CN113092459A

CN113092459A - Gel reagent and kit for fast screening of urease activity and fast screening method

Info

Publication number: CN113092459A
Application number: CN202110384243.XA
Authority: CN
Inventors: 许奕鹏; 杨晓辉
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-04-09
Filing date: 2021-04-09
Publication date: 2021-07-09

Abstract

The invention discloses a gel reagent and a kit for rapidly screening urease activity and a rapid screening method, and belongs to the field of medical detection. The gel reagent for fast screening of urease activity comprises 0.2-0.3 volume part of urea; 4-8 parts by volume of phenol red; 0.02-0.1 volume part of proclin 300; 2-10 parts by volume of a pH buffer solution; 5-15 parts by volume of agar gel, wherein the concentration of urea is more than or equal to 7mg/ml, and the concentration of phenol red is more than or equal to 30 multiplied by 10^‑3mg/ml. The detection method utilizes the reaction product of urease and urea in saliva or biopsy specimen to initiate corresponding index change to determine the activity of the urease. The detection steps are as follows: and (3) collecting a proper amount of sample by using an applicator device, pushing the whole sample to the lower part of the surface of the gel in the kit from the tweezers by using the applicator device, exposing the sample in the gel as much as possible, standing for 2-3 min, observing the color change of the gel by a visual method, and determining the sample to be urease-positive if the sample turns red and urease-negative if the sample does not turn color.

Description

Gel reagent and kit for fast screening of urease activity and fast screening method

Technical Field

The invention belongs to the field of medical detection, and particularly relates to a gel reagent and a kit for rapidly screening urease activity and a rapid screening method.

Background

This bacterium was first cultured in 1982 by Warren and Marshall, the Robin physician, from a antral biopsy, and was called Campylobacter-like bacterium. Later, this genus was named helicobacter pylori, meaning a spiral or helical bacterium. Helicobacter pylori has been shown to cause active chronic gastritis, a risk factor for gastric cancer and mucosa-associated lymphoid (MALT) lymphoma. Helicobacter pylori inhabits the human digestive tract, usually in the gastric mucosal epithelium, and may also multiply in the oral cavity. Urease produced by the helicobacter pylori can rapidly carry out enzymolysis on urea to produce alkaline ammonia, so that the helicobacter pylori is less susceptible to gastric acid in the stomach.

Urease is a nickel-containing oligomeric enzyme, and can be produced by bacteria such as streptococcus salivarius, actinomyces naeslundii, staphylococcus wowensis, helicobacter pylori and the like which may be contained in oral cavity. Urease can cause a variety of human diseases, including caries, periodontitis, etc., associated with the oral cavity. At present, the detection methods of urease mainly comprise a Nassner reagent colorimetric method, a Raman spectroscopy method, a phenol red indicator method, a colloidal gold immunochromatography method, a precision test paper method and the like. The nano-silver reagent contains mercury, toxic reagents such as silver nitrate and the like are needed for Raman spectroscopy, and the equipment popularization rate is low, so that the two methods are not widely applied. The qualitative detection of urease in saliva can be realized by a saliva test board and a precision test paper method based on the colloidal gold immunochromatography. The urease has high homology, urea can be rapidly hydrolyzed into carbon dioxide and ammonia, and solution pH changes are caused, and based on the principle, the phenol red indicator method can rapidly characterize the urease in living tissues such as gastric mucosa and the like. Therefore, the invention provides a gel reagent for rapid screening by taking phenolphthalein as an indicator, a method for rapid screening of urease activity and a detection kit for rapid chromogenic time of urease activity detection, which have important significance.

Disclosure of Invention

The invention aims to: the urease activity rapid screening method and the urease activity rapid screening kit are improved, have improved detection performance, are convenient and simple to operate and are provided; according to the method, the collected sample can be visually and rapidly tested and detected only by wiping the collected sample in the gel of the detection reagent; the helicobacter pylori is rapidly screened by using the corresponding index change caused by the reaction product of urease and urea in the sample.

In order to achieve the purpose, the invention adopts the following technical scheme:

a gel reagent for rapidly screening urease activity comprises the following components in parts by volume:

in the invention, the concentration of urea in the gel reagent is more than or equal to 7mg/ml, preferably 7-20 mg/ml, and further preferably 7 mg/ml; phenol red concentration is greater than or equal to 30 x 10^-3mg/ml, preferably 30X 10^-3～50×10^-3mg/ml, further preferably 30X 10^- ³mg/ml。

In the invention, the pH buffer solution comprises at least one acid and at least one alkali which are mixed, and the acid and the alkali are both soluble acid and alkali, and no precipitate is generated during reaction and no chemical reaction is generated with urea. Further, it is preferable that the pH buffer is a mixture of 2- (N-morpholine) ethanesulfonic acid monohydrate and sodium hydroxide.

In the invention, the pH value of the pH buffer solution is adjusted to 5-8, and the activity detection effect of urease is good under the condition.

