A test strip for CAMP experiment and preparation method thereof

Abstract

The present invention provides a test strip for CAMP experiment and a preparation method thereof, which relates to the field of biomedical technology. The method comprises: mixing β-Staphylococcus aureus with a diluent to obtain a bacterial solution; ultrasonically crushing the bacterial solution to obtain a crushed solution; mixing the crushed solution with bovine serum albumin and sucrose to obtain a protective solution; mixing the protective solution with a film-forming pore-forming solution to obtain a working solution; the components of the film-forming pore-forming solution include PVA124 and CMC-Na; coating the working solution on a polyester fiber film and drying to obtain a coated fiber film; gluing the back of the coated fiber film to a plastic backing pad to obtain a test strip. When the test strip is used for CAMP test, the problem of long-term storage and management of laboratory strains in traditional methods can be solved, and the problem of room temperature storage and transportation of reagents in existing solutions can also be solved, and the problem of standardization of the experimental process can also be solved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a test strip for CAMP experiment and a preparation method thereof. Background Art

[0002] Group B Streptococcus can produce CAMP factor, which can promote the hemolytic activity of β-hemolytic Staphylococcus aureus, enhance the hemolytic ability, and cause a sagittal enhanced transparent hemolysis area to appear on the blood agar plate. This characteristic of Group B Streptococcus is used clinically to identify Group B Streptococcus. This method is called the CAMP test.

[0003] The traditional laboratory CAMP test method is to first draw a horizontal line on a sheep blood or horse blood agar plate with β-hemolytic Staphylococcus aureus, and then vertically inoculate the bacteria to be tested with the previous line. The presence of an arrow-shaped hemolytic area at the intersection of the two bacterial lines is positive. This method requires the use of freshly cultured Staphylococcus aureus for testing. The problem is that the detection time needs to be extended by 24 to 48 hours. At the same time, Staphylococcus aureus is a Class II pathogenic microorganism and needs to be operated in a Class II biosafety laboratory, which poses certain biosafety risks. At the same time, this operation depends on the activity of Staphylococcus aureus and operating experience, with a low degree of standardization, poor test repeatability, and is time-consuming and labor-intensive.

[0004] There are also some CAMP product solutions on the market that use purified β-hemolysin instead of Staphylococcus aureus for CAMP tests. The disadvantage of this solution is that β-hemolysin needs to be stored at low temperatures, which is not conducive to long-distance transportation and long-term storage. At the same time, because the CAMP test reaction is a complex hemolytic process, the purified β-hemolysin does not have some natural and necessary ingredients, so the test effect is poor. In addition, the pure reagent is more soluble, and its diffusion rate is faster than the diffusion rate after normal secretion of bacteria. The arrow-shaped hemolysis zone formed is not obvious and is easy to misjudge. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a test strip for CAMP experiment and a preparation method thereof. When the test strip is used for CAMP experiment, the problem of long-term storage and management of laboratory strains in traditional methods can be solved, and the problem of room temperature storage and transportation of reagents in existing schemes can also be solved, and the problem of standardization of the experimental process can also be solved.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a test strip for a CAMP experiment, comprising the following steps:

[0008] 1) mixing β-Staphylococcus aureus with a diluent to obtain a bacterial solution;

[0009] 2) Ultrasonic disruption of the bacterial solution obtained in step 1) to obtain a disrupted solution;

[0010] 3) mixing the crushing solution obtained in step 2) with bovine serum albumin and sucrose to obtain a protective solution;

[0011] 4) mixing the protective solution obtained in step 3) with the film-forming pore-forming solution to obtain a working solution;

[0012] The components of the film-forming pore-forming fluid include PVA124 and CMC-Na;

[0013] 5) coating the working solution obtained in step 4) on a polyester fiber membrane and drying to obtain a coated fiber membrane;

[0014] 6) The back side of the coated fiber membrane obtained in step 5) is adhered to a plastic backing pad to obtain a test strip.

[0015] Preferably, the bacterial solution in step 1) is 4.0 MCF.

[0016] Preferably, the solvent of the diluent in step 1) is PBS buffer, and the solute is Triton-100; the mass percentage of Triton-100 in the diluent is 0.1%.

[0017] Preferably, the conditions for ultrasonic crushing in step 2) include: ultrasonic power 200 W, crushing for 10 s, rest for 10 s, and crushing for 20 min.

[0018] Preferably, the mass percentage of bovine serum albumin in the protective solution in step 3) is 0.5%.

[0019] Preferably, the mass percentage of sucrose in the protective solution in step 3) is 10%.

[0020] Preferably, the volume ratio of the protective solution to the film-forming pore-forming solution in step 4) is 1:2.

[0021] Preferably, in the film-forming pore-forming solution of step 4), the mass percentage of PVA124 is 10%, and the mass percentage of CMC-Na is 30%.

[0022] Preferably, the specifications of the polyester fiber film in step 5) are: length 3 cm, width 0.5 cm;

[0023] Each polyester fiber membrane was coated with 100 μl of the working solution.

[0024] The present invention provides a test strip for CAMP experiment prepared by the preparation method described in the above technical scheme.

