate. After the antigen-antibody reaction, we separate those that bind and those that do not, and then apply a secondary antibody labeled with an enzyme or fluorescent substance. Detection is performed at the gene level using the sandwich antibody method, which identifies r f411, or by transferring it to the colony t-membrane filter and after lysis, allowing the specific NA fragment to interact with a t-labeled DNA probe such as an enzyme or a radioactive substance. There is the DNA 7" lobe method. This method uses the fact that when there is a base sequence complementary to the DNA probe in the DNA in the colony, a hydrogen bond is created between the two molecules. This method is used to detect the target food poisoning bacteria, etc. It is.

(c) Problems to be Solved by the Invention However, the above-mentioned method for obtaining bacterial cell components requires a considerable amount of time for culturing, and therefore, it is difficult to obtain bacterial cell components quickly. In particular, in the case of causative bacteria that cause 11k in food, identifying the causative bacteria early and administering antibiotics etc. will have a great effect on preventing the spread of the pathogen and treating it. The proposed nucleic acid purification method requires expensive equipment such as centrifuges and dangerous organic solvents such as phenol and chloroform, and is not applicable to actual clinical settings. The purpose of the present invention is to provide a bacterial testing method that can easily, safely, and inexpensively identify all bacteria that cause food poisoning. It is about providing.

ni) Assignment? Means for Solving the Problem In order to solve the above-mentioned problems, the present invention provides a suspension containing food poisoning bacteria in a first tube of different diameter. Pass it through the second filter sequentially,
After removing the insoluble solids, the bacterial components are captured by a third filter having a diameter different from that of the filter, and the captured bacterial components are collected in a solution containing Kurayoshi. By subjecting it to polymerase chain reaction and amplifying the desired bacteria by 2 times, the desired food poisoning bacteria can be detected.

(E) Function In the present invention, 1) the suspension containing bacteria is passed through a coarse first filter 4) to separate insoluble solids. Next is a bacteriolytic agent for ag?
In addition, bacterial cell components are released.

Then, ICm-insoluble solids are further removed by a second filter. To the filtrate obtained here, a sulfuric acid component suspect agent is added to precipitate the filtrate, which is then passed through a third filter to capture the nucleic acid component. Kon'
? Wash with 70% ethanol aqueous solution and further
% ethanol aqueous solution, water and surfactant aqueous solution flowed into the third filter, and the nucleic acid component? Elute.

The overall test method for amplifying the eluted nucleic acid component using polymerase chain reaction is quick and simple. In addition, since results are obtained quickly, it can be performed by administering an antibiotic that specifically acts on pathogenic bacteria.

- 夾mgAl (Preparation of sample liquid
152i) 10 pieces, 10 hE, i fc-tri
lo'4fllj/l gltilk 50atn
IJ: y-hydrochloric acid buffer (pH 7.0) (hereinafter referred to as pB) 4
-IKtJIL.

I got it.

(Rough to uneven processing) The sample solution obtained above was then used for treatment.

Attach a nylon mesh filter with a pore size of 10p to a PP-47 filter holder made by Atoppanchik. 1
For 0-1/Pour the sample liquid into the ring, connect it to the filter holder, and pass it through.

(Bacterial lysis treatment) The above-obtained solution KN-7 cetylmuramid
Nu'? 60μg/■J1 Acromope τ titase 21m
g 7 ml, and incubated at 37° C. for 10 minutes. Further, 21 g of VCBDs and proteinase Kk1/dK were added and incubated at 60'C for 30 minutes. For another 95 minutes, the proteinase was inactivated.

(Filtration treatment) Add 1/10 g of 2.5 M KCl aqueous solution to the obtained lf' solution to aggregate the SDS component, and then add solution 2-I.
K/) and MILLEX -PF O,B pm yi
lter Llnit MILLE! GV O, 22p
The insoluble fraction and 8Di9 components were removed by filtering for one day.

(Ethanol precipitation treatment) To the obtained filtrate, 1/10 amount of 3M sodium chloride (pd
5.2), add 7Xl to twice the volume of 99% cold ethanol.

(Capture of nucleic acid components) GF/D (WHA
Glass fiber filter paper manufactured by TMAN Corporation) is installed, and the liquid obtained above is turned into a liquid! I met you. Further, this filter was washed with 70% cold ethanol and 99% cold ethanol.

