RU2540473C2 - Method of detecting urease-producing microorganisms "rapid urease test helicobacter test" and device for its application - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and can be used for detection of urease-producing microorganisms, in particular Helicobacter pylori. For this purpose express-analysis is carried out on diagnostic discs to determine urease activity of samples. Discs are made from filter paper, on which urea, acid-base indicator, muromidase and conditioning substances are preliminarily applied. In case if urease activity is present in sample colouration of disc takes place.

EFFECT: invention provides increased sensitivity of method of Helicobacter pylori diagnostics.

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Description

The invention relates to the section of analytical chemistry used in biomedical research, namely in the identification of microorganisms for the determination of urease activity, and can be used both in microbiology and in medicine, in particular, for the diagnosis of Helicobacter pylori. This microorganism is characterized by extremely high urease activity, performed in vivo: to control the content of equilibrium ammonia.

Urease activity, both in vivo and in vitro, is usually measured by the kinetics of urea decomposition and, in particular, by the amount of ammonia or carbon dioxide (JB Sumner, GF Somers, “Enzyme Chemistry and Methods of Their Research,” under Edited by V.A. Engelhardt, Foreign Literature, Moscow, 1948, 584 p., p. 178), which stand out as a result of the enzymatic hydrolysis reaction. Liquid media prepared ex tempore from solutions containing urea in water, 50% ethanol, or a phosphate buffer with an acid-base indicator, usually phenol red (transition pH 6.8-8.4), are conventionally used to determine the urease activity of biopsy samples. For example, the Christyansen medium used in microbiology: urea 20 g / l, phenol red 0.012 g / l, KH2PO4 2 g / l, peptone 1 g / l, NaCl 5 g / l, glucose 10 g / l. It was this medium that was first used to determine HP in biopsy specimens of the gastric mucosa. Or a medium containing 2 g of urea, 10 mg of phenol red and sodium azide in 100 ml of 0.01 M phosphate buffer pH 6.5 (N.V. Safonova, A.V. Zhebrun "Gastritis, peptic ulcer disease and helicobacteriosis." Recommendations for doctors, Research Institute named after Pasteur, St. Petersburg, 1993, 40 p., s.32). Urease commercial tests are known where gels are used as the reaction medium. "Pyloritek" (BJ Marshall and coil. Rapid urease test in the management of Campylobacter pyloridis-associated gastritis. Am. J. Gastroenterol. 1987; v.82, p.200-210; Westblom TU and coll. Evaluation of a rapid urease test to detect Campylobacter pylori infectim. J. Clin. Microbiol., 1988, v. 26, p. 1393-1394) and commercial CLOtest sold by Delta West (Australia) in conjunction with Tri-Med Specialites Inc. (USA), tests of the Finnish company BIOCHIT.

The USSR author's certificate 1564192, C12Q 1/04 dated 04/18/08 "Method for the determination of Campylobacter pyloridis in gastric ulcer and duodenal ulcer" is known, where dense gel-like medium carriers made in the form of tablets are used. Agar is usually used as a solid carrier, and sodium azide as a preservative. For one analysis, a nutrient medium is used in an amount of 0.05-0.15 ml, containing urea 15-25 g / l, phosphate 0.01 M pH 6.5 buffer and phenol red indicator 500 mg / ml. The reaction time is tens of minutes. The invention is known (RF patent 97117123.13), selected as a prototype, involving the reaction of indicating the urease activity of the sample on the basis of a solid hygroscopic porous or granular support, and the decomposition reaction of urea and the subsequent release of ammonia, which causes the color reaction of the indicator, occurs when a natural amount of liquid is used the medium in the sample. The reaction time is quite short - up to 5 minutes.

The use of all the above methods, as well as chosen as the quality of the prototype, involves the analysis of the urease activity of enzymes located on the surface of the cells of microorganisms cells for the decomposition of conditioned enzymes with an indicator of the substrate (urea).

In this case, the sensitivity of the method is of fundamental importance, since in the analysis of biopsy samples or other samples on the content of microorganisms, it is possible that a very small number of microbial bodies get into the sample, which with insufficient sensitivity can give a false negative result.