[0025] The invention extracts liquid from freshly cultured β-hemolytic Staphylococcus aureus by ultrasonic crushing and filtration enrichment, uses PVA124 as a film-forming agent and CMC-Na as a pore-forming agent, prepares a membrane-controlled sustained-release system, applies it to a polyester fiber membrane strip (1), undergoes low-temperature drying, and then sticks it to a plastic backing pad (2) to prepare a test strip, wherein the edge of the backing pad is 3 mm away from the edge of the membrane strip. When in use, the test strip is placed with the membrane strip facing downward and horizontally stuck on a sheep blood or horse blood agar plate, and then the bacteria to be tested and the test strip are vertically inoculated, one end of the inoculation line (3) is flush with the edge of the test strip, and an arrow-shaped hemolysis area appears at the junction of the bacteria to be tested and the membrane strip, indicating that the test strip is positive.

[0026] The present invention relates to a test strip for CAMP experiment and a preparation method thereof. When the test strip is used for CAMP experiment, the reagent strip is attached to a blood plate to replace the streaking of Staphylococcus aureus in the CAMP experiment of group B streptococcus. The reagent can solve the problem of long-term storage and management of laboratory strains in traditional methods; because a membrane-controlled slow-release system is adopted and a stabilizer is added at the same time, the problem of room-temperature storage and transportation of the reagent in the existing scheme can also be solved; because a slow-release material is adopted and the inoculation position is fixed at the same time, the reaction time and distance of the two bacteria are also standardized. The test strip standardizes the experimental process while reducing the complexity of detection, greatly improving the success rate of the test and the consistency of the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0028] Figure 1 1 is a polyester fiber film strip, 2 is a plastic backing pad, and 3 is an inoculation line. DETAILED DESCRIPTION

[0029] The present invention provides a method for preparing a test strip for a CAMP experiment, comprising the following steps:

[0030] 1) mixing β-Staphylococcus aureus with a diluent to obtain a bacterial solution;

[0031] 2) Ultrasonic disruption of the bacterial solution obtained in step 1) to obtain a disrupted solution;

[0032] 3) mixing the crushing solution obtained in step 2) with bovine serum albumin and sucrose to obtain a protective solution;

[0033] 4) mixing the protective solution obtained in step 3) with the film-forming pore-forming solution to obtain a working solution;

[0034] The components of the film-forming pore-forming fluid include PVA124 and CMC-Na;

[0035] 5) coating the working solution obtained in step 4) on a polyester fiber membrane and drying to obtain a coated fiber membrane;

[0036] 6) The back side of the coated fiber membrane obtained in step 5) is adhered to a plastic backing pad to obtain a test strip.

[0037] The present invention mixes β-Staphylococcus aureus with a diluent to obtain a bacterial solution. The present invention preferably revives and rejuvenates β-hemolytic Staphylococcus aureus (such as ATCC25923), and then uses conventional culture to obtain fresh β-hemolytic Staphylococcus aureus colonies within 24 hours of culture, and mixes the β-hemolytic Staphylococcus aureus colonies with a diluent. In the present invention, the bacterial solution is 4.0 MCF. In the present invention, the solvent of the diluent is preferably PBS buffer, and the solute is preferably Triton-100; the mass percentage of Triton-100 in the diluent is preferably 0.1%.

[0038] The present invention ultrasonically crushes the obtained bacterial liquid to obtain a crushed liquid. The present invention preferably performs ultra-temperature crushing at a low temperature, such as 2-8° C. In the present invention, the conditions for the ultrasonic crushing preferably include: ultrasonic power 200W, crushing for 10s, rest for 10s, and crushing for 20min.

[0039] The present invention mixes the obtained crushing liquid with bovine serum albumin and sucrose to obtain a protective solution. In the present invention, the mass percentage of bovine serum albumin in the protective solution is preferably 0.5%. In the present invention, the mass percentage of sucrose in the protective solution is preferably 10%.

[0040] The present invention mixes the obtained protective solution with a film-forming pore-forming solution to obtain a working solution; the components of the film-forming pore-forming solution include PVA124 and CMC-Na. In the present invention, the volume ratio of the protective solution to the film-forming pore-forming solution is preferably 1:2. In the present invention, the mass percentage of PVA124 in the film-forming pore-forming solution is preferably 10%, and the PVA124 is a film-forming agent. In the present invention, the mass percentage of CMC-Na in the film-forming pore-forming solution is preferably 30%.

[0041] The present invention applies the obtained working solution on a polyester fiber membrane and dries it to obtain a coated fiber membrane. In the present invention, the specifications of the polyester fiber membrane are preferably: 3 cm in length and 0.5 cm in width. In the present invention, each polyester fiber membrane is preferably evenly coated with 100 μl of the working solution.

[0042] The invention adheres the back side of the obtained coated fiber membrane to a plastic backing pad to obtain a test paper strip.

