阅读说明：本技术 一种快速v-p试验法以及用于该方法的试剂盒 (Rapid V-P test method and kit used for same ) 是由 马玥 周浩 于 2019-08-20 设计创作，主要内容包括：本发明公开了一种快速V-P试验法以及用于该方法的试剂盒。所述的快速V-P试验法,包括以下步骤：(1)将试验菌种接种于改良MR-VP肉汤培养基中,37℃保温3-4h,哈夫尼亚菌在22-25℃保温；(2)保温结束后往培养基中依次加入A液2滴,B液3滴,C液2滴,充分混合2-5min,呈现红色判定为阳性；若无红色出现,置于室温或37℃恒温箱,15min内仍不显现红色、判定为阴性。其中,A液为0.5％w/w肌酸溶液；B液为含5％w/wα-萘酚(1-naphthol)的95％(w/w)酒精溶液；C液为40％w/v氢氧化钾水溶液。该方法加快了出现阳性反应的速度,3-4h即可判读结果。增强显色效果,鲜明直观。可同时适用于肠杆菌科和微球菌科细菌的检测,没有对待检菌落的特殊要求。培养基、试剂用量少,价格低廉；配方简单,性能稳定。(The invention discloses a rapid V-P test method and a kit used for the method. The rapid V-P test method comprises the following steps: (1) inoculating the test strain into improved MR-VP broth culture medium, and maintaining the temperature at 37 deg.C for 3-4h, and maintaining the temperature at 22-25 deg.C for Hafnia bacteria; (2) after the heat preservation is finished, adding 2 drops of the solution A, 3 drops of the solution B and 2 drops of the solution C into the culture medium in sequence, fully mixing for 2-5min, and judging the culture medium to be positive when the culture medium is red; if no red appears, the sample is placed in a constant temperature cabinet at room temperature or 37 ℃, and the sample still does not appear red within 15min and is judged to be negative. Wherein the solution A is 0.5% w/w creatine solution; the solution B is a 95% (w/w) alcohol solution containing 5% w/w alpha-naphthol (1-naphthol); the solution C is 40% w/v potassium hydroxide aqueous solution. The method accelerates the speed of positive reaction, and the result can be judged within 3-4 h. The color development effect is enhanced, and the color developing effect is bright and visual. Can be simultaneously suitable for detecting the bacteria in enterobacteriaceae and micrococcaceae, and has no special requirement on the colony to be detected. The culture medium and the reagent are less in dosage and low in price; simple formula and stable performance.)

1. A rapid V-P test method, comprising the steps of:

(1) Inoculating the test strain into improved MR-VP broth culture medium, and maintaining the temperature at 37 deg.C for 3-4h, and maintaining the temperature at 22-25 deg.C for Hafnia bacteria;

The improved MR-VP broth is prepared by the following method: 1g of yeast extract, 5g of plant peptone, 7g of polypeptone, 5g of sodium chloride, 10g of glucose and 5g of agar, adding 1000ml of distilled water, adjusting the pH value to 7.0, sterilizing at 121 ℃ for 15min, and subpackaging in test tubes;

(2) After the heat preservation is finished, adding 2 drops of the solution A, 3 drops of the solution B and 2 drops of the solution C into the culture medium in sequence, fully mixing for 2-5min, and judging the culture medium to be positive when the culture medium is red; if no red appears, placing the mixture in a constant temperature box at room temperature or 37 ℃, and judging that the red does not appear and the mixture is negative within 15 min;

wherein the solution A is 0.5% w/w creatine solution; the solution B is a 95% w/w alcohol solution containing 5% w/w alpha-naphthol (1-naphthol); the solution C is 40% w/v potassium hydroxide aqueous solution.

2. The rapid V-P assay of claim 1, wherein said rapid V-P assay is useful for intergeneric identification of enterobacter and escherichia, staphylococcus and micrococcus; or identifying strains among Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella odorifera, and Klebsiella rhinosclerosus; or identifying the species of Hafnia alvei, Yersinia enterocolitica and Listeria monocytogenes.

3. A kit for use in a rapid V-P assay, said kit comprising:

modified MR-VP broth: 1g of yeast extract, 5g of plant peptone, 7g of polypeptone, 5g of sodium chloride, 10g of glucose, 5g of agar, 1000ml of distilled water, pH7.0, 15min at 121 ℃ for sterilization, and subpackaging according to 0.2ml per unit;

Solution A: 0.5% w/w creatine solution;

And B, liquid B: 95% w/w alcoholic solution containing 5% w/w alpha-naphthol (1-naphthol);

And C, liquid C: 40% w/v aqueous potassium hydroxide solution.

4. Use of the kit of claim 3 for rapid V-P assay detection.

Technical Field

The present invention relates to a V-P assay and to kits for use in the method. The invention belongs to the technical field of microorganism detection.

Background

the bacterial species of enterobacteriaceae and micrococcaceae are various, widely distributed and have a large host range, dozens of medical-related bacterial species account for 50% of the total number of clinical isolates, nearly 50% of septicemia and more than 70% of urinary tract infection are caused by the bacteria, and food poisoning events caused by the bacteria become public health problems worldwide, are potential killers of human health, avoid the limitation of equipment conditions, realize rapid identification of the bacterial species of enterobacteriaceae and micrococcaceae by using rapid and simple biochemical tests, and are important subjects needing to be researched and broken.

the V-P test, also known as the diacetyl test. The principle is that certain bacteria decompose glucose to generate pyruvic acid in the sugar metabolism process, the pyruvic acid decarboxylates to generate the acetyl methyl carbinol, the acetyl methyl carbinol is oxidized into diacetyl by oxygen in the air in an alkaline environment, and then the acetyl methyl carbinol reacts with guanidino contained in arginine in peptone in a culture medium to generate a red compound, and the V-P test is positive. The V-P assay can be used for the following purposes:

The intergeneric identification of enterococcus and Escherichia, staphylococcus and micrococcus;

identifying the strains among Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella odorifera and Klebsiella rhinosclerosus;

③ the identification of the species of Hafnia alvei, Yersinia enterocolitica and Listeria monocytogenes.

