RU1824446C - Method of differential diagnosis of gas gangrene pathogen - Google Patents

Abstract

Usage: microbiologists, the use of high performance liquid chromatography for the diagnosis of anaerobic infections. SUMMARY OF THE INVENTION: for the differential diagnosis of causative agents of gas gangrene, clostridia is grown on meat broth, an internal standard is introduced to obtain derivatives of aminoprograms of culture fluid and control medium, proteins are precipitated with perchloric acid, the precipitate is centrifuged, the centrifugate is treated with a solution of dansyl chloride in acetone and the obtained amine derivatives are separated by HPLC, and the type of clostridia is determined by the ratio of the content of amine derivatives in the culture fluid and the control th nutrient srsdo, 1,6-diaminohexane is used as the internal standard. 1 Clostrdiumi perfringcns, Clostrlclium hlstolytlum, Clostridium oedematlens, Clostrldlum septicum are determined in the presence of / phenylethylamine, putrescine, cadaverine and shstamine in the culture liquid in certain ratios. 5 cp f-ly, 5 ill, 1 tab. next to

Description

The invention relates to chromatographic research methods, in particular to the field of application of high performance liquid chromatography in bacteriology.

The aim of the invention is to improve the accuracy of the method.

This is achieved by the fact that in the method, including growing clostridia on meat broth, obtaining amine derivatives and their chromatographic determination, an internal standard is introduced into the sample of the culture liquid, proteins are precipitated with perchloric acid, the protein precipitate is separated by centrifugation, the centrifugate is treated with a solution of dansyl chloride in acetone , the reaction products are separated by high performance liquid chromatography, and the type of clostridia is determined by the ratio of the amines in the culture fluid and the culture medium ovany.

Kl. perfringence is determined by the presence of / - phenylethylamine, putrescine, cadaverine and histamine in the sample in the following proportions:

 -40-100: d-2-1ЈeP kg -4-14.

NYKm2) / M3k NV

where NI, N2, N3 and N4 - the ratio of the areas of chromatographic peaks of / -phenylethylamine, putrescine, cadaverine and histamine

00

Yu

Јь

4

ABOUT

the peak area of the internal standard in the ultural fluid, respectively:

Mi, N2, Mzk and Mi are the ratios of the area of the chromatographic peaks of / phenyl-ylamine, putrescine, cadaverine and istamine to the peak area of the internal standard in Weinberg medium, respectively.

C. edematiens is determined by the presence of a sample of / -phenylethylamine, putrescine, adaverine and histamine in the following ratios;

N4-4n-mn-- -2 I

   I UU, TPG I iTTP I miI

N "NZK, Nj

Cl. Histolyticum is determined when p-phenylethylamine, putrescine, cadaverine and histamine are present in the sample in the following proportions:

N, 1 0N, N, N.ZKN. "Nuk

C. septicum is determined by the presence of / 3-phenylethylamine, putrescine, cadaverine and histamipe in the sample about the following ratios:

N / L N2 O / irvN3 -1 fi.4, tgg -1 tg 40 tg 1.

N "

The introduction of an internal standard (1,6-diaminohexane) into the sample allows the results to be expressed in terms of the ratio of the peak area of a specific amine to the peak area of the internal standard, etc. to obtain relative quantitative indicators of the amine content in culture liquids of clostridia.

The ability to take into account the differences between species, not only qualitative (the presence or absence of an amine), but also quantitative (the amine content in relation to the internal standard) increases the specificity of differentiation of species.

The use of an internal standard also improves other characteristics of the method;

the accuracy of determination is improved by eliminating errors associated with sample preparation, introducing the sample into the chromatograph, instability of the operating conditions of the chromatography;

analysis and processing of results is simplified by reducing the number of informative components considered to four instead of 18 in the prototype.

A significant difference of the proposed method is the use of high-performance liquid chromatography (HPLC) for the analysis of amines in culture fluids for the differential diagnosis of pathogens of gas gangrene

The determination of amines as specific metabolites of clostridia was previously carried out by gas chromatography (GC) or GC methods in combination with mass spectrometry. The aim of the work was to identify the spectrum of amines produced by different species in the culture medium. The work was carried out at a high level.

The use of the HPLC method for the analysis of amines in culture fluids, together with the rest of the traits, improves the reproducibility and specificity of the determination of clostridia.

