JPH08149974A - Bacterium-multiplying culture medium for pcr - Google Patents

Abstract

PURPOSE: To enable to directly add a bacterium-multiplied medium to a reaction solution for the PCR for the smooth proceeding of the PCR reaction, and unnecessitate a complicated bacterium-collecting work, etc., before the PCR reaction, by subjecting the bacterium to a bacterium-multiplying treatment in the bacterium-multiplying medium not containing a phosphate salt and used for the PCR. CONSTITUTION: A desired bacterium is subjected to a bacterium-multiplying treatment in a bacterium-multiplying medium used for PCR, containing a nitrogen raw material such as peptone, a salt, a buffering agent, etc., and not containing a phosphate salt. Even when the bacterium-multiplied medium as such is added to a reaction solution for the PCR and subsequently subjected to the PCR reaction for a replication treatment, the PCR reaction proceeds smoothly.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a culture medium for PCR.

[0002]

2. Description of the Related Art Generally, a DNA chain of a bacterium contains a DNA component specific to the bacterium. Therefore,
Attach two primers (a kind of key) to both ends (start end and end end) of the unique DNA constituent part of the DNA chain, and
The so-called PCR method, which is characterized in that the DNA at the site sandwiched by both primers is copied and produced in large quantities by the action of NA polymerase, is a DNA (component) that is unique to the bacterium in the gene DNA chain of the bacterium. Copy in large quantities (reproduction)
After carrying out the treatment described above, it can be used in a method for identifying a bacterium for inspecting the presence or absence of the bacterium depending on whether or not the specific DNA is detected. In this PCR method, after subjecting the bacteria to growth treatment in a normal medium, the bacteria are collected by a centrifugation method, a membrane separation method or the like, suspended in distilled water or a buffer solution,
It is general that this suspension is added to a reaction solution for PCR, and this is subjected to a PCR thermal cycler to carry out a replication treatment of a target DNA constituent part. The reason why the bacteria are separated from the medium by the cell-harvesting treatment is that the addition of the bacteria together with the medium to the PCR reaction solution often inhibits the PCR reaction.

[0003]

However, the above-mentioned work of collecting bacteria is troublesome and complicated. An object of the present invention is to provide a PCR-enriched culture medium in which the PCR reaction is not inhibited even when the culture medium subjected to bacterial growth treatment is directly added to the PCR reaction solution.

[0004]

The present inventor has arrived at the present invention as a result of various studies for achieving the above object. That is, the PCR enrichment medium of the present invention is characterized by not containing phosphate.

The present invention will be described in detail below. In the present invention, all "%" mean "% by weight". First, the PCR enrichment medium of the present invention is used for the growth of bacteria,
Moreover, it refers to a medium that can be directly added to the PCR reaction solution after enrichment. A medium that inhibits the PCR reaction when added directly is used as P in the present invention.
It does not serve as a CR enrichment medium. Specifically, the PCR enrichment medium of the present invention is composed of a nitrogen raw material such as peptone, salts (such as sodium chloride), and a buffer. In this medium, nutrients such as glucose and yeast extract, and agents that inhibit the growth of bacteria other than the bacteria to be tested (selective bacteria inhibitors), for example, nalidixic acid, acriflavine hydrochloride,
Crystal violet, brilliant green, etc. may optionally be added. In addition, p is somehow
Add buffer for H adjustment. As a buffer, TRIS
(Tris aminomethane), HEPES (N-2-hydroxyeth
ylpiperazinesurufonic acid), MOPS (N-morpholi
no propanesulfonic acid) and the like.

Phosphate is a typical buffering agent, but the medium of the present invention does not include it. This is because the inclusion of phosphate inhibits the PCR reaction when the medium after the bacterium enrichment is added as it is to the PCR reaction solution and used. The pH of the medium is adjusted with a buffer, and the pH is usually adjusted to the optimum value for the growth of the desired bacteria.

The enrichment medium for PCR of the present invention is used as a medium for growing the bacteria to be inspected for the purpose of use in the PCR method.

[0008]

The PCR-enriched culture medium of the present invention does not inhibit the PCR reaction when it is directly added to the PCR reaction solution, but its action is not clear.

Examples and test examples of the present invention will be described below.

