Semi-quantitative detection kit for clostridium perfringens, detection method and application

Abstract

The invention belongs to the technical field of microbiological detection, and particularly provides a clostridium perfringens semi-quantitative detection kit, a detection method and application, the clostridium perfringens semi-quantitative detection kit comprises a liquid reagent A and a solid reagent B; the liquid reagent A comprises an aqueous solution in which tryptone, soy peptone, yeast extract powder and ammonium ferric citrate are dissolved; the solid reagent B comprises sodium pyrosulfite. According to the clostridium perfringens semi-quantitative detection kit provided by the invention, the specific liquid culture medium with an optimized formula is adopted, and the reagent A and the reagent B adopt a liquid-solid split charging design, so that sodium pyrosulfite is prevented from being degraded in a liquid environment, and the shelf life is prolonged. And a standard colorimetric card and a time comparison table can be prepared, so that subjective errors are reduced. In addition, the device can be integrated with a disposable sampling tool, a portable constant-temperature water bath cup and a timing prompt assembly to form a complete detection system which can be used after being opened, and the single detection cost is low.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to a semi-quantitative detection kit, detection method, and application of Clostridium perfringens. Background Technology

[0002] Clostridium perfringens is an important zoonotic pathogen that is widely present in the environment and animal intestines. It is a major pathogen causing diseases such as necrotic enteritis in poultry and enterotoxemia in piglets, and has become one of the main health threats under antibiotic-free farming models.

[0003] Currently, the detection of Clostridium perfringens mainly relies on traditional microbial culture and counting methods or molecular biological methods, such as PCR. However, microbial culture and counting methods require anaerobic culture on selective media, selection of typical colonies, and biochemical identification. The process is cumbersome, usually taking 48-72 hours, and is time-consuming. It also requires highly skilled operators and cannot meet the needs of rapid diagnosis in aquaculture sites.

[0004] While molecular biology methods are sensitive and specific, they require complex nucleic acid extraction from samples, expensive PCR instruments, and specialized laboratory personnel, making them difficult to apply in aquaculture fields. Most existing commercially available reagent kits are only suitable for laboratory environments and cannot meet the immediate screening needs of farms.

[0005] Although some rapid testing products have been launched, there is still a lack of portable solutions that integrate the entire process of sampling, processing, culture, and interpretation, and are suitable for operation by non-professionals, resulting in inconvenience for field use. Therefore, there is an urgent need to develop a field testing kit system that is easy to operate, responds quickly, provides intuitive results, is cost-effective, and highly integrated. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of long detection cycles, strong equipment dependence, complex operation, and unsuitability for field application of Clostridium perfringens in the prior art.

[0007] Therefore, the present invention provides a semi-quantitative detection kit for Clostridium perfringens, comprising liquid reagent A and solid reagent B; wherein liquid reagent A comprises an aqueous solution containing tryptone, soybean peptone, yeast extract, and ferric ammonium citrate; and solid reagent B comprises sodium metabisulfite.

[0008] Specifically, the concentrations of tryptone in the above aqueous solution are 10-20 g / L, soybean peptone concentration is 5-10 g / L, yeast extract concentration is 5-10 g / L, and ferric ammonium citrate concentration is 0.5-1 g / L.

[0009] Specifically, the pH of the above aqueous solution is 7-8.

[0010] Specifically, the above-mentioned semi-quantitative detection kit for Clostridium perfringens also includes a standard colorimetric card; the standard colorimetric card displays the color development time and color development status of different concentrations of Clostridium perfringens.

[0011] This invention also provides a semi-quantitative detection method for Clostridium perfringens, based on the above-mentioned semi-quantitative detection reagent for Clostridium perfringens, comprising the following steps:

[0012] Add solid reagent B to liquid reagent A and mix well to obtain the reaction reagent;

[0013] Prepare Clostridium perfringens bacterial suspensions with different concentration gradients, add them to the reaction reagents respectively, incubate at constant temperature, record the time point when the first uniform black color appears, and establish the relationship between bacterial concentration and color development time;

[0014] Add the sample to be tested into the reaction reagent, incubate at a constant temperature under the same conditions, and record the time point at which a uniform black color first appears;

[0015] The Clostridium perfringens load in the test sample was assessed based on the relationship between the sample blackening time, control bacterial concentration, and color development time.

[0016] Specifically, in the above reaction reagents, the amount of solid reagent B added is 1 g / L based on the volume of liquid reagent A.

