Food-poisoning bacteria detection carrier, and method for detecting food-poisoning bacteria

一种食物中毒菌、检测方法的技术，应用在的载体领域，能够解决难以识别菌株、测序无迅速性、鉴定困难等问题

Inactive Publication Date: 2012-12-26
TOYO SEIKAN KAISHA LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, when using electrophoresis detection, there is such a problem: it is difficult to distinguish substances with similar amplified sizes, so it is difficult to identify strains, and its identification is difficult
In addition, in the case of detection by electrophoresis, there are problems such as complicated operations such as gel preparation, and staining with carcinogenic substances (ethidium bromide, etc.)
In addition, there is a problem that sequencing is not rapid and difficult to operate.

Method used

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  • Food-poisoning bacteria detection carrier, and method for detecting food-poisoning bacteria
  • Food-poisoning bacteria detection carrier, and method for detecting food-poisoning bacteria
  • Food-poisoning bacteria detection carrier, and method for detecting food-poisoning bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0184] Next, using these PCR amplification products, an experiment for confirming whether detection target food poisoning bacteria can be detected by the probe immobilized on the carrier for food poisoning bacteria detection of this embodiment was performed.

[0185] Such as Figure 5 As shown, the carrier for detecting food-poisoning bacteria of this embodiment uses a carrier immobilized with a probe. That is, probes for detecting heat shock protein genes of Escherichia coli of SEQ ID NOS: 1 to 4 were immobilized at positions 1 to 4. At the positions 5 and 6, probes for detecting the uridine monophosphate kinase gene of Escherichia coli of SEQ ID NO: 5 and 6 were immobilized. At positions 7 to 13, probes for detecting heat shock protein genes of Listeria of SEQ ID NOS: 7 to 13 were immobilized. At positions 14 to 19, probes for detecting ribosomal genes of Campylobacter sequence numbers 14 to 19 were immobilized. At positions 20 to 24, probes for detecting the heat-resista...

Embodiment 2

[0221] Next, a test was conducted to verify that the carrier for detecting food-poisoning bacteria according to the present embodiment can simultaneously detect seven kinds of food-poisoning bacteria to be detected.

[0222] As the PCR reaction solution, materials having the following composition were used. Primers synthesized by Life Technologies Japan Co., Ltd. were used. In addition, materials made by Takara Bio Co., Ltd. were used.

[0223] Buffer 10×Ex Taq buffer (20mM Mg 2+plus) 2.0μl

[0224] Nucleic acid synthesis substrate dNTP Mixture (dATP, dCTP, dGTP, dTTP each 2.5mM) 1.6μl

[0225] Primer F (10ng / μl, final conc. 2ng) 0.2μl×7

[0226] ・Primer R (10ng / μl, final concentration (final conc.) 2ng) 0.2μl×7

[0227] ・Primer F for detection of Vibrio 0.4μl

[0228] ・Primer R for detection of Vibrio 0.4μl

[0229] · Nucleic acid synthetase EX Taq (5U / μl) 0.1μl

[0230] ・Labeling component Cy5 0.2μl

[0231] ・Sample DNA 1.0μl×7

[0232] ·Sterilized water 5.5μl

[0...

Embodiment 3

[0268] Next, an experiment was performed to confirm that the probe immobilized on the carrier for detecting food-poisoning bacteria according to the present embodiment does not generate a false positive reaction.

[0269] In this example, a material containing genomic DNA of six kinds of food poisoning bacteria was prepared except that genomic DNA of one kind of food poisoning bacteria was removed from the PCR reaction liquid of Example 2. conduct experiment.

[0270] Then, it was confirmed whether or not fluorescence was detected at the site to which the probe for food poisoning bacteria whose genomic DNA was not contained in the PCR reaction liquid was immobilized. The results of the fluorescence photographs are shown in Figure 13 and Figure 14 , the fluorescence intensity values ​​are shown in Figure 15 and Figure 16 .

[0271] exist Figure 13 and Figure 14 , each column 1 to 7 represents the number of each experiment performed, and the food-poisoning bacteria ...

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Abstract

The disclosed method simultaneously and specifically detects at least two types of food-poisoning bacteria from among Escherichia coli, Listeria, Campylobacter, Vibrio parahaemolyticus, Staphylococcus aureus, Salmonella, and Bacillus cereus. The food-poisoning bacteria detection carrier immobilizes one or at least two probes selected from each of at least two groups from among: an E. coli-detecting first probe group comprising the probes of sequence numbers 1-6 and probes (hereinafter written as "and the like") that are complementary thereto; a Listeria-detecting second probe group comprising the probes of sequence numbers 7-13, and the like; a Campylobacter-detecting third probe group comprising the probes of sequence numbers 14-19, and the like; a Vibrio parahaemolyticus-detecting fourth probe group comprising the probes of sequence numbers 20-24, and the like; a Staphylococcus aureus-detecting fifth probe group comprising the probes of sequence numbers 25-29, and the like; a Salmonella-detecting sixth probe group comprising the probes of sequence numbers 30-40, and the like; and a Bacillus cereus-detecting seventh probe group comprising the probes of sequence numbers 41-49.

Description

technical field [0001] The present invention relates to a carrier such as a microarray for detecting food poisoning bacteria, and particularly relates to a carrier for detecting food poisoning bacteria capable of specifically and simultaneously detecting a plurality of food poisoning bacteria, and a method for detecting food poisoning bacteria. Background technique [0002] Conventionally, the presence or absence of food-poisoning bacteria has been inspected in food inspections, environmental inspections, clinical trials, and animal hygiene. In such inspections, in recent years, DNA contained in inspection samples is amplified by PCR (polymerase chain reaction), and target bacteria are detected from the obtained amplification products. [0003] At this time, electrophoresis is widely used in the detection of food-poisoning bacteria, thereby determining the presence or absence of target bacteria. In addition, the detected amplification product was purified and sequenced to i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12M1/00C12M1/34C12N15/09
CPCC12Q1/689C12Q2600/16Y02A50/30
Inventor 山崎隆明原田天章猿渡由宽龟井修一
Owner TOYO SEIKAN KAISHA LTD
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