Figure 1 shows a chromatogram of dan-5 strong derivatives of amines from Weinberg's medium, where 1 is ft-phenylethylamine, 2 is putrescine, 3 is cadaverine, 4 is histamine, 5 is an internal standard; figure 2 is a chromatogram of dansyl-derivatives of amines of culture liquid Cl. perfringens; Fig. 3 is a chromatogram of dansyl derivatives of amines of the culture fluid of Cl. edematiens; figure 4 is a chromatogram of dansyl derivatives of amines of culture 5 liquid Cl. histolyticum; figure 5 is a chromatogram of dansyl derivatives of amines of the culture fluid of Cl. Septicum.

Example 1. The method for identifying C. perfringens is carried out as follows.

The culture is incubated in Weinberg medium. containing 1% glucose, under vaseline oil at 37 ° C for 24 hours,

The culture fluid is centrifuged for 5 minutes at 5000 rpm.

An internal standard (1,6-diaminohexane) in an amount of 5-10 mol is added to 1 ml of the centrifugate.

Proteins are precipitated with 1 N perchloric acid, 0 then in a ratio of 1: 2 to the volume of the sample.

Protein precipitate was separated by centrifugation for 15 minutes at 8,000 rpm. The centrifuge is neutralized with a saturated solution of potassium carbonate to a pH of 7.0. 5 The precipitate of potassium perchlorate is separated by centrifugation for 10 minutes at 8000 rpm.

Obtaining dansyl derivatives of amines.

To 0.5 ml of the centrifugate in dark flask 0, 30 mg of dry potassium carbonate and 1 ml of dansyl chloride in acetone (5 mg / ml) were added. The vessel is screwed on with a cap and thermostated for 30 minutes at a temperature of 60 ° C.

After 30 minutes, 10 mg of proline was added to the reaction mixture 5 to bind the excess reagent and thermostated for another 10 minutes at a temperature of 60 ° C. 20 µl or more of the reaction mixture was taken and submitted to a chromatograph. Chromatography is carried out on an inverted-phase column (C k1) e chop | , "H-th

(by volume) acetonitrile water solution at a rate of 1 ml / min.

The output of amines from the column is recorded by a photometric detector at a wavelength of 254 nm.

In the chromatograms (FIGS. 1-5), peaks of / 9-phenylethylamine 1, putrescine 2, cadaverine 3 and histamine 4 are identified by the retention time obtained for standard substances and the ratios of the peak area of each amine to the peak area of the internal standard are calculated which are designated as NI. N2. N3 and N4, respectively.

In parallel, an analysis of the Weinberg medium is carried out, the results of which serve as a control. The control is characterized by a low content of all four of the amines in question. Calculated to control peak area ratios of / - phenylethylamine. putrescine, cadaverine and histamine to the peak area of the internal standard, are designated as NiK, No., No. K and

N4.

The experimental data are shown in the table and on the chromatograms (Fig. 1-5). C. perfringens determined at

N4

Nt

j ± - 40-100 (ft-phenylethylamine 1) and -j

4-14 (histamine 4), putrescine 2 and cadaverine 3 are contained at the control level (tab., Figures 1 and 2).

Example 2. The method for identifying C. edematiens is carried out as follows.

Culture growth and culture fluid analysis were performed in accordance with the conditions given in Example 1.

Amine peaks are identified on the chromatograms and the ratios of the areas of these peaks to the peak area of the internal standard are calculated. The results are compared with control (culture medium).

Cl. Edematiens is determined at t °

40-100 (ft-phenylethylamine 1), putrescine 2. cadaverine 3 and histamine 4 are contained at the control level (tab., Figures 1 and 3).

Example 3. The identification method of Cl. Histolyticum is carried out as follows.

Culture growth and culture fluid analysis are performed according to conditions. given in example 1.

Amine peaks are identified on the chromatogram and the ratio of the areas of these peaks to the peak area of the internal standard is calculated. The results are compared with control (culture medium).

N

Ri NM

Cl. Histolyticum determined

60-300 (putrescin 2) and (kadzpeNIK

rin 3), P-phenylethylamine 1 and histamine 4 are contained at the control level (tab., figures 1 and 4).

Example 4. The method for identifying Cl. Septicum is as follows.

Culture growth and culture fluid analysis were performed in accordance with the conditions given in Example 1.

Peaks of amines are identified on chromatograms and peak area ratios are calculated. The results are compared with the control.

