A rapid detection kit for fecal spore group content

Abstract

The present application is a kind of rapid detection kit for fecal spore group content, belonging to the technical field of microorganism detection. The kit includes DPA standard curve preparation reagent, spore standard curve preparation reagent, feces pretreatment reagent and fecal spore group content detection reagent. DPA in the reagent is a special component in bacterial spores, which can rapidly react with lanthanide compound terbium ion to produce fluorescence. By detecting the fluorescence intensity, bacterial spores are quantified, so that donor feces with high spore content can be quickly screened. In addition, the kit contains a purification reagent to remove the interference in the feces, which can remove a large amount of impurities in the feces that interfere with subsequent spectral absorption, thereby making the detection more accurate. Based on this principle, the kit developed can quickly screen donor feces with high content of spore group. At the same time, it can also detect various spores and be applied to other fields.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically a rapid detection kit for fecal spore count. Background Technology

[0002] Spores are dormant forms of certain bacteria that develop under adverse conditions such as nutrient deficiency, exhibiting remarkable resilience. Common spore-forming bacteria include *Bacillus* and *Clostridium* genera. Under certain conditions, these bacteria can form spores. Spores differ significantly from vegetative cells in structure (e.g., multi-layered thick-walled structures) and chemical composition (e.g., rich in pyridine dicarboxylic acid and special small-molecule acid-soluble proteins), giving them unique biological characteristics. The most crucial characteristic of spores is their extremely strong resilience, exhibiting high tolerance to high temperatures, dryness, ultraviolet radiation, ionizing radiation, and various chemical disinfectants and toxic substances. Crucially, spores also possess strong tolerance to gastric acid, bile salts, and various digestive enzymes in the digestive tract. This characteristic makes the spores of certain spore-forming anaerobic bacteria (especially some *Clostridium* spores) a key vector for their long-term survival in the environment, their ability to overcome the host's digestive tract barrier, and their eventual germination and colonization in the intestines, ultimately facilitating human-to-human transmission. The spore core is rich in a large amount of characteristic pyridine dicarboxylic acid, which can serve as a spore-specific biomarker, providing a theoretical basis for establishing a rapid and specific quantitative detection method for spores.

[0003] The detection of spore-forming bacteria in feces has irreplaceable value in clinical medicine, public health, and microecological research. The exceptional tolerance of spores to the harsh environment of the digestive tract (gastric acid, bile salts, digestive enzymes) makes them a core indicator and transmission vector for difficult-to-culture spore-forming bacteria (especially anaerobic spore-forming bacteria like Clostridium difficile). Quantitative analysis of spore groups in fecal samples can effectively reflect: First, the colonization dynamics and ecological functions of intestinal spore-forming bacteria: Spore formation is a survival strategy for bacteria in response to adverse environments (such as antibiotic exposure and host immunity), and changes in its abundance can reveal the activity status and community fluctuations of related bacterial communities (especially anaerobic bacteria that are difficult to culture in vitro). Second, the risk of carrying and transmitting important pathogens: Taking Clostridium difficile infection as an example, its spores are the only effective form of human-to-human transmission and a key factor in causing nosocomial infections, community-acquired diarrhea, and antibiotic-associated enteritis. Rapid and accurate quantification of Clostridium difficile spores or total spore load in feces is crucial for accurately identifying sources of infection, implementing effective isolation and control measures, assessing patient recurrence risk, and guiding clinical decision-making. Third: Efficacy evaluation of probiotic spore preparations: For probiotic preparations administered in spore form (such as Bacillus subtilis and Bacillus licheniformis), the number of germinating spores surviving in feces is a core indicator for evaluating the product's actual survival rate, colonization ability, metabolic activity, and final effective dose in the intestine, directly affecting the determination of the probiotic intervention effect.