[0043] In the present invention, the method for using the test strip preferably includes: sticking the test strip to the center of a sheep blood agar plate, vertically inoculating the bacteria to be tested and the test strip, with one end of the inoculation line flush with the edge of the test strip, culturing at 37 degrees for 12-24 hours and observing the results. The presence of an arrow-shaped hemolysis area at the junction of the bacteria to be tested and the membrane strip is positive.

[0044] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] A method for preparing a test strip for a CAMP experiment, comprising the following steps:

[0047] (1) resuscitating and rejuvenating β-hemolytic Staphylococcus aureus (ATCC25923);

[0048] (2) Take a freshly cultured β-hemolytic Staphylococcus aureus colony within 24 hours and make a 4.0 MCF bacterial suspension with PBS + 0.1% Triton-100 dilution solution;

[0049] (3) The bacterial liquid was ultrasonically disrupted at low temperature to disrupt the bacterial cells, with an ultrasonic power of 200 W, disruption for 10 seconds, rest for 10 seconds, and disruption for 20 minutes;

[0050] (4) After disruption, add 0.5% BSA and 10% sucrose as protective agents to prepare a solution;

[0051] (5) mixing the solution in (4) with a solution containing 10% by mass of PVA 124 and 30% by mass of CMC-Na in a volume ratio of 1:2 to prepare a working solution;

[0052] (6) Cut the polyester fiber membrane into rectangular strips of 0.5 cm × 3 cm and apply the working solution at 100 μl / strip;

[0053] (7) Drying the film strips by low temperature drying;

[0054] (8) Glue the reverse side of the film strip to the center of the plastic backing pad coated with glue and fix it;

[0055] (9) The formed film strips are placed with a desiccant and individually packaged to make test strips specifically for the CAMP test.

[0056] (10) When in use, stick the above test strip on the center of the sheep blood agar plate, then vertically inoculate the bacteria to be tested with the test strip, with one end of the inoculation line flush with the edge of the test strip, and incubate at 37 degrees for 12-24 hours to observe the results.

[0057] Comparative Example 1

[0058] Traditional CAMP test:

[0059] (1) resuscitating and rejuvenating β-hemolytic Staphylococcus aureus (ATCC25923);

[0060] (2) Take an inoculation loop of the bacteria in (1) and inoculate it in the center of the sheep blood agar plate. Then inoculate the bacteria to be tested vertically with a distance of 3 mm between them. Incubate at 37 degrees for 12-24 hours and observe the results.

[0061] Comparative Example 2

[0062] β-hemolysin replacement method:

[0063] (1) Spread β-hemolysin on the center of the sheep blood agar plate using an inoculation loop;

[0064] (2) Then inoculate the bacteria to be tested vertically with a distance of 3 mm between them and culture at 37 degrees for 12-24 hours to observe the results.

[0065] The test effects and efficiency results are shown in Table 1.

[0066] Table 1 Test results

[0067]

[0068] Note: A good standard is a typical sagittal arrow, easy to identify, and good stability.

[0069] The test strips or reagents were tested for stability at 25°C. The results are shown in Table 2.

[0070] Table 2 Stability results

[0071] 0d 1d 3d 7d 1 month 3 months Example 1 + + + + + + Comparative Example 2 + + ± - - -

[0072] Note: +: positive, ±: weak positive, -: negative

[0073] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a test strip for a CAMP experiment, characterized in that: The following steps are involved: 1) mixing β-Staphylococcus aureus with a diluent to obtain a bacterial solution; 2) Ultrasonic disruption of the bacterial solution obtained in step 1) to obtain a disrupted solution; 3) mixing the crushing liquid obtained in step 2) with bovine serum albumin and sucrose to obtain a protective solution, wherein the mass percentage of bovine serum albumin in the protective solution is 0.5%, and the mass percentage of sucrose is 10%; 4) mixing the protective solution obtained in step 3) with the film-forming pore-forming solution to obtain a working solution; Wherein, the components of the film-forming pore-forming fluid include PVA124 and CMC-Na; The mass percentage of PVA124 in the film-forming pore-forming liquid is 10%, and the mass percentage of CMC-Na is 30%; The volume ratio of the protective liquid to the film-forming pore-forming liquid is 1:2; 5) coating the working solution obtained in step 4) on a polyester fiber membrane and drying to obtain a coated fiber membrane; 6) The back side of the coated fiber membrane obtained in step 5) is adhered to a plastic backing pad to obtain a test strip.

2. The preparation method according to claim 1, characterized in that: The bacterial liquid in step 1) is 4.0 MCF.

3. The preparation method according to claim 1, characterized in that: The solvent of the diluent in step 1) is PBS buffer, and the solute is Triton-100; the mass percentage of Triton-100 in the diluent is 0.1%.

4. The preparation method according to claim 1, characterized in that: The conditions of ultrasonic crushing in step 2) include: ultrasonic power 200W, crushing for 10s, rest for 10s, and crushing for 20min.

5. The preparation method according to claim 1, characterized in that: The specifications of the polyester fiber membrane in step 5) are: 3 cm in length and 0.5 cm in width; each polyester fiber membrane is coated with 100 μl of the working solution.

6. A test strip for CAMP experiment prepared by the preparation method according to any one of claims 1 to 5.

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