(Elution of nucleic acid components) Using the above filter Vcl syringe, 25μ
I 0.5% N0NID, IEτP-40, 0,5
% Tween 20 aqueous solution was poured into the ink, 50"C was inked for 10 minutes, and the nucleic acid components were collected using a 1-liter syringe.

(Amplification of DNA) Take 5 μl of the above elution & i-,- and 100-fold diluted solution and perform polymerase chain reaction (PCB 65alk1i,
x, et al, 5science 239487-
491 (1988)) for 4 hours. Here, the above P
The CR method was carried out under the following conditions. That is, distilled water 26.
5 μl, dNTP 8p I (1,25mM each dNT
P), primer 2.5μ1 (20μM) 2 types, 10
Double buffer solution 5μl (manufactured by Perkinilmartheta 2),
2.5 units of Choaq polymerase (from the same company) total of 5
Overlay 0 μl with 100 μm of mineral oil as an evaporation inhibitor, and add DNA
(manufactured by the same company).

What is meant by 10 times buffering here?
gCl2.

As primers, we used the NA Hanawa sequence described in #Nyohei 1-185682 of Jinlin et al. and specific for Bacillus cerenu. Each primer was synthesized using a DNA automatic articulation device 1NS-1 (manufactured by Atsu Seisakusho) and purified by high-performance liquid talomadography equipped with a reverse phase column.

The temperature cycle for the PCB reaction was denaturation at 94°C for 1 minute, annealing at 37°C for 1 minute, chain elongation at 60°C for 1 minute, and 1 cycle/L.
15.7 minutes, 42 cycles.

(Detection) Add 20μm of ethidium bromide to the solution after PCR using 2g6 agarose gel at 100V for 35 minutes.
! After performing pneumophoresis, apply gel lv on a transilluminator.
’ and irradiated the entire PC with UV light with a wavelength of 320 nm.
The DNA amplified by the R reaction emitted fluorescence. Figure 1 shows the results taken with a camera equipped with the fluorescent instant film. Bacillus cereus in the sample (in stool X)
A suspension containing VC is shown in lanes 1 to 9 of the first section of the electrophoresis pattern of Shun, who carried out the method according to the present invention. Lane 1.2.3 has 106 bacteria, lane 4.5.6 has 10 bacteria, lane 7.8.9 has 108 bacteria! 2
X stool contained in 1g! ! This is the pattern of the present invention's kW application. Lane 1, 4.7 is the elution stock solution, lane 2.5.8d is the 10-fold dilution of Soko et al., lane 3.6.
9 was subjected to a 100-fold diluted solution 1PcR. Lane 10 fl is a positive control for Bacillus cereus. M to X174 DN A k@limited enzyme Hinc
nfP: is a molecular weight marker obtained by incubation with
A, B.

This is the number of base pairs corresponding to C and D.

As a result, a band appeared at a position of about 232 base pairs. This fL and I at the same time! Since it is in the same position as the positive control sample (Lane 10 in Section 1) that was electrophoresed, is it Bacillus cereus? It can be seen that specific detection was possible. The positive control referred to here is a pure culture of Bacillus cereus (JCM2152 strain) in a liquid medium.
A DNA solution obtained by lysis with enzymes and surfactants, extraction with phenol and chloroform, and precipitation with ethanol was used.

From the above results, according to the bacterial testing method of this invention, 1
It has been found that if a gram contains at least 106 Bacillus cereus bacteria, the above method can be detected easily and within a short time of 5 hours.

(g) Effects According to the method of the present invention, bacterial components containing DNA contained in biological samples such as feces, which are extremely contaminated, can be obtained quickly, easily, safely, and inexpensively. Moreover, since polymerase chain reaction is used, the entire calibration method is simple, and the target pathogen WII can be identified in a short time of less than 5 hours.

[Brief explanation of drawings]

The first problem is the electrophoresis pattern diagram after implementing the method of this invention.

Claims (1)

[Claims] 1. After passing the food poisoning-containing suspension through first and second filters with different diameters to remove insoluble fixed components, the bacterial components are captured by a third filter with a different diameter from the filter, and the captured bacterial components are removed. 1. A method for testing food poisoning bacteria, which comprises testing for desired bacteria by subjecting a solution containing bacterial body components to polymerase chain reaction to amplify the DNA of the desired bacteria.