A kit and method for detecting clostridium perfringens in the field

Abstract

The present application relates to the technical field of microorganism detection, and particularly relates to a kit and method for detecting Clostridium perfringens in situ. The method comprises the following steps: placing a sample to be detected into a sample dilution tube, diluting and standing, then dipping an upper liquid with a sampling cotton swab, subsequently vertically inserting the cotton swab into a detection tube, leaving the cotton swab rod in the tube, standing and recondensing to form a double anaerobic sealing system; selecting room temperature culture or 37 DEG C constant temperature culture for 2-6 hours according to the ambient temperature; observing the blackening of the culture medium to determine the result, and the blackening is positive and no blackening is negative. The method does not need professional equipment such as an anaerobic incubator and a sterile operation table, simplifies the operation, does not need complex pretreatment, can quickly determine the result in 2-6 hours, can complete the whole process detection in situ at a farm, outdoors and other places without a laboratory, effectively solves the problems of high dependence of the existing detection method on equipment, complicated operation, long period, insufficient anaerobic guarantee and the like, and is suitable for rapid screening application at the grassroots and in situ.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, specifically to a kit and method for on-site detection of Clostridium perfringens. Background Technology

[0002] Clostridium perfringens is a Gram-positive anaerobic spore-forming bacillus widely distributed in nature and the intestines of humans and animals. As an important zoonotic pathogen, it can cause sudden-death intestinal diseases in animals and food poisoning in humans, posing a serious threat to livestock and poultry farming safety and public health control. In livestock production, diseases caused by this bacterium, such as sudden anthrax in sheep, enterotoxemia, and lamb dysentery, are characterized by rapid onset, short course, and high mortality, often resulting in significant economic losses. Establishing efficient and convenient detection methods is a key link in the prevention and control of these diseases.

[0003] Currently, the mainstream techniques for detecting Clostridium perfringens include traditional isolation and culture, immunological detection, and molecular biological detection. Traditional isolation and culture is considered the "gold standard." Its principle lies in the fact that the bacterium can decompose sulfites in the culture medium to produce sulfides. These sulfides react with iron salts (such as ferric ammonium citrate) to form a black ferrous sulfide precipitate, resulting in the characteristic black color of the colonies or culture medium. While reliable, this method requires complex procedures such as enrichment and purification, streak plating, and biochemical identification, taking several days and relying on specialized equipment like anaerobic jars or incubators, making it unsuitable for rapid on-site diagnosis. Immunological detection methods, such as enzyme-linked immunosorbent assay (ELISA), are relatively simple to operate, but have limited sensitivity, high cost, and require specialized equipment like microplate readers. In recent years, molecular biological detection methods such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) have been widely used due to their high sensitivity, but they rely on nucleic acid extraction and isothermal / variable-temperature amplification instruments, requiring specific facilities and personnel, making them difficult to conduct on-site without laboratory facilities. Overall, existing technologies cannot achieve rapid on-site detection that integrates sampling, cultivation, and interpretation.

[0004] Existing publicly available detection technologies generally suffer from common drawbacks such as high equipment dependence, cumbersome operation, insufficient anaerobic support, and poor portability, making them unsuitable for direct use in field settings such as farms, slaughterhouses, feed processing plants, or outdoor epidemic areas. To address these technical bottlenecks, developing a field detection method for Clostridium perfringens that is easy to operate, highly efficient, portable, and capable of creating a self-sufficient anaerobic environment through structural design has become an urgent technical challenge in this field. Summary of the Invention

[0005] To address the technical problems of existing Clostridium perfringens detection methods, such as high equipment dependence, cumbersome operation, insufficient anaerobic protection, poor portability, and long detection cycle, this invention provides a kit and method for on-site detection of Clostridium perfringens.

[0006] The technical solution of this invention is as follows: In a first aspect, the present invention provides a method for on-site detection of Clostridium perfringens, comprising the following steps: (1) Pretreatment of test tubes: Immerse the test tubes in a mini constant temperature water bath and rehydrate them at 70°C for 10-15 minutes before use; the test tubes contain a semi-solid modified culture medium with a volume of 4±0.2 mL, which includes the following raw materials at the following mass-volume concentrations: tryptone 14-16 g / L, soybean peptone 4-6 g / L, yeast extract powder 4-6 g / L, ferric ammonium citrate 1.1-1.3 g / L, sodium metabisulfite 1.1-1.3 g / L, agar 4-5 g / L, polymyxin B 11-13 mg / L and sodium sulfadiazine 0.11-0.13 g / L; (2) Sampling: After placing the sample to be tested into the sample dilution tube, shake it by hand for 10~20s and let it stand for 5~10min to complete the sample dilution; then immerse a cotton swab in the upper liquid of the diluted sample to be tested for sampling. (3) Inoculation and culture: Insert the sampling swab vertically into the test tube, leaving the swab rod inside the tube, tighten the tube cap, let stand for 5~10 minutes, and culture for 2~6 hours; (4) Result determination: Observe the color of the semi-solid modified culture medium. The presence of black precipitate indicates a positive result, while the absence of black precipitate indicates a negative result.