A kit for rapidly screening urease activity comprises a plastic glass sheet, wherein a concave plastic glass reagent cup is arranged in the center of the glass sheet, a gel reagent for rapidly screening the urease activity is filled in the reagent cup, and the gel reagent is formed by pouring urea, a pH indicator phenol red, a bacteriostatic agent proclin300 and a pH buffer solution into agar gel and uniformly mixing; the upper surface of the plastic glass sheet is also pasted with a label for filling information of a detected person, and the concave plastic glass reagent cup is sealed and covered by the label.

The invention also comprises a method for rapidly screening the activity of urease, which comprises the following specific steps:

a. collecting an appropriate amount of sample with an applicator device;

b. pushing the sample from the applicator device with forceps below the surface of the gel reagent in the kit, exposing the sample as much as possible to the gel reagent and ensuring that the biopsy specimen is completely immersed in the gel reagent;

c. standing for 2-3 min, observing the color change of a gel reagent in the kit by a visual method, and if the color change is red, determining that the kit is urease positive; if the color is not changed, the color is negative to urease.

In the invention, the applicator device is a toothpick, a cotton swab or the like, and the collected sample is any one of saliva, tartar, biopsy stomach and the like.

The bacteriostatic agent proclin300 is used for preventing the growth of pollution urease positive bacteria.

The combined use of the bacteriostatic agent and the buffer can effectively improve the detection performance of urease activity.

The invention has the following advantages and beneficial effects:

according to the invention, the sample is not required to be processed, the sample collected by the applicator device is pushed into the gel, and the reaction product of urease and urea in saliva is used for triggering corresponding index change, so that visual and rapid detection can be realized by a visual method. Compared with the conventional mode, the method has the advantages of no pretreatment process, simple and convenient operation, convenient carrying and transportation, lower cost and higher convenient application performance.

Detailed Description

The present invention is further illustrated by the following examples, but is not limited thereto, and any variations or modifications based on the teachings of the present invention are within the scope of the present invention.

Examples

In the embodiment, 1-25 gel reagents composed of different urea concentrations and different phenol concentrations are provided, the concentrations are measured in mg/ml, the formula of each component is shown in table 1, and the volume is measured in ml.

TABLE 1 examples 1-25 gel reagent compositions

In this embodiment, a method for rapidly screening urease activity is performed according to the following steps:

a. collecting appropriate amount of sample (saliva, tartar, biopsy stomach, etc.) with toothpick or cotton swab;

b. pushing the sample from the forceps with a toothpick or cotton swab below the surface of the gel reagent in the kit, exposing as much of the sample to the gel reagent as possible and ensuring that the biopsy specimen is fully immersed in the gel reagent;

c. standing for 2-3 min, observing the color change of the gel reagent in the kit by a visual method, wherein if the color change is red, the kit is urease-positive, and if the color change is not red, the kit is urease-negative.

Color development test

According to the above steps, cotton swabs are respectively placed in the oral cavities of several patients with positive helicobacter pylori (i.e. containing urease in the oral cavity) and several normal persons with negative helicobacter pylori (i.e. without urease in the oral cavity) to be wiped to respectively obtain 25 parts of saliva samples, each part of the gel reagent in the examples 1-25 is divided into two parts, the 25 parts of the samples with positive helicobacter pylori are respectively pushed to the lower part of the surface of one part of the gel reagent in the examples 1-25 by using tweezers, the 25 parts of the samples with negative helicobacter pylori are respectively pushed to the lower part of the surface of the other part of the gel reagent in the examples 1-25, and after standing for 2-3 min, the color change of the gel reagent in the examples is observed by a visual method as shown in table 2.

TABLE 2 examination of positive and negative coloration of H.pylori with the gel reagents of examples 1-25

As can be seen from the data in Table 2, examples 13, 14, 15, 18, 19, 20, 23, 24 and 25 showed red color when they were positive for H.pylori, while the other examples showed no red color when they were positive for H.pylori; all examples did not show a red color when H.pylori was negative. It can be seen that, within the scope of the above examples, the urea concentration is 7mg/ml or more and the phenol red concentration is 30X 10 or more^-3The two conditions of mg/ml reach the gel reagent at the same time, and the red color is developed.

(II) color development time test

2ml of urea solution with the concentration of 7, 20, 35, 70 and 140mg/ml and the concentration of 30 multiplied by 10 respectively^-36ml of phenolphthalein, 0.06ml of proclin300 solution, 5ml of pH buffer solution (adjusted to 5-8) and 10ml of agar gel are respectively prepared into the 5 gel reagents capable of positively discoloring the helicobacter pylori. And color development time tests were performed for the different dominant quality controls of H.pylori, as shown in Table 3 below. In the table, the strong positive quality control refers to saliva of the same patient in which helicobacter pylori dominance is "positive + + +", and the weak positive quality control refers to saliva of the same patient in which helicobacter pylori dominance is "positive +".