[0017] Specifically, the above-mentioned establishment of the bacterial concentration-color development time relationship involves: obtaining the corresponding data of bacterial concentration and color development time, and establishing a bacterial concentration-color development time model through nonlinear regression analysis.

[0018] Specifically, the above bacterial concentration-color development time model is log 10 (N) = 8.5 - 1.2 × T; where N is the bacterial concentration and T is the color development time.

[0019] Specifically, the constant temperature for incubation is 40-45℃.

[0020] The semi-quantitative detection kit or method for Clostridium perfringens provided by this invention can be used for the detection of livestock and poultry feces or intestinal contents.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] The semi-quantitative detection kit for Clostridium perfringens provided by this invention uses a specific liquid culture medium with an optimized formula. Reagents A and B are packaged in a liquid-solid aliquot design to avoid degradation of sodium metabisulfite in the liquid environment, thus extending shelf life. It can also be equipped with a standard colorimetric card and a time reference table to reduce subjective errors. Furthermore, it can be integrated with disposable sampling tools, portable constant temperature water baths, and timing reminder components to form a complete out-of-the-box detection system with low cost per test.

[0023] The detection method of this kit is simple to operate, and ordinary farmers can complete it independently after simple training. By establishing a bacterial concentration-color development time correlation model, the entire process from sampling to result interpretation can be completed within 5 hours, truly achieving zero-threshold, high-efficiency, and repeatable rapid on-site screening in grassroots farms. It can be widely applied to the detection of fecal samples from various livestock and poultry such as chickens, ducks, pigs, and sheep.

[0024] The present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the color development of the experimental group and the control group at different times in Example 4 of the present invention.

[0026] Figure 2 This is a schematic diagram of the color development of Clostridium perfringens at different concentration gradients in Example 5 of the present invention.

[0027] Figure 3 This is a schematic diagram of the color development of broiler chicken samples in Embodiment 7 of the present invention.

[0028] Figure 4 This is a schematic diagram of the color development of the laying hen sample in Embodiment 8 of the present invention. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0030] This invention provides a semi-quantitative detection kit for Clostridium perfringens, comprising a liquid reagent A and a solid reagent B. Liquid reagent A comprises an aqueous solution containing tryptone, soybean peptone, yeast extract, and ferric ammonium citrate. Solid reagent B comprises sodium metabisulfite, preferably lyophilized powder. The kit employs a separate solid and liquid reagent design to avoid degradation of sodium metabisulfite in a liquid environment, extending shelf life. It also eliminates the need for preparing solid plates or performing solid-liquid separation operations, avoiding problems such as uneven agar coagulation and separation failure due to improper temperature control. The concentrations of tryptone, soybean peptone, yeast extract, and ferric ammonium citrate in the aqueous solution are 10-20 g / L, 5-10 g / L, and 1 g / L, respectively. The pH of the aqueous solution is adjusted to 7-8, preferably using NaOH solution.

[0031] To reduce subjective error, the Clostridium perfringens semi-quantitative detection kit also includes a standard colorimetric card; the standard colorimetric card displays the color development time and color development status of different concentrations of Clostridium perfringens. This allows operators to quickly assess the infection status by referring to the standard colorimetric card.

[0032] Furthermore, the kit also includes timing stickers or electronic timers to help record the start time of the culture, as well as a marker pen for easy sample numbering and labeling.

[0033] In a detailed implementation, the kit also includes a sampling module, which comprises a sterile shovel, a sampling box (with a shovel), and sterile gloves to ensure hygienic operation, prevent cross-contamination, seal and preserve samples, and facilitate transportation. The number of each device in the sampling module can be designed according to the actual size of the kit.

[0034] Preferably, the kit also includes a temperature-adjustable constant-temperature water bath, powered by a power cord or with its own power supply, to adapt to use in environments without AC power, such as farms, ranches, and temporary sites in epidemic areas.

[0035] To facilitate quick mastery of the kit's usage by personnel without prior experience, an instruction manual and a result interpretation table can be included in the kit, clearly defining the correspondence between the blackening time and the infection level.

[0036] In one implementation, the result interpretation table is shown in Table 1.