[0007] Furthermore, in step (4), blackening occurs when the culture time is ≤2h, and the concentration of Clostridium perfringens in the sample is ≥1×10⁻⁶. 7 CFU / mL; blackening occurred after 2-6 hours of incubation, and the concentration of Clostridium perfringens in the sample was 1×10⁻⁶. 5 ~1×10 7 CFU / mL; where CFU / mL is the number of colony-forming units per milliliter of sample.

[0008] Furthermore, in step (2), when the sample to be tested is fecal pellets or feces, the dilution factor is 5 times; when the sample to be tested is a diarrhea sample, the dilution factor is 2 times; when the sample to be tested is any one of intestinal contents, milk sample or blood, no dilution operation is required; the dilution factor is calculated based on the volume of the sample to be tested.

[0009] Furthermore, in step (3), when the ambient temperature is ≥25℃, the inoculated test tube is placed at room temperature for static incubation; when the ambient temperature is <25℃, the inoculated test tube is placed in a mini constant temperature water bath and incubated at 37℃±0.5℃.

[0010] Furthermore, the mini constant temperature water bath has a volume of ≤4L, is equipped with a test tube rack inside, which can hold 1 to 10 test tubes, and has a temperature adjustment range of 30 to 80℃.

[0011] Furthermore, the water bath adopts a dual-mode power supply design, supporting independent power supply from external power source and lithium battery, with a battery life of ≥8 hours, adapting to different on-site testing scenarios where no external power source is available.

[0012] Furthermore, in step (1), the test tube includes a transparent test tube body, a sterile screw cap, and a semi-solid modified culture medium pre-encapsulated in the transparent test tube body. The total volume of the test tube is 10 mL, and the upper layer of the semi-solid modified culture medium is pre-coated with a paraffin film with a thickness of 0.5~1 mm.

[0013] Secondly, the present invention provides a kit for on-site detection of Clostridium perfringens for use in the above-described method, comprising a detection tube, a disposable sampling swab, a sample dilution tube, and a sterile portable packaging box.

[0014] Furthermore, the sample dilution tubes are filled with PBS buffer, and are available in two sizes: 2 mL and 5 mL, depending on the buffer volume.

[0015] Furthermore, the kit has a shelf life of at least 3 months at 4°C.

[0016] The beneficial effects of this invention are as follows: 1. The method for on-site detection of Clostridium perfringens provided by this invention simplifies the sampling, dilution, inoculation, culture and result interpretation operations. The rapid culture design allows for result interpretation within 2-6 hours of culture, solving the problems of complex operation, high professional threshold and long detection cycle of existing detection methods. It significantly reduces the difficulty of on-site operation, improves detection efficiency and meets the actual needs of rapid screening at the grassroots level and on-site.

[0017] 2. The method for on-site detection of Clostridium perfringens provided by the present invention uses a semi-solid modified culture medium and a low-melting-point paraffin film to form a double anaerobic sealing structure, combined with the design of self-sealing of the culture medium after swab inoculation. This solves the problems of the existing technology having a single anaerobic protection method and poor sealing effect, which easily leads to deviation in detection results. It can provide a stable anaerobic growth environment for Clostridium perfringens and effectively improve the accuracy and reliability of detection results.

[0018] 3. The kit for on-site detection of Clostridium perfringens provided by this invention solves the problem that existing detection technologies are highly dependent on laboratory instruments and cannot be carried out in farms and outdoor settings through the combination design of integrated pre-packaged detection tubes, disposable sampling components, sample dilution tubes and mini constant temperature water baths, significantly improving the portability and applicability of the detection. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a diagram showing the on-site test results of Example 7.

[0021] Figure 2 This is a diagram showing the on-site test results of Example 8.

[0022] Figure 3 Figure 9 shows the sensitivity test results of the kit in Example 9; Figure a is the agarose gel electrophoresis detection result, where M represents the DNA molecular weight standard (Marker) used for band size reference, and NC represents the negative control; Figure b is the kit detection result.

[0023] Figure 4 These are the stability test results for the kit in Example 10. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0025] Example 1 A kit for on-site detection of Clostridium perfringens includes a test tube, a disposable sampling swab, a sample dilution tube, and a sterile portable packaging box; The preparation method of the reagent kit includes the following steps: (1) Prepare semi-solid modified culture medium: Weigh 15.0g tryptone, 5.0g soybean peptone, 5.0g yeast extract powder, 1.2g ferric ammonium citrate, 1.2g sodium metabisulfite and 4.5g agar, add 1L purified water and stir to dissolve, adjust pH to 7.4±0.1, autoclave at 121℃ for 15min; when cooled to 50℃, add 12mg polymyxin B and 0.12g sodium sulfadiazine, mix well to obtain semi-solid modified culture medium; (2) Prepare test tubes: While the above culture medium is still hot, dispense 4±0.2 mL into each 10 mL transparent polypropylene test tube; after cooling to room temperature, drop low melting point paraffin onto the upper layer of the culture medium to form a paraffin film 0.7±0.1 mm thick, and cover with a sterile screw cap to obtain pre-sealed test tubes. (3) Prepare sample dilution tubes: Prepare sample dilution tubes of two sizes, 2 mL and 5 mL, and add 2 mL and 5 mL of PBS buffer respectively; (4) Kit assembly: Pack the pre-packaged test tubes, disposable sampling swabs and two sizes of sample dilution tubes into a sterile portable packaging box to assemble a kit for on-site detection of Clostridium perfringens.