The V-P test method commonly used at home and abroad comprises the following steps: o 'Meara's method, Barrit's method, Coblentz's method, etc., wherein the detection result can be obtained as soon as 24 hours as possible and 4 days as slow as possible; when detecting other bacteria such as bacillus, staphylococcus and the like, phosphate in the general culture medium can block the generation of the acetyl methyl methanol, so that a false negative result is caused; other methods only detect acid-producing cultures. Not only the detection time is long, the reagent is unstable, and the application range is limited.

In view of the current situation, the invention establishes a rapid V-P test method, and solves the problems and the defects of the prior method while ensuring the reliability of the result. The rapid V-P test method has the advantages that: 1. the speed of positive reaction is increased, and the result can be judged within 3-4 h. 2. The color development effect is enhanced, and the color developing effect is bright and visual. 3. Meanwhile, the method is suitable for detecting the bacteria in the enterobacteriaceae and the micrococcaceae, and has no special requirement on the colony to be detected. 4. The culture medium and the reagent are less in dosage and low in price; simple formula and stable performance.

Disclosure of Invention

the invention aims to establish a rapid V-P test method and a kit used for the method.

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

The invention relates to a rapid V-P test method, which comprises the following steps:

(1) Inoculating the test strain into improved MR-VP broth culture medium, and maintaining the temperature at 37 deg.C for 3-4h, and maintaining the temperature at 22-25 deg.C for Hafnia bacteria;

The improved MR-VP broth is prepared by the following method: 1g of yeast extract, 5g of plant peptone, 7g of polypeptone, 5g of sodium chloride, 10g of glucose and 5g of agar, adding 1000ml of distilled water, adjusting the pH value to 7.0, sterilizing at 121 ℃ for 15min, and subpackaging in test tubes;

(2) After the heat preservation is finished, adding 2 drops of the solution A, 3 drops of the solution B and 2 drops of the solution C into the culture medium in sequence, fully mixing for 2-5min, and judging the culture medium to be positive when the culture medium is red; if no red appears, placing the mixture in a constant temperature box at room temperature or 37 ℃, and judging that the red does not appear and the mixture is negative within 15 min;

Wherein the solution A is 0.5% w/w creatine solution; the solution B is a 95% w/w alcohol solution containing 5% w/w alpha-naphthol (1-naphthol); the solution C is 40% w/v potassium hydroxide aqueous solution.

wherein, preferably, said rapid V-P assay is useful for intergeneric identification of Enterobacter and Escherichia, Staphylococcus and Micrococcus; or identifying strains among Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella odorifera, and Klebsiella rhinosclerosus; or identifying the species of Hafnia alvei, Yersinia enterocolitica and Listeria monocytogenes.

Further, the present invention also provides a kit for a rapid V-P assay, the kit comprising:

Modified MR-VP broth: 1g of yeast extract, 5g of plant peptone, 7g of polypeptone, 5g of sodium chloride, 10g of glucose, 5g of agar, 1000ml of distilled water, pH7.0, 15min at 121 ℃ for sterilization, and subpackaging according to 0.2ml per unit;

Solution A: 0.5% w/w creatine solution;

And B, liquid B: 95% w/w alcoholic solution containing 5% w/w alpha-naphthol (1-naphthol);

And C, liquid C: 40% w/v aqueous potassium hydroxide solution.

Furthermore, the invention also provides application of the kit in rapid V-P test detection.

the rapid V-P test is based on the examination of the production of acetomethylmethanol by certain bacteria in the glucose metabolism. The acetyl methyl methanol can be oxidized into butanedione by the oxygen in the air in an alkaline environment, and the butanedione and guanidino contained in arginine in the peptone react to generate a red compound, namely the positive V-P test. The key technical means adopted by the invention comprise:

1. free NH2 of the guanidino moiety is a component required to produce color. Creatine or creatinine can supply more guanidino than arginine, and has the effect of accelerating the positive reaction of V-P test.

2. the addition of alpha-naphthol can make the V-P test more sensitive, raise the sensitivity of V-P test by about 50 times without reducing the specificity of test, and its alcohol solution also has the function of strengthening colour.

3. potassium hydroxide as an oxidant accelerates the oxidation of the acetomethyl methanol to butanedione and aids in the absorption of carbon dioxide.

4. In the test, alpha-naphthol was first added to bind the guanidino complex of butanedione, and then 40% potassium hydroxide solution was added. If potassium hydroxide is added in reverse order, it may react with some components of the peptone to produce an orange-red color, resulting in false positives.

5. The sodium chloride is used for replacing phosphate in the universal culture medium, so that the phenomenon that the phosphate in the culture medium obstructs the production of the acetyl methyl alcohol to cause false negative results when other bacteria such as bacillus, staphylococcus and the like are detected can be avoided. Thus really realizing intuition, rapidness, convenience, accuracy, economy and applicability.

compared with the prior art, the invention has the beneficial effects that:

1. The speed of positive reaction is increased, and the result can be judged within 3-4 h.

2. The color development effect is enhanced, and the color developing effect is bright and visual.

3. Meanwhile, the method is suitable for detecting the bacteria in the enterobacteriaceae and the micrococcaceae, and has no special requirement on the colony to be detected.

4. the culture medium and the reagent are less in dosage and low in price; simple formula and stable performance.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.