Therefore, in order to further expand our analytical capabilities, we propose a quick urease test based on a solid hygroscopic carrier containing a substrate (urea) in an amount of 1.0 kg to 2.5.0 kg per 1 m2, one of the acid-base indicators with a transition interval in the region from pH 3.2-5.0 to 8.2-9; 0 in an amount of 21 g to 50 g per 1 m2 (e.g. (e.g. bromothymol blue), buffer (e.g. phosphate) to pH 7.0, increasing the osmotic pressure agent (e.g. sodium chloride) 1200 kg per 1 m2, an enzyme that destroys the cell meme the brane of a microorganism (for example, muromidase) - 0.001 kg per m2 The test is made by successive crystallization of the solutions of the above substances on filter paper in a fume hood when using water and isopropyl alcohol as a solvent.

During the analysis, the biopsy is placed on the indicator disc and the indicator effect is evaluated within 3-4 minutes. In the presence of urease-producing microorganisms, the cell membrane is destroyed due to the increased osmotic gradient and the action of enzymes. The releasing intracellular pool of urease causes an immediate reaction of urea cleavage with the release of ammonia, which changes the pH of the medium, which causes color staining of the indicator disc. Thus, there is a sharp increase in the sensitivity of the test, which allows to identify subthreshold amounts of microorganisms by other methods. The application of the method makes possible the diagnosis of gastroduodenal pathology of a person in a clinical and bacteriological laboratory of medical institutions.

In general, the method consists in the fact that the reaction medium is hypertonic and contains substances of organic and inorganic origin that cause damage to the cell wall and release of endogenous urease into the reaction medium.

Example 1

Patient V. was admitted to the clinic at the age of 28 with complaints of pain in the epigastrium. In the process of fibrogastroscopy in the antrum of the stomach, complete erosion was detected, and biopsy material was taken. After dividing the biopsy specimen into 3 parts, one of them was placed on a prototype test, the second was sent for microscopic examination, the third was placed on a disk according to the method described above.

After 3 minutes, the prototype test was negative, with microscopy with staining single Helicobacter pylori microbial bodies, the proposed test was positive after 28 s, on the basis of which it was concluded that HP was present in the material, and therefore the laboratory diagnosis was confirmed - pyloric helicobacteriosis , and the corresponding etiotropic therapy of the disease was started.

Example 2

Patient B., aged 35, was admitted to the clinic for further examination because of epigastric pain. When fibrogastroscopy revealed chronic subatrophic gastritis with duodeno-gastric reflux IV century. and material was taken for research.

The biopsy is divided into 3 parts. In the process of testing the biopsy according to the variant of the prototype of the microscopic method and the method proposed above, no microorganisms were detected - it was concluded that there was no HP.

Example 3

Patient F., aged 48, was admitted to the clinic with complaints of epigastric pain. In the process of fibrogastroscopy in the stomach, chronic atrophic gastritis, chronic ulcer of the bulb 1.0 × 0.8 cm, cicatricial deformity of the bulb 12 p of the intestine. Biopsy material taken. After dividing the biopsy specimen into 3 parts, one of them was placed on a prototype test, the second was sent for microscopic examination, the third was placed on a disk according to the method described above.

After 3 minutes, the prototype test was negative, with microscopy with staining single Helicobacter pylori microbial bodies, the proposed test was positive after 14 s, on the basis of which it was concluded that HP was present in the material, and therefore laboratory diagnosis was confirmed - Helicobacteriosis, and the corresponding etiotropic therapy of the disease was started.

Claims (1)

A method for detecting urease-producing microorganisms, which consists in introducing chemicals into the medium containing urea, conditioning the reaction medium and an indicator that reacts with a color change to a pH shift caused by the release of ammonia during the decomposition of urea with urease of the tested microorganisms, and made in the form of diagnostic disks from natural or synthetic polymers, characterized in that in order to increase the sensitivity of the method, the reaction medium is hypertonic and contains organic substances and inorganic origin, causing damage to the cell wall and exit into the reaction medium of endogenous urease.