[0026] Example 2 A mini constant-temperature water bath, with a volume ≤4L, external dimensions of 20cm×20cm×10cm, and a total weight ≤2kg, is made of 304 stainless steel. It has an internal test tube rack capable of holding 10 test tubes, a temperature adjustment range of 30~80℃, a temperature control accuracy of ±0.5℃, and features anti-dry-burn protection and a temperature alarm function. The mini constant-temperature water bath employs a dual-mode power supply design, supporting both external power and independent lithium battery power. When connected to a 220V AC power supply, the heating element operates at 600W. When switched to built-in backup battery power, the water bath temperature can be maintained at 70±1℃ for at least 30 minutes and at 37±1℃ for at least 2 hours, facilitating continuous on-site testing operations in different locations.

[0027] Example 3 Screening test for optimal reconstitution temperature of semi-solid modified culture medium: 1. Experimental Materials and Grouping: The kit from Example 1 was used, with a concentration of 1.0 × 10⁻⁶. 7 CFU / mL Clostridium perfringens suspension was used as the indicator strain, and a constant temperature water bath with a temperature control accuracy of ±0.5℃ was used as the temperature control environment. Five reconstitution temperature test groups were set up at 60℃, 65℃, 70℃, 75℃ and 80℃, with 6 test tubes in each group.

[0028] 2. Experimental methods: 2.1 Reconstitution treatment: Immerse the bottom of the test tube of each test group vertically into the corresponding constant temperature water bath, start timing, and continue until the paraffin film is completely melted and the culture medium is uniformly softened. Record the time taken for complete reconstitution. Remove the test tube immediately after the complete reconstitution is achieved. 2.2 Swab Insertion and Index Recording: Take 3 test tubes from each experimental group, and vertically insert disposable sampling swabs into the softened culture medium to the bottom of the tube, leaving the swab stem outside the tube. Observe and record the smoothness of swab insertion (no resistance / slight resistance / significant resistance) and the generation of air bubbles in the culture medium (no air bubbles / few tiny air bubbles / many air bubbles / large air bubbles). Start timing immediately after insertion and record the time it takes for the gap at the swab insertion point to completely close and fill. Use a high-definition camera to record the gap closure status at 0 min, 1 min, 3 min, 5 min, and 10 min after insertion. 2.3 Verification of anaerobic sealing effect: After inserting the cotton swab, let it stand for 10 minutes to allow the culture medium to completely recombine and form a self-sealing structure. Invert the test tube for 10 minutes and observe whether there is leakage or loosening of the cotton swab. If there is no leakage or loosening, the seal is qualified; otherwise, it is unqualified. 2.4 Verification of detection validity: Take another 3 test tubes from each test group, inoculate 100 μL of Clostridium perfringens indicator strain with a cotton swab, tighten the cap and incubate at 37℃ for 6 h, observe the specific blackening of the culture medium (obvious blackening / slight blackening / no blackening) to verify that the reconstitution temperature has no effect on the detection results; 2.5 Quantitative Judgment Criteria for Indicators: Complete reconstitution time: ≤15min is excellent, 15-20min is medium, >20min is poor; Bubble generation in the culture medium: no bubbles is excellent, a few tiny bubbles (diameter <0.5mm) is medium, a large number of bubbles / large bubbles (diameter ≥0.5mm) is poor; Smooth swab insertion: no resistance is excellent, slight resistance is medium, significant resistance is poor; Time for the swab insertion site to completely close and fill the gap: ≤5min is excellent, 5-10min is medium, >10min without closure is poor; Anaerobic sealing effect: qualified is excellent, unqualified is poor; Validity verification: obvious blackening is excellent, all others are poor.

[0029] 3. Experimental Results The results of the screening test for the optimal reconstitution temperature of the semi-solid modified culture medium are shown in Table 1: Table 1 Results of the screening test for the optimal reconstitution temperature of the semi-solid modified culture medium

[0030] In terms of reconstitution efficiency, the time required for complete reconstitution gradually decreases with increasing temperature from 60 to 80°C, with 80°C having the shortest time. However, the reconstitution time at 70°C (12 minutes) already meets the superior standard. Regarding anaerobic sealing effect, 70°C is the only reconstitution temperature that achieves bubble-free, resistance-free, and rapid closure. 60°C and 65°C exhibit insufficient softening, operational resistance, and slow closure. 75°C and 80°C show varying degrees of bubble generation. Bubbles are a significant potential factor that disrupts the anaerobic environment and affects detection accuracy; bubble-free operation is a key indicator for the formation of a self-sealing structure. Clostridium perfringens showed significant blackening at all temperature gradients, proving that the semi-solid modified culture medium of this invention maintains stable activity within the 60–80°C range. This experiment, through multi-dimensional index screening, clarified the optimality of the 70°C reconstitution temperature, ensuring the stable formation of the self-sealing anaerobic structure and the accuracy and repeatability of the detection results.