[0037] Table 1 Result Interpretation Table

[0038]

[0039] This invention also provides a semi-quantitative detection method for Clostridium perfringens, based on the above-mentioned semi-quantitative detection reagent for Clostridium perfringens, comprising the following steps:

[0040] Solid reagent B is added to liquid reagent A and mixed well to obtain the reaction reagent; the amount of solid reagent B added in the reaction reagent is 1 g / L based on the volume of liquid reagent A.

[0041] Prepare Clostridium perfringens bacterial suspensions with different concentration gradients, add them to the reaction reagents respectively, and incubate at a constant temperature, preferably 40-45℃. Record the time point when the first uniform black color appears, and establish the relationship between bacterial concentration and color development time.

[0042] Specifically, obtain the data corresponding to bacterial concentration and color development time, and establish a bacterial concentration-color development time model through nonlinear regression analysis: log 10 (N) = 8.5 - 1.2 × T; where N is the bacterial concentration and T is the color development time;

[0043] Add the sample to be tested into the reaction reagent, incubate at a constant temperature under the same conditions, and record the time point at which a uniform black color first appears;

[0044] The Clostridium perfringens load in the test sample was assessed based on the relationship between the sample blackening time, control bacterial concentration, and color development time.

[0045] The following specific embodiments illustrate the effects of the Clostridium perfringens semi-quantitative detection kit, detection method, and application of the present invention.

[0046] Example 1:

[0047] This embodiment provides a semi-quantitative detection kit for Clostridium perfringens, comprising liquid reagent A and solid reagent B.

[0048] The preparation method of liquid reagent A is as follows: Weigh 15 g of tryptone, 5 g of soybean peptone, 5 g of yeast extract powder, and 1 g of ferric ammonium citrate, dissolve them in 1 L of distilled water, stir thoroughly until completely dissolved, and adjust the pH value to 7.6 using 1 mol / L NaOH solution.

[0049] Place the prepared liquid reagent A in an autoclave and heat for 15 minutes to sterilize and remove dissolved oxygen. After removing it, allow it to cool naturally to room temperature (about 25°C), then dispense it into sterile centrifuge tubes, 10 mL per tube, and store it in the dark for later use.

[0050] The preparation method for solid reagent B is as follows: Weigh 10 mg of sodium metabisulfite, accurately place it into a 1.5 mL EP tube, vacuum dry it, seal and store it. Each EP tube is for one test.

[0051] Example 2:

[0052] This embodiment provides a semi-quantitative detection kit for Clostridium perfringens, comprising liquid reagent A and solid reagent B.

[0053] The preparation method of liquid reagent A is as follows: Weigh 10g of tryptone, 5g of soybean peptone, 5g of yeast extract powder, and 0.5g of ferric ammonium citrate, dissolve them in 1L of distilled water, stir thoroughly until completely dissolved, and adjust the pH value to 7 using 1mol / L NaOH solution.

[0054] Place the prepared liquid reagent A in an autoclave and heat for 15 minutes to sterilize and remove dissolved oxygen. After removing it, allow it to cool naturally to room temperature (about 25°C), then dispense it into sterile centrifuge tubes, 10 mL per tube, and store it in the dark for later use.

[0055] The preparation method for solid reagent B is as follows: Weigh 10 mg of sodium metabisulfite, accurately place it into a 1.5 mL EP tube, vacuum dry it, seal and store it. Each EP tube is for one test.

[0056] Example 3:

[0057] This embodiment provides a semi-quantitative detection kit for Clostridium perfringens, comprising liquid reagent A and solid reagent B.

[0058] The preparation method of liquid reagent A is as follows: Weigh 20g of tryptone, 10g of soybean peptone, 10g of yeast extract powder, and 1g of ferric ammonium citrate, dissolve them in 1L of distilled water, stir thoroughly until completely dissolved, and adjust the pH value to 8 using 1mol / L NaOH solution.

[0059] Place the prepared liquid reagent A in an autoclave and heat for 15 minutes to sterilize and remove dissolved oxygen. After removing it, allow it to cool naturally to room temperature (about 25°C), then dispense it into sterile centrifuge tubes, 10 mL per tube, and store it in the dark for later use.

[0060] The preparation method of solid reagent B is as follows: Weigh 10 mg of sodium metabisulfite, accurately dispense it into 1.5 mL EP tubes, vacuum dry, seal and store. Each EP tube is for one test.

[0061] Example 4:

[0062] This embodiment is based on the Clostridium perfringens semi-quantitative detection kit provided in Example 1. Common livestock and poultry intestinal microorganisms Escherichia coli and Salmonella enterica were selected for cross-reactivity testing to verify the specificity of the kit.