TABLE 3 analysis of the development time for screening helicobacter pylori with different urea concentrations

Respectively at a concentration of 30 × 10^-3、50×10^-3、70×10^-3、100×10^-36ml of phenolphthalein with the concentration of 7mg/ml, 2ml of urea solution with the concentration of 7mg/ml, 0.06ml of proclin300 solution, 5ml of pH buffer solution (adjusted to 5-8) and 10ml of agar gel, and the 4 gel reagents capable of positively discoloring the helicobacter pylori are respectively prepared. And color development time tests were performed for the different dominant quality controls of H.pylori, as shown in Table 4 below. In the table, the strong positive quality control refers to saliva of the same patient in which helicobacter pylori dominance is "positive + + +", and the weak positive quality control refers to saliva of the same patient in which helicobacter pylori dominance is "positive +".

TABLE 4 analysis of the development time for screening helicobacter pylori with different phenolphthalein concentrations

As can be seen from tables 3 and 4, when the concentration of urea is 7mg/ml or more and the concentration of phenolphthalein is 30X 10 or more^-3The color development time observed by naked eyes is less than 2min in both strong positive quality control and weak positive quality control at mg/ml(so the observation time is 2-3 min, the gel reagent can be developed), and the development time is reduced along with the increase of the concentration of urea and phenolphthalein, but the reason of the high concentration of urea and phenolphthalein can not be selected, firstly, from the quality control, the color change range of phenolphthalein is pH 8.2-10.0, the phenolphthalein changes to red color when meeting the alkali solution, however, the phenolphthalein rapidly fades to colorless from red in the strong alkali, when the concentration of urea is too high, the urea reacts with urease to generate NH₃The phenolic phthalein is easy to dissolve in water, so that the alkalinity of the reagent is increased, and the phenolphthalein is possible to turn red and then returns to be colorless, thereby causing inaccurate observation results; secondly, the proportion of each reagent component is compatible, excessive phenolphthalein is dripped into a dilute acid solution to cause precipitation, so that the solution becomes white and turbid, the pH of the gel reagent in the embodiment of the invention is adjusted to 5-8 by using a pH buffer solution, namely the solution is likely to be weakly acidic, so that the reagent components with too high phenolphthalein concentration are incompatible and the judgment of the discoloration phenomenon is influenced; thirdly, the raw material cost required for high concentration of urea and phenolphthalein is also increased in combination with the consideration of production cost. Therefore, the concentration of the urea is preferably 7-20 mg/ml, and more preferably 7 mg/ml; the concentration of phenolphthalein is preferably 30X 10^-3～50×10^-3mg/ml, further preferably 30X 10^- ³mg/ml。

Claims

1. The gel reagent for rapidly screening the urease activity is characterized by comprising the following components in parts by volume:

2. the gel reagent for rapid screening of urease activity according to claim 1, wherein the urea concentration is not less than 7mg/ml, and the phenol red concentration is not less than 30 x 10^-3mg/ml。

3. The gel reagent for rapid screening of urease activity according to claim 1, wherein the urea concentration is 7-20 mg/ml, and the phenol red concentration is 30 x 10^-3～50×10^-3mg/ml。

4. The gel reagent for rapid screening of urease activity according to claim 1, wherein the urea concentration is 7mg/ml, and the phenol red concentration is 30 x 10^-3mg/ml。

5. The gel reagent for rapid screening of urease activity according to claim 1, wherein the pH buffer solution comprises at least one acid and at least one alkali which are mixed, the pH is adjusted to 5-8, the acid and the alkali are both soluble acid and alkali, no precipitate is generated during reaction, and no chemical reaction is generated with urea.

6. The gel reagent for rapid screening of urease activity according to claim 1, wherein the pH buffer solution is prepared by mixing 2- (N-morpholine) ethanesulfonic acid monohydrate and sodium hydroxide, and the pH is adjusted to 5-8.

7. A kit for rapidly screening urease activity is characterized by comprising a plastic glass sheet, wherein a concave plastic glass reagent cup is arranged in the center of the glass sheet, a gel reagent for rapidly screening urease activity is filled in the reagent cup, and the gel reagent is formed by pouring urea, a pH indicator phenol red, a bacteriostatic agent proclin300 and a pH buffer solution into agar gel and uniformly mixing; the upper surface of the plastic glass sheet is also pasted with a label used for filling information of a detected person, and the concave plastic glass reagent cup is sealed and covered by the label.

8. A method for rapid screening of urease activity according to any one of claims 1-8, comprising the steps of:

a. collecting an appropriate amount of sample with an applicator device;

9. The method for rapid screening of urease activity according to claim 8, wherein the applicator device is a toothpick, a cotton swab, or the like.

10. The method for rapid screening of urease activity according to claim 8, wherein the collected sample is any one of saliva, tartar, biopsy stomach and the like.