[0031] Example 4 Antibiotic combination screening test in semi-solid modified culture medium: 1. Experimental Materials and Grouping 1.1 Semi-solid culture medium: Weigh 15.0g tryptone, 5.0g soybean peptone, 5.0g yeast extract powder, 1.2g ferric ammonium citrate, 1.2g sodium metabisulfite and 4.5g agar, add 1L purified water and stir to dissolve, autoclave at 121℃ for 15min, and adjust the pH to 7.4±0.1; 1.2 Indicator strain: concentration of 1×10 7 Clostridium perfringens CFU / mL, total bacterial concentration 1×10⁻⁶ 7 CFU / mL of common mixed bacteria in farm feces (including Escherichia coli, Staphylococcus aureus and Enterococcus faecium). 1.3 Experimental Groups: Six antibiotic combinations were set up, with six test tubes in each group, each pre-filled with 4 ± 0.2 mL of semi-solid culture medium. In experimental group 1, 12 mg / L polymyxin B and 0.12 g / L sodium sulfadiazine were added to 4 mL of semi-solid culture medium, and the upper layer was coated with a 0.7 ± 0.1 mm paraffin film and sealed. In experimental group 2, 0.5 g / L of cycloserine (TSC medium core) was added to 4 mL ± 0.2 mm of semi-solid culture medium, and the top layer was coated with a 0.7 ± 0.1 mm paraffin film and sealed. In experimental group 3, 0.5 g / L cycloserine and 50 mg / L neomycin sulfate were added to 4 ± 0.2 mL of semi-solid culture medium, and the upper layer was coated with a 0.5 mm paraffin film and sealed. In experimental group 4, 12 mg / L polymyxin B and 50 mg / L neomycin sulfate were added to 4 ± 0.2 mL of semi-solid culture medium, and the upper layer was coated with a 0.7 ± 0.1 mm paraffin film and sealed. In experimental group 5, 20 mg / L vancomycin and 12 mg / L polymyxin E were added to 4 ± 0.2 mL of semi-solid culture medium, and the upper layer was coated with a 0.7 ± 0.1 mm paraffin film and sealed. The blank control group consisted of 4 ± 0.2 mL of semi-solid culture medium, with a 0.5 mm paraffin film applied to the top layer for sealing.

[0032] 2. Experimental methods: (1) Pretreatment of test tubes: Immerse the test tubes of each test group in a water bath and rehydrate them at 70°C for 10 minutes before use; (2) Sampling: Immerse the sampling swabs in a mixture of Clostridium perfringens and common bacteria for 5 minutes to fully absorb the bacteria. (3) Inoculation and culture: The sampling swabs adsorbed with Clostridium perfringens were vertically inserted into the three test tubes of each test group, and the sampling swabs adsorbed with the mixed solution of common bacteria were vertically inserted into the other three test tubes of each test group; after inoculation, the swab rod was left in the tube, the tube cap was tightened, and the tube was left to stand for 10 minutes. Then, each test group was placed in a constant temperature incubator at 37℃ and cultured for 6 hours. (3) Indicator Judgment The inhibition of contaminant bacteria was determined by testing tubes inoculated with a mixture of common contaminants: if the semi-solid culture medium was clear, it indicated that the contaminants were completely inhibited and the antibiotic activity was retained; if the semi-solid culture medium was slightly turbid, it indicated that some contaminants had grown and the antibiotic activity was partially lost; if the semi-solid culture medium was turbid, it indicated that a large number of contaminants had grown and the antibiotic was completely inactivated. The inhibition of the target bacteria was determined by inoculating the test tubes with Clostridium perfringens: if obvious blackening of the semi-solid culture medium was observed, it indicates that Clostridium perfringens is growing normally, the color reaction of the semi-solid culture medium is normal, and the antibiotic has no inhibitory effect on the target bacteria; if slight blackening or no blackening was observed in the semi-solid culture medium, it indicates that the growth of Clostridium perfringens is inhibited and the antibiotic is not suitable.

[0033] 3. Experimental Results The results of the antibiotic combination screening test in the semi-solid modified culture medium are shown in Table 2: Table 2 Results of antibiotic combination screening test in semi-solid modified culture medium

[0034] The blank control group showed no inhibitory effect on contaminants, proving that antibiotics are the core component of the culture medium for contaminant screening. Without antibiotics, contaminants would severely interfere with the detection results, making effective screening impossible. Traditional classic antibiotic combinations (decycloserine + neomycin sulfate or decycloserine alone) are not compatible with the 70°C water bath wax melting pretreatment process, which is an important reason why traditional culture media cannot be used for rapid on-site detection. Both experimental groups 4 and 5 showed decreased activity and weakened contaminant inhibition, and even slightly inhibited the growth of Clostridium perfringens. Vancomycin is also expensive and not suitable for large-scale on-site detection kit preparation. The combination of polymyxin B and sulfadiazine sodium used in experimental group 1 can achieve complete contaminant inhibition and has no inhibition on the growth of Clostridium perfringens or its specific black color development. It is the optimal antibiotic combination for the 70°C water bath pretreatment process of the detection tubes of this invention.