【Example】

 Example 1 Enrichment medium for PCR of Salmonella consisting of the following formulation Type of raw material Blending ratio Peptone 10.0 g / l (liter) Sodium chloride 5.0 g / l TRIS 6.5 g / l (pH 7.2)

Example 2 Enrichment medium for PCR of Salmonella having the following formulation Type of raw material Blending ratio Peptone 10.0 g / l Sodium chloride 5.0 g / l HEPES 10.5 g / l (pH 7.2)

Example 3 Enrichment medium for PCR of Salmonella having the following composition: Type of raw material Mixing ratio Peptone 10.0 g / l Sodium chloride 5.0 g / l MOPS 11.9 g / l (pH 7.2)

Example 4 Enrichment medium for PCR of toxigenic Escherichia coli consisting of the following formulation Type of raw material Blending ratio Peptone 10.0 g / l Lactose 10.0 g / l Sodium chloride 5.0 g / l TRIS 3.0 g / l (pH 7.3)

Example 5 Enrichment medium for PCR of Listeria monocytogenes having the following formulation Type of raw material Blending ratio Peptone 5.0 g / l Tryptone 5.0 g / l Yeast extract 5.0 g / l Sodium chloride 20.0 g / l TRIS 15.0 g / l nalidixic acid 20 mg / l (selective bacterium inhibitor) acriflavine hydrochloride 12. mg / l (selective bacterium inhibitor) (pH 7.2)

[0014]

[Test example]

<Test method> Three types of medium sterilized at 121 ° C (Example 1
~ 3) for each of the
lerae serovar Enteritidis) 2.8 × 10 / ml
And incubate at 35 ° C for 16 hours, then
It sterilized by heating at 00 degreeC for 10 minutes. This sterilized medium (liquid) was subjected to a test using the PCR method to examine the presence or absence of a DNA component peculiar to Gertonel bacterium. For comparison,
The same treatment as described above was performed on the phosphate-containing culture medium for enrichment.

<Test Results> The results are shown in the table below. That is, the DNA component specific to the bacterium was detected in the phosphate-free medium of the present invention, whereas the specific DNA component was not detected in the phosphate-containing medium. It can be understood that is not progressing smoothly.

<Test Result Table> Present Invention Comparative Example Sample No. 1 2 3 4th item (Example 1) (Example 2) (Example 3) -------------------------- --- Concentration of bacteria in medium before sterilization 2.3 3.6 2.3 3.0 (cells / ml) x 10 ⁸ x 10 ⁸ x 10 ⁸ x 10 ⁸ -------- −−−−−−−−−−−−−−−−−−−−− Detection Yes (○) No (x) ○ ○ ○ × −−−−−−−−−−−−−−− −−−−−−−−−−−−−−−−−

Note 1) The test method using the PCR method was as follows. Prepare a reaction solution for PCR with the following formulation and add it to P
The target DNA component is increased by applying a CR thermal cyclizer, and this is separated by gel electrophoresis to determine the presence or absence of the DNA component.

<Blending of PCR reaction solution> Bacterial fluid (medium sterilized after culturing) 5.0 μl (microliter) Water 23.5 μl PCR buffer 5.0 μl 25 mM magnesium chloride 3.0 μl Primer 1 5.0 μl Primer 2 5.0 μl dNTP mixed solution 3.0 μl Taq polymerase 0.5 μl

<Treatment conditions in PCR thermal cycler> (90 ° C. × 1 minute, 50 ° C. × 1 minute, 72 ° C. × 1 minute) × 30
cycle

Note 2) The composition of the phosphate-containing medium used as a comparative example is as follows: Type of raw material Mixing ratio Peptone 10.0 g / l Sodium chloride 5.0 g / l Disodium phosphate 3.5 g / l Phosphoric acid Monopotassium 1.5g / l (pH 7.2)

[0021]

As described above, when the PCR-enriched culture medium of the present invention is used, the PCR reaction is not inhibited even when the culture medium after the bacterial culture is added directly to the PCR reaction solution, and therefore, It is possible to eliminate the troublesome work such as the bacteria collection process before proceeding to the PCR reaction.

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Claims (2)

[Claims] 1. P that is free of phosphate
CR enrichment medium. 2. TRIS, HEPES as a pH buffering agent
Alternatively, the enrichment medium for PCR according to claim 1, which contains MOPS.