[0063] Add solid reagent B to liquid reagent A at a concentration of 1 g / L and mix well to obtain the reaction reagent for later use.

[0064] Clostridium perfringens (C. perfringens) (two replicates), Escherichia coli (E. coli), and Salmonella enterica were inoculated into the reaction reagent at a concentration of 10 μL each. 5 CFU / mL, incubate at 45℃ for 5 hours.

[0065] The results are as follows Figure 1 As shown, only the *Clostridium perfringens* group showed a distinct black precipitate within 5 hours, while the other strains did not show any color change throughout the entire culture period, and the culture medium remained brownish-yellow and transparent. This indicates that the kit provided by this invention has high specificity for *Clostridium perfringens* and can effectively exclude interference from other common enteric bacteria.

[0066] Example 5:

[0067] This embodiment, based on the Clostridium perfringens semi-quantitative detection kit provided in Example 1, provides a method for establishing the relationship between bacterial concentration and color development time, including the following steps.

[0068] (1) Gradual dilution of bacterial culture

[0069] The standard strain of *Clostridium perfringens* was taken, and after anaerobic activation culture, it was serially diluted 10-fold with phosphate-buffered saline (PBS, pH 7.2) to obtain the following final concentration gradient: 10 8 CFU / mL, 10 7 CFU / mL, 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL.

[0070] (2) Inoculation and culture

[0071] Add solid reagent B to liquid reagent A at a concentration of 1 g / L and mix well to obtain the reaction reagent;

[0072] Take 0.25 mL of each concentration of bacterial culture and add it to 10 mL of reaction reagent. Mix gently. Set up 3 parallel replicates for each group. Place all test tubes in a 45℃ constant temperature water bath and incubate in the dark.

[0073] (3) Dynamic observation and data recording

[0074] From the start of the culture, the tubes were taken out for observation at regular intervals. The time point at which the first uniform, visible black color appeared in each test tube was recorded. Observation continued until 5 hours later. The results are as follows: Figure 2 .

[0075] (4) Data Analysis and Results

[0076] The bacterial concentration-color development time data were obtained, as shown in Table 2. A bacterial concentration-color development time model was established using nonlinear regression analysis: log 10 (N) = 8.5 − 1.2 × T (R) 2 =0.987); where N is the bacterial concentration (CFU / g) and T is the color development time (h).

[0077] Table 2. Effect of bacterial concentration-color development time on results

[0078]

[0079] There is a significant negative correlation between the blackening time and the initial bacterial concentration, which can be used to semi-quantitatively assess the Clostridium perfringens load level in samples.

[0080] Example 6:

[0081] This embodiment, based on the semi-quantitative detection kits for Clostridium perfringens in Examples 1-3, provides a semi-quantitative detection method for Clostridium perfringens, including the following steps:

[0082] (1) Pre-test preparation

[0083] Open the thermos water bath lid and add purified or distilled water to the 250 mL mark.

[0084] Press and hold the power button to start the device, set the temperature to 45℃ (default operating temperature), and start using it after the display screen stabilizes and shows the target temperature.

[0085] Take a test tube containing liquid reagent A, open the cap, pour all the powder from a test tube containing solid reagent B into it, and gently shake to mix well to obtain the reaction reagent. Prepare and use immediately.

[0086] (2) On-site sampling

[0087] Wearing sterile gloves, use a disposable sterile shovel to collect a mid-section internal sample of fresh animal feces (within 1 hour of defecation);

[0088] Transfer approximately 0.5 g of feces (about one full scoop with a shovel) into the pre-mixed reaction reagent;

[0089] Tighten the test tube cap and gently invert it several times to mix, ensuring the sample is fully dispersed;

[0090] Use a marker to mark the sample number on the test tube wall.

[0091] (3) Constant temperature incubation and colorimetric observation

[0092] Place the test tube with the sample added vertically into the constant temperature water bath and start timing;

[0093] From the start of the culture, the color change of the culture medium is observed at regular intervals.

[0094] Record the time point (in hours) when a uniform, visible black color first appears.

[0095] Continue observation until the end of 5 hours. Based on the relationship between the sample blackening time, control bacterial concentration, and color development time, assess the Clostridium perfringens load in the sample to be tested. If no blackening is observed, it is judged as negative or the bacterial count is extremely low.