[0035] Example 5 Experiment on the effect of semi-solid modified culture medium on the detection rate of Clostridium perfringens: 1. Experimental Materials and Grouping 1.1 Indicator strain: concentration of 1×10 5 Clostridium perfringens CFU / mL; concentrations were 1×10⁻⁶. 5 A mixture of Clostridium perfringens and Escherichia coli at CFU / mL; concentrations of 1×10⁻⁶. 5 A mixture of Clostridium perfringens, Escherichia coli, Salmonella, Staphylococcus aureus, and Enterococcus faecalis at CFU / mL; 1.2 Culture medium type: Semi-solid culture medium: Weigh 15.0g tryptone, 5.0g soybean peptone, 5.0g yeast extract powder, 1.2g ferric ammonium citrate, 1.2g sodium metabisulfite and 4.5g agar, add 1L purified water and stir to dissolve, adjust pH to 7.4±0.1, autoclave at 121℃ for 15min, and seal the upper layer with a 0.7±0.1mm paraffin film; Semi-solid modified culture medium: Weigh 15.0g tryptone, 5.0g soybean peptone, 5.0g yeast extract powder, 1.2g ferric ammonium citrate, 1.2g sodium metabisulfite and 4.5g agar, add 1L purified water and stir to dissolve, adjust pH to 7.4±0.1, autoclave at 121℃ for 15min; add 12mg polymyxin B and 0.12g sodium sulfadiazine, and seal the upper layer with a 0.7±0.1mm paraffin film; 1.3 Experimental Groups: Six experimental groups were set up, with three test tubes in each group. Each tube was pre-filled with 4 mL of culture medium. Control group I was dispensed with 4 ± 0.2 mL of semi-solid culture medium, with Clostridium perfringens as the indicator strain; In experimental group I, 4 ± 0.2 mL of semi-solid modified culture medium was dispensed, and the indicator strain was Clostridium perfringens; In control group II, 4 ± 0.2 mL of semi-solid culture medium was dispensed, and the indicator strain was a mixture of Clostridium perfringens and Escherichia coli. In experimental group II, 4 ± 0.2 mL of semi-solid modified culture medium was dispensed, and the indicator strain was a mixture of Clostridium perfringens and Escherichia coli. In control group III, 4 ± 0.2 mL of semi-solid culture medium was dispensed, and the indicator strains were a mixture of Clostridium perfringens, Escherichia coli, Salmonella, Staphylococcus aureus and Enterococcus faecalis. In experimental group III, 4 ± 0.2 mL of semi-solid modified culture medium was dispensed, and the indicator strains were a mixture of Clostridium perfringens, Escherichia coli, Salmonella, Staphylococcus aureus, and Enterococcus faecalis.

[0036] 2. Experimental methods: (1) Pretreatment of test tubes: Immerse the test tubes of each test group in a water bath and rehydrate them at 70°C for 10 minutes before use; (2) Sampling: Immerse the sampling swabs into the corresponding indicator strains according to the test groups and allow them to fully absorb for 5 minutes; (3) Inoculation and culture: Insert the sampling swabs vertically into the three test tubes of each test group. After inoculation, leave the swab stick inside the tube, tighten the tube cap, let stand for 10 minutes, and then place each test group in a 37℃ constant temperature incubator for 6 hours. (3) Indicator Judgment Record the degree of blackening of the culture medium in each experimental group within 6 hours: Severe blackening, with the entire medium clearly blackened, is rated as "++++"; moderate blackening, with large localized blackening, is rated as "+++"; slight blackening, with punctate / linear blackening, is rated as "++"; slight blackening, with only a few black spots, is rated as "+"; no blackening, not detected, is rated as "-". Clostridium perfringens detection rate = (number of blackened samples / total number of samples in the group) × 100%; where the total number of samples in the group is 3.

[0037] 3. Experimental Results The results of the experiment on the effect of semi-solid modified culture medium on the detection rate of Clostridium perfringens are shown in Table 3: Table 3. Effect of semi-solid modified culture medium on the detection rate of Clostridium perfringens. Experimental results.

[0038] The experimental group, due to the addition of polymyxin B and sulfadiazine sodium antibiotics, exhibited targeted inhibitory effects on Staphylococcus aureus, Enterococcus faecium, and Gram-negative bacteria (Escherichia coli, Salmonella). This effectively reduced nutrient competition for Clostridium perfringens among bacteria in clinical fecal samples, significantly improving the detection rate of Clostridium perfringens. Although the degree of blackening decreased with the increase in the number of mixed bacteria, Clostridium perfringens could still be detected 100%, which was far superior to the control group. In the control group, no antibiotics were added, and bacteria multiplied in large numbers, competing with Clostridium perfringens for nutrients. As the number of mixed bacteria increased, the detection rate dropped sharply, and in some cases, it was completely undetectable.

[0039] Example 6 Experiment on the effect of swab retention and removal after sample inoculation on the detection rate of Clostridium perfringens: 1. Experimental Materials and Grouping 1.1 Indicator strain: concentration of 1×10 5 CFU / mL Clostridium perfringens; 1.2 Test Groups: Two test groups were set up, with 10 test tubes in each group: Experimental group A: Swabs were left in place after sample inoculation; Experimental Group B: The swab was removed after the sample was inoculated.