Cl.speakum determined at r1-f 2-40

If

(putrescine 2). cadaverin 3 is at the level of control or exceeds it, but not

more than six times -yy. ft-feniNi;

ethylamine 1 and histamine 4 are at the control level (table, figures 1 and 5).,

In the proposed method, in comparison with the prototype, the preparation of a sample of the culture fluid consists of simple steps of protein precipitation with perchloric acid and staining of amines with dansyl chloride (5-NI.N-dimethylamiponaphthalene-1, sulfonyl chloride). The absence of the stage of extraction of organic amines with organic solvents eliminates possible losses due to the high solubility of diamines in water. Dansylamnes are stable for several days and have the ability to fluoresce and absorb ultraviolet rays at a wavelength of 254 nm, which makes it possible to use both fluorescence and photometric detectors for recording amines in chromatography. The sensitivity of the method (the limit of detection by absorption is about 10 mol; by fluorescence by an order of magnitude or more) allows you to analyze the reaction mixture after dancing directly without prior concentration.

Simplification of operations and the use of fewer DO amines to identify the type of clostridia can reduce time and increase the specificity of the assay. The volume of sample to be loaded into the chromatograph can range from 1 to 100 µl, depending on the concentration of amines. Chromatography takes 30 minutes

Since the outcome of the disease with gas gangrene largely depends on the timeliness and purposefulness of SPRICFIC treatment, it is vital to quickly establish the type of the leading pathogen.

A drawback of most bacteriological and biochemical methods for differentiating species is the lack of specificity and the need to use test biological materials that require high initial purity, as well as to control and update them during storage of experimental animals.

The proposed method has high specificity and reliability of species differentiation; does not require specific biological test material (enzymes, serums, indicator media, diagnosticums, experimental animals) to perform diagnostics; reagents are universal and allow long-term storage; the methodology for preparing the sample for analysis, as well as the analysis itself, are simple and well reproducible, can be easily standardized and unified.

The introduction of the proposed method on the practice of bacteriological laboratories in addition to traditional methods will increase the reliability of differential diagnosis and reduce the time of analysis (test).

FORMULA AND SECTION

Claims (5)

1. A method for differential diagnosis of gas gangrene pathogens, including growing clostridia on meat broth, obtaining amine derivatives from the culture fluid, separating them by chromatographic method and determining the type of clostridia by their content, characterized in that, in order to increase the accuracy of the method, in culture samples the liquid and the control medium are introduced an internal standard, proteins are precipitated with perchloric acid, the precipitate formed is centrifuged, the centrifugate is treated with a dansyl chloride solution horide in acetone, the resulting amine derivatives are separated by high performance liquid chromatography and the type of clostridia is determined by the ratio of the amines in the culture fluid and the control culture medium. 2. The method according to claim 1, characterized by that. that 1.6-diaminohexane is used as an internal standard. 3. The method according to claim 1. This is due to the fact that Cloctrldlum perfrlngens defines 0 in the presence of fl-phenylethylamine, putrescine, cadaverine and histamine in the culture fluid in the following proportions: N, 40-100; Ј- Ф 1; st- 4-14, N / kNZK NjK NM 5 where Ni, N2, N3 and N4 are the ratio of the areas of chromatographic peaks of / -phenylethylamine. putrescine, cadaverine and histamine to the peak area of the internal standard in the culture medium, respectively. NIK. N2. N3 and - the ratio of the areas of chromatographic peaks / 3-phenylethylamine, putrescine, cadaverine and histamine to the peak area of the internal standard in the control nutrient medium, respectively. 4. The method according to claim 1, characterized in that Clostrlslum hlstolyticum is determined when the sample contains / -phenylethylamine, putrescine, cadaverine and histamine in the following proportions: 0 5 0 N4, N cn olp NJ . tg 60-300: -yy NIK.NK 5-15: y-N // 1. 5. The method according to claim 1, characterized the fact that Clostrldlum oedematiens is determined when the sample contains / 9-phenylethylamine, putrescine, cadaverine and histamine b the following ratios: Ј- 40-100; .N3 N. N / xN / k Ni (NM b. A method according to claim 1, characterized in that Clostridium septicum is determined by the presence of / -phenylethylamine, putrescine in the sample. cadaverine and histamine in the following ratios: NjLv Nf 2-40-3 N "nN3 1-6: d -1. N  TO The relative content of amines in the culture Clostridium fluids after 24 hours growth on Weinberg environment Note: the amines in the control are designated NIK, M2k, Mz, N4 g-o -g10 FIG. I Continuation of the table. 20 minutes nii Q2 nii 02 i i. 01 ABOUT 9WfrZ8L Vj -G 10 20 minutes