JPH06245793A - Method for detecting helicobacter pylori - Google Patents

Abstract

(57) [Summary] [Objective] To provide a method for specifically and simply detecting Helicobacter pylori. [Structure] A sample containing Helicobacter pylori was subjected to acid methanolysis, (In the formula, n is 5 or 7.) A method for detecting Helicobacter pylori, which comprises identifying 11-methoxy fatty acid methyl ester represented by the formula.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting microorganisms, and more particularly to Helicobacter pylori (Helic).
bacterium pylori; hereinafter sometimes referred to as Hp. ) Detection method.

[0002]

2. Description of the Related Art Hp is detected in the mucous membrane of human active chronic gastritis at a high rate, and when it is inoculated into the monkey stomach, Hp colonizes and produces active gastritis similar to human. It is known to be related to. Recently, this bacterium has been listed as one of several factors involved in the development of gastric cancer [Am. J. Gastroenterol. , 84th
Volume 775-781 (1989), Cancer,
66, 2569-2574 (1990), etc.].

[0003] Conventionally, the identification of Hp was carried out by culturing a tissue collected from the gastric mucosa, and then culturing the tissue.
Confirmed by microscopic examination after staining with any of the silver dyes of Giemsa, Hematoxylin, Eosin or Acridine Orange. Since Hp produces a large amount of urease, a method of detecting Hp by detecting the presence of urease has been studied. further,
In order to clarify epidemiologic Hp infection in patients with gastritis, gastric / duodenal ulcer, gastric cancer such as gastric cancer, and especially gastric cancer, a method of demonstrating the presence of antibodies serologically has been adopted.

However, both the direct staining method and the urease activity method require a long time, and satisfactory sensitivity and specificity have not yet been shown. Even in the method of proving the existence of antibodies, the antigenicity of Hp is extremely extensively used, and even with the Western kits that have already been developed, sufficient specificity cannot be obtained. False positives are expressed.

[0005]

An object of the present invention is to provide a method for specifically and simply detecting Hp.

[0006]

Means for Solving the Problems The present inventor has, as a result of diligent studies on a substance producing Hp in view of the above-mentioned object, found for the first time that 11-methoxy fatty acid is produced from Hp. So far, methoxy fatty acid has been Mycobacterium
It was found only in certain bacteria of ium, but the position of the methoxy group in Hp is Mycobacterium
It was different from that. Thus, the presence of 11-methoxy fatty acids is characteristic of Hp taxonomy.

The present invention has been completed based on such findings, and the present invention will be described below. The present invention is acid methanolysis of a sample containing Helicobacter pylori,
formula

[0008]

A method for detecting Helicobacter pylori, which comprises identifying an 11-methoxy fatty acid methyl ester represented by the formula (n is 5 or 7).
Hereinafter, the present invention will be described in detail step by step. In the present invention, the sample containing HP is gastric mucosa, gastric juice and the like of higher animals, which are optionally cultured. The culture may be performed by any method as long as it can grow Hp. For example, Fukami's method [Inspection and Technology, Volume 17, 1389-1394 (1989)], Brucel
There is a method using a la agar medium. Then, the cultured bacteria can be collected from the culture by a conventional method, washed and centrifuged to obtain bacterial cells.

In the present invention, it is preferable to use the extracted lipid component as the sample for acid methanolysis. In order to extract lipids, for example, a method of extracting gastric juice with an appropriate solvent, a method of leaving the above-mentioned cells at room temperature overnight using a mixed solvent of chloroform-methanol, etc. The present invention is not limited to this. As the extraction solvent, other than the above, solvents that do not react with lipids such as methylene chloride, ethyl acetate, ethyl ether, tetrahydrofuran, and ethanol can be used alone or in combination. The extraction conditions may be heating depending on the solvent, and may be for several minutes to several hours.

The acid methanolysis is preferably carried out using a 5% anhydrous methanol-hydrochloric acid mixture at 60 to 100 ° C.
It is carried out by heating or refluxing for 4 hours. After acid methanolysis, a crude product containing fatty acid methyl ester is obtained by extraction with a solvent such as hexane in the same manner as in ordinary chemical means.

Acid alcoholysis can be carried out with other alcohols. For identification of 11-methoxy fatty acid methyl ester, for example, gas liquid chromatography (GLC), gas chromatography mass spectrometry (GC
-MS), infrared spectrum, analysis by scientific instruments such as NMR, as well as enzymatic method, chemical reaction analysis method and the like are used, but the present invention is not limited to these means.

[0013]

INDUSTRIAL APPLICABILITY The present invention provides a method for detecting Hp which is extremely specific and simple as compared with the conventional method.

[0014]

The present invention will be described in more detail below.

EXAMPLE A standard strain of Hp (ATCC43504) was treated with 10% CO ₂ ,
No antibiotics under 10% O ₂ and 80% N ₂ conditions 10
The cells were cultured at 37 ° C. for 5 days in brucella agar medium supplemented with% horse serum. The cultured bacteria were collected, washed with physiological saline, and centrifuged for 15 minutes (9 × 10 ³ g) to obtain 1.6 g of bacterial cells.

Mixing ratio (volume / volume) 2: 1, 1: 1,
Using 50 ml of each 1: 2 chloroform-methanol mixture, the cells were left overnight at room temperature to extract lipids. The extracts were combined and evaporated to dryness in a rotary evaporator, which was then suspended in 10 ml of distilled water,
It was dialyzed against distilled water to finally obtain 40 mg of a dried solid matter (crude lipid). Of this, 1 mg was subjected to methanolysis at 100 ° C. for 3 hours using 1 ml of a 5% anhydrous methanol-hydrochloric acid mixture. The reaction solution was extracted three times with 1.5 ml of hexane, the hexane layers were combined, and evaporated to dryness in a nitrogen stream (sample 1).

Some of them were subjected to trimethylsilylation by stirring with a mixture of 5: 2: 1 (volume ratio) of pyridine / hexamethyldisilazane / trimethylchlorosilane for 20 minutes at 60 ° C. (Sample 2). .

Identification by GLC and GC-MS; Specimen 1 and Specimen 2 were dissolved in 0.1 ml of hexane, and DB
-1 Fused silica column (0.25mm × 25m, J & W
Scientific, California, U.S.A.
S. A. ) From 180 ℃ to 250 ℃ at 5 ℃ / min
The temperature was raised by and GLC analysis was performed.

The analysis result of the sample 1 is shown in FIG. According to FIG. 1, peaks A and B of unknown structure were found.
This peak is in trimethylsilylated sample 2,
No change was seen.

The peaks for A and B are from Shima.
zu QP-1000 (Kyoto) Mass spectrometer (Carri
er gas: subjected to GC-MS analysis by N _2).
The respective results are shown in FIGS. 2 and 3. A and B
The GC-MS analysis results of the above show that these are typical 11-methoxy fatty acid methyl esters.

[0021]

[Brief description of drawings]

FIG. 1 shows a GLC analysis result of Sample 1.

FIG. 2 shows the GC-MS analysis result of peak A.

FIG. 3 shows the GC-MS analysis result of peak B.

Claims (1)

[Claims] 1. A sample containing Helicobacter pylori is subjected to acid methanolysis, (In the formula, n is 5 or 7.) A method for detecting Helicobacter pylori, which comprises identifying 11-methoxy fatty acid methyl ester represented by the formula.