[0040] 2. Experimental methods: (1) Pretreatment of detection tubes: Immerse the detection tubes in a water bath and rehydrate them at 70°C for 10 minutes before use; (2) Sampling: Immerse the sampling swab in 1×10 5 In a CFU / mL Clostridium perfringens bacterial suspension, fully adsorb for 5 min; (3) Inoculation and culture: The sampling swabs were vertically inoculated into the bottom of the test tubes of each experimental group; for experimental group A, the tube cap was directly closed and left to stand for 8 minutes to allow the culture medium to cool and recrystallize completely, and the swabs were left in place throughout the process; for experimental group B, the tube cap was closed and left to stand for 3 minutes, then the swabs were slowly pulled out, the tube cap was closed again, and the tube was left to stand for another 5 minutes to allow the culture medium to recrystallize; the tubes were placed in a water bath and incubated at a constant temperature of 37°C. The time was started from the beginning of the incubation and the observation was continued for 6 hours. (4) Result determination: Three repeated experiments were conducted, and the time when the first blackening of each test tube was recorded as a reference index for detection sensitivity. After 6 hours of incubation, the detection rate of each group was calculated. Detection rate = (number of blackened samples / total number of samples in the group) × 100%; the total number of samples in the group was 30 tubes.

[0041] 3. Experimental Results The results of the experiment on the effect of swab retention and removal after sample inoculation on the detection rate of Clostridium perfringens are shown in Table 4. Table 4. Effects of swab retention and removal after inoculation on the detection rate of Clostridium perfringens. Results of the experiment.

[0042] The first blackening time of experimental group A was significantly earlier than that of experimental group B, and the detection rate reached 100% at 6 hours. In the three repeated experiments, eight test tubes of experimental group B showed no blackening, indicating that removing the swab would lead to sample loss and damage to the double anaerobic seal, thus affecting the growth and blackening reaction of Clostridium perfringens. In contrast, when the swabs of experimental group A were left in place, the culture medium tightly wrapped the swab, there was no sample loss, the anaerobic environment was stable, the target bacteria could concentrate and multiply at the inoculation point, and the blackening reaction was more obvious.

[0043] Example 7 A method for on-site detection of Clostridium perfringens in a livestock farm, using the kit from Example 1, includes the following steps: (1) Samples to be tested: Five fresh sheep manure samples (manure balls) were collected from a sheep farm and numbered 1 to 5; (2) Pretreatment of test tubes: Take 5 test tubes and immerse them in a mini constant temperature water bath. Re-dissolve them at 70°C for 15 minutes and then set them aside. (2) Sampling: Add samples numbered 1 to 5 to be tested into 5 mL sample dilution tubes, shake for 15 seconds, and let stand for 5 minutes; immerse 5 sampling swabs into the upper liquid of 5 diluted samples to be tested to complete the sampling work. (3) Inoculation and culture: Insert five sampling swabs vertically into the softened culture medium, leaving the swab stem inside the tube, tighten the tube cap, let stand for 10 minutes, and place the test tube at room temperature for 2-6 hours at an ambient temperature of 30℃. (4) Result determination: such as Figure 1 As shown, after 2 hours of incubation, samples 1 and 3 showed obvious blackening and were therefore deemed positive, with a concentration ≥1×10⁻⁶. 7 CFU / mL; After 4 hours of incubation, blackening was observed in sample 2, indicating a positive result (concentration 1×10⁻⁶). 5 ~1×10 7 CFU / mL; after 6 hours of incubation, samples 4 and 5 showed no blackening and were therefore determined to be negative.

[0044] Example 8 A method for outdoor field detection of Clostridium perfringens, using the kit from Example 1, includes the following steps: (1) Samples to be tested: Three samples of sheep diarrhea feces were collected from an outdoor breeding base and numbered A to C; (2) Pretreatment of test tubes: Take 3 test tubes and immerse them in a mini constant temperature water bath. Redissolve them at 70°C for 15 minutes and then set them aside. (2) Sampling: Add the samples to be tested numbered A to C into 2 mL sample dilution tubes, shake for 15 seconds, and let stand for 5 minutes; immerse 3 sampling swabs into the upper liquid of 3 diluted samples to be tested. (3) Inoculation and culture: Insert three sampling swabs vertically into the softened culture medium, leaving the swab stem inside the tube, tighten the tube cap, let stand for 6 minutes, and place the test tube in a mini constant temperature water bath at 37℃ for 2~6 hours. (4) Result determination: such as Figure 2 As shown, after 2.5 hours of incubation, sample A showed a blackening and was therefore identified as positive, with a concentration of 1×10⁻⁶. 5 ~1×10 7 CFU / mL; After 3 hours of incubation, sample B showed blackening and was therefore deemed positive, with a concentration of 1×10⁻⁶. 5 ~1×10 7 CFU / mL; after 6 hours of incubation, it was observed that item C did not turn black and was therefore determined to be negative.