[0004] Traditional fecal spore counts rely on dilution plating, which has several drawbacks: First, the pretreatment process is cumbersome and destructive, requiring multiple centrifugation and washing processes, ethanol inactivation of non-spore-forming bacteria, heat treatment (e.g., 65°C for 30 minutes) to kill vegetative cells and activate spores, followed by the addition of germination agents for 24-72 hours of anaerobic / aerobic culture. The procedures are complex. Second, the accuracy and systematicity are relatively low: heat treatment cannot completely eliminate heat-resistant bacteria (such as enterococci), and relying on selective media can exclude some target spore-forming bacteria, affecting the count. Some spores lack germination signal molecules and are difficult to germinate; furthermore, heat treatment can damage the viability of fragile spores, further reducing the detectable proportion. Third, the timeliness and applicability are severely lacking: strict aseptic operation is required throughout the process, resulting in a high technical threshold; the detection cycle is as long as 48-72 hours, which cannot meet the needs of rapid clinical diagnosis, let alone support real-time quality control for large-scale epidemiological screening or probiotic preparation production.

[0005] Spores differ significantly from vegetative cells in both structure (e.g., multi-layered thick-walled structure) and chemical composition (e.g., rich in pyridine dicarboxylic acid and special small-molecule acid-soluble proteins), endowing them with unique biological characteristics. It is recognized in this art that spore wall components—DPA and terbium ions (Tb)—are important. 3+In solution, DPA efficiently chelates to form a stable fluorescent complex (DPA-Tb), emitting strong green fluorescence (peak at 544 nm) under UV excitation. The fluorescence intensity is linearly positively correlated with DPA concentration (i.e., spore count). However, applying the DPA-Tb fluorescence method to the rapid detection of spore content in fecal samples faces two major technical bottlenecks. First, matrix interference is uncontrollable: even after multiple centrifugations and washings to enrich spore populations, residual pigment impurities (such as bilirubin and heme) significantly quench the DPA-Tb fluorescence signal, leading to systematic deviations in the detection values. Second, there is a purification-time constraint: to reduce matrix interference, purification needs to be enhanced (e.g., increasing the number of washes or sonication), but this simultaneously causes spore loss and prolongs sample pretreatment time, thus negating the advantages of rapid detection. These bottlenecks severely restrict the practical application of the DPA-Tb fluorescence method in the quantification of fecal spore populations, necessitating the development of an innovative technology that can simultaneously overcome both matrix interference and time constraints. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a rapid detection kit for fecal spore content. The kit includes reagents for detecting the content of DPA (pyridine dicarboxylic acid), a representative component of bacterial spores, thereby quantifying the spores. DPA is a special component in bacterial spores that reacts rapidly with terbium ions (a lanthanide compound) to produce fluorescence. The fluorescence intensity is used to quantify the bacterial spores, allowing for rapid screening of donor feces with high spore content. Furthermore, the kit contains purification reagents to remove interference from the feces, eliminating a large number of impurities that interfere with subsequent spectral absorption, thus improving detection accuracy. Using this kit, donor feces can be screened and unqualified donor feces can be discarded within 2 hours, with high accuracy and a wide detection range.

[0007] To achieve the above-mentioned objectives, the specific technical solution of this invention is as follows: This invention first provides a strain of double-fermented Clostridium parasiticum ( Paraclostridium bifermentans Application of S1-GT1 in quantitative detection of spore content or quantitative detection of fecal spore population content.

[0008] This strain is an existing strain (already described in application number 202510074022.0), and was deposited at the Guangdong Provincial Microbial Culture Collection Center on May 27, 2024, with accession number GDMCCNO:64683. In the disclosed prior art, *Clostridium paragallinarum* undergoing double fermentation... Paraclostridium bifermentansS1-GT1 exhibits the absence of virulence factors and is genomically safe; direct intraperitoneal or tail vein injection of live bacterial cells or spores into mice does not cause animal death, demonstrating good safety. Furthermore, this strain possesses superior spore-producing ability; the spores are obligate anaerobic, capable of germinating, colonizing, and growing in hypoxic tumor areas, inducing oncolytic necrosis in solid tumor tissue, showing great promise and application potential in the treatment of solid tumors. However, further research by the applicant revealed that this strain is also easily purified, and the spore component DPA reacts with lanthanide compounds to produce fluorescence, making it suitable for rapid detection of fecal spore populations