[0096] Example 7:

[0097] In this embodiment, the method of Example 6 and the kit of Example 1 were used to test four fresh fecal samples collected from a large-scale broiler farm in Weifang, Shandong Province. Two samples were from the experimental group (suspected necrotizing enteritis group), numbered E1-E2; and two samples were from the control group (healthy group), numbered K1-K2.

[0098] Experimental results are as follows Figure 3 As shown in Table 3, the blackening time of each sample was recorded, and the results were classified according to the interpretation criteria established in Example 5.

[0099] Table 3. Sample blackening time and estimated bacterial count

[0100]

[0101] Necropsy of individuals E1–E2 in the experimental group revealed typical necrotic enteritis lesions, including thickened intestinal walls, mucosal necrosis, and foul-smelling contents. These results were highly consistent with the findings of this detection method. This method demonstrates good applicability and relevance in real-world aquaculture environments.

[0102] Example 8:

[0103] This embodiment uses the method of Example 6 and the kit of Example 1 to test 6 fresh fecal samples collected from a large-scale egg-laying chicken farm in Jingzhou, Hubei Province. Among them, there are 3 samples of 370-day-old Hainan Grey chickens, numbered Y1-Y3, and 3 samples of 325-day-old Jinghong chickens, numbered H1-H3.

[0104] Experimental results are as follows Figure 4 As shown, the blackening time of each sample was recorded, and the results were classified according to the interpretation criteria established in Example 5. The results are shown in Table 4.

[0105] Table 4. Sample blackening time and estimated bacterial count

[0106]

[0107] This method has good applicability and relevance in real aquaculture environments.

[0108] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A semi-quantitative detection kit for Clostridium perfringens, characterized in that: It includes liquid reagent A and solid reagent B; liquid reagent A includes an aqueous solution containing tryptone, soybean peptone, yeast extract, and ferric ammonium citrate; solid reagent B includes sodium metabisulfite.

2. The semi-quantitative detection kit for Clostridium perfringens as described in claim 1, characterized in that: The aqueous solution contains 10-20 g / L of tryptone, 5-10 g / L of soybean peptone, 5-10 g / L of yeast extract, and 0.5-1 g / L of ferric ammonium citrate.

3. The semi-quantitative detection kit for Clostridium perfringens as described in claim 1, characterized in that: The pH of the aqueous solution is 7-8.

4. The semi-quantitative detection kit for Clostridium perfringens as described in claim 1, characterized in that: It also includes a standard colorimetric card; the standard colorimetric card shows the color development time and color development state of different concentrations of Clostridium perfringens.

5. A semi-quantitative detection method for Clostridium perfringens, characterized in that, The semi-quantitative detection reagent for Clostridium perfringens according to any one of claims 1-4 includes the following steps: Add solid reagent B to liquid reagent A and mix well to obtain the reaction reagent; Prepare Clostridium perfringens bacterial suspensions with different concentration gradients, add them to the reaction reagents respectively, incubate at constant temperature, record the time point when the first uniform black color appears, and establish the relationship between bacterial concentration and color development time; Add the sample to be tested into the reaction reagent, incubate at a constant temperature under the same conditions, and record the time point at which a uniform black color first appears; The Clostridium perfringens load in the test sample was assessed based on the relationship between the sample blackening time, control bacterial concentration, and color development time.

6. The semi-quantitative detection method for Clostridium perfringens as described in claim 5, characterized in that: The amount of solid reagent B added is 1 g / L, based on the volume of liquid reagent A.

7. The semi-quantitative detection method for Clostridium perfringens as described in claim 5, characterized in that, Establishing the relationship between bacterial concentration and color development time involves obtaining data corresponding to bacterial concentration and color development time, and then establishing a bacterial concentration-color development time model through nonlinear regression analysis.

8. The semi-quantitative detection method for Clostridium perfringens as described in claim 7, characterized in that: The bacterial concentration-color development time model is log 10 (N) = 8.5 - 1.2 × T; where N is the bacterial concentration and T is the color development time.

9. The semi-quantitative detection method for Clostridium perfringens as described in claim 5, characterized in that: The constant temperature for incubation is 40℃-45℃.

10. The application of the semi-quantitative detection kit for Clostridium perfringens as described in any one of claims 1-4 or the semi-quantitative detection method for Clostridium perfringens as described in any one of claims 5-9 in the detection of livestock and poultry feces or intestinal contents.

Images