[0045] Example 9 Reagent kit sensitivity testing: 1. Experimental materials: for a concentration of 1.4 × 10⁻⁶ 8 CFU / mL Clostridium perfringens culture was serially diluted tenfold, starting from a concentration of 1.4 × 10⁻⁶. 8 CFU / mL Clostridium perfringens culture: 100 μL of bacterial culture was added to 900 μL of physiological saline to obtain a concentration of 1.4 × 10⁻⁶. 7 CFU / mL Clostridium perfringens suspension was diluted sequentially using this method to obtain concentrations of 1.4 × 10⁻⁶. 6 CFU / mL, 1.4×10 5 CFU / mL, 1.4×10 4 CFU / mL, 1.4×10 3 CFU / mL and 1.4×10 2 CFU / mL Clostridium perfringens suspension; 2. Experimental methods: 2.1 Using each diluted bacterial culture as a template, conventional PCR amplification was performed on the 16S rRNA gene of *Clostridium perfringens*. The expected amplified fragment size was 279 bp (the detection method was based on the literature published in FEMS Microbiology Letters: The use of PCR to monitor the population abundance of six human intestinal bacterial species in an in vitro semicontinuous culture system). The amplified products were separated and identified by agarose gel electrophoresis. The detection results are as follows: Figure 3 As shown in Figure a.

[0046] 2.2 Perform kit sensitivity testing on the kit from Example 1, including the following steps: (1) Samples to be tested: diluted bacterial solutions; (2) Pretreatment of test tubes: Take 7 test tubes and immerse them in a mini constant temperature water bath. Redissolve them at 70°C for 15 minutes. After the paraffin film is completely melted and the culture medium is softened, they are ready for use. (2) Sampling: Immerse 7 sampling swabs into the bacterial solution sample to be tested; (3) Inoculation and culture: Insert 7 sampling swabs vertically into the softened culture medium, leaving the swab stick inside the tube, tighten the tube cap, let stand for 6 minutes, and put the test tube into a mini constant temperature water bath and set it to 37℃ for constant temperature culture. (4) Experimental results: The color change of the culture medium was observed and recorded at 0.5-hour intervals. The time point at which black colonies first appeared in each diluted bacterial solution was counted and recorded. The test results are as follows: Figure 3 As shown in b.

[0047] Figure 3 Image a shows the target fragment at position 279bp, from 1.4×10. 8 CFU / mL up to 1.4 × 10⁻⁶ 4 Clear and bright specific amplification bands were observed at all CFU / mL dilution gradients; at 1.4 × 10⁻⁶ CFU / mL... 3 At concentrations of CFU / mL and below, the bands gradually fade until they disappear, indicating that the detection limit for nucleic acid amplification can reach 1.4 × 10⁻⁶. 4 CFU / mL.

[0048] Figure 3Figure b shows that the effective detection threshold of the kit gradually decreases with increasing incubation time. At 9 hours, the sensitivity is comparable to that of PCR. Given that *Clostridium perfringens* is a common bacterium in the intestines of animals such as cattle and sheep, the detection time of the kit was set at 6 hours to better guide clinical disease diagnosis, with a detection sensitivity of approximately 1.4 × 10⁻⁶. 5 CFU / mL.

[0049] Example 10 The reagent kit stability test includes the following steps: 1. Experimental Groups: Experimental group: Two kits from Example 1 were stored at 4°C for 3 months and labeled as I and II respectively; Control group: Two kits from the newly prepared kit in Example 1 were taken and labeled III and IV, respectively; 2. Experimental methods: (1) Sample to be tested: concentration of 1×10 5 CFU / mL Clostridium perfringens suspension; mixed bacterial suspension consisting of Clostridium perfringens, Escherichia coli, Salmonella, Staphylococcus aureus, and Enterococcus faecalis (all at a concentration of 1×10⁻⁶). 5 CFU / mL (2) Pretreatment of test tubes: Take the test tubes of the above experimental group and control group, immerse them in a mini constant temperature water bath, re-dissolve at 70℃ for 15 minutes, and after the paraffin film is completely melted and the culture medium is softened, they are ready for use; (2) Sampling: Immerse four sampling swabs into the two types of samples to be tested, respectively; (3) Inoculation and culture: Insert four sampling swabs vertically into the softened culture medium, leaving the swab stem inside the tube. Tighten the tube cap, let stand for 6 minutes, and then place the test tube in a mini constant temperature water bath and incubate at 37°C for 5 hours. Among them, the sample to be tested in No. I is Clostridium perfringens, the sample to be tested in No. II is a mixed bacterial solution, the sample to be tested in No. III is Clostridium perfringens, and the sample to be tested in No. IV is a mixed bacterial solution. (4) Result determination: Observe the blackening of each test tube.

[0050] 3. Test Results: All test tubes showed no signs of cracking or liquefaction, and the paraffin film seal remained intact. After inoculation with Clostridium perfringens standard or mixed bacterial suspensions, the test results were as follows: Figure 4 As shown, the color development time was no different from that of the fresh kit, and the culture media numbered II and IV were both clear, indicating that the contaminating bacteria were completely inhibited and the antibiotic activity was retained, proving that the kit has good stability when stored at 4°C.

[0051] Example 11 The kit specificity test includes the following steps: 1. Experimental Materials and Grouping 1.1 Samples to be tested: all concentrations are 1×10⁻⁶ 7 CFU / mL of Clostridium perfringens, Escherichia coli, Salmonella enterica, Enterococcus faecalis, Staphylococcus aureus, Bacteroides fragilis, and Prevotella ruminicola bacterial suspension; 1.2 Test grouping: The test was divided into 7 groups, numbered 1 to 7. Each group had 3 test tubes, and each test tube in the group corresponded to one of the above-mentioned samples to be tested. 2. Experimental Methods (1) Pretreatment of test tubes: Immerse the test tubes of each test group in a mini constant temperature water bath and re-dissolve them at 70°C for 15 minutes. After the paraffin film is completely melted and the culture medium is softened, they are ready for use. (2) Sampling: Immerse 21 sampling swabs into the samples to be tested, and take samples 3 times for each sample to be tested; (3) Inoculation and culture: Insert 21 sampling swabs vertically into the softened culture medium, leaving the swab stem inside the tube, tighten the tube cap, let stand for 6 minutes, and then place the test tube into a mini constant temperature water bath and incubate at 37℃ for 2~6 hours. (4) Result determination: Observe whether the test tube turns black.

[0052] The test results are shown in Table 5. Only Clostridium perfringens turned black, while the other bacterial strains did not turn black, indicating that the test kit has good specificity.

[0053] Table 5. Results of reagent kit specificity test

[0054] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A method for on-site detection of Clostridium perfringens, characterized in that, Includes the following steps: (1) Pretreatment of test tubes: Immerse the test tubes in a mini constant temperature water bath and rehydrate at 70°C for 10-15 min; the test tubes contain semi-solid modified culture medium; the volume of the semi-solid modified culture medium is 4±0.2 mL, including the following raw materials at the following mass-volume concentrations: tryptone 14-16 g / L, soybean peptone 4-6 g / L, yeast extract powder 4-6 g / L, ferric ammonium citrate 1.1-1.3 g / L, sodium metabisulfite 1.1-1.3 g / L, agar 4-5 g / L, polymyxin B 11-13 mg / L and sodium sulfadiazine 0.11-0.13 g / L; (2) Sampling: Place the sample to be tested into the sample dilution tube, shake it by hand for 10-20 seconds, let it stand for 5-10 minutes to complete the sample dilution, and then immerse the sampling swab into the upper liquid in the sample dilution tube to complete the sampling. (3) Inoculation and culture: Insert the sampling swab vertically into the test tube, leaving the swab rod inside the tube, tighten the tube cap, let stand for 5~10 minutes, and culture for 2~6 hours; (4) Result determination: Observe the color of the semi-solid modified culture medium. The presence of black precipitate indicates a positive result, while the absence of black precipitate indicates a negative result.

2. The method as described in claim 1, characterized in that, In step (4), blackening occurs when the culture time is ≤2h, and the concentration of Clostridium perfringens in the sample is ≥1×10⁻⁶. 7 CFU / mL; blackening occurred after 2-6 hours of incubation, and the concentration of Clostridium perfringens in the sample was 1×10⁻⁶. 5 ~1×10 7 CFU / mL; where CFU / mL is the number of colony-forming units per milliliter of sample.

3. The method as described in claim 1, characterized in that, In step (2), when the sample to be tested is fecal pellets or feces, the dilution factor is 5 times; when the sample to be tested is a diarrhea sample, the dilution factor is 2 times; when the sample to be tested is any of intestinal contents, milk or blood, no dilution operation is required.

4. The method as described in claim 1, characterized in that, In step (3), when the ambient temperature is ≥25℃, the inoculated test tube is placed at room temperature for static culture; when the ambient temperature is <25℃, the inoculated test tube is placed in a mini constant temperature water bath and cultured at 37℃±0.5℃.

5. The method as described in claim 1, characterized in that, The mini constant temperature water bath has a volume of ≤4L and is equipped with a test tube rack inside. The test tube rack can hold 1 to 10 test tubes and the temperature adjustment range is 30 to 80℃.

6. The method as described in claim 5, characterized in that, The water bath adopts a dual-mode power supply design, which can be powered by an external power source or by a built-in lithium battery.

7. The method as described in claim 1, characterized in that, In step (1), the test tube includes a transparent test tube body, a sterile screw cap and a semi-solid modified culture medium. The total volume of the test tube is 10 mL. The upper layer of the semi-solid modified culture medium is pre-coated with a paraffin film with a thickness of 0.5~1 mm.

8. A kit for on-site detection of Clostridium perfringens used in the method according to any one of claims 1 to 7, characterized in that, It includes test tubes, disposable sampling swabs, sample dilution tubes, and sterile portable packaging boxes.

9. The reagent kit as described in claim 8, characterized in that, The sample dilution tubes are filled with PBS buffer, and are available in two sizes: 2 mL and 5 mL, depending on the buffer volume.

10. The kit according to claim 8, characterized in that, The kit has a shelf life of at least 3 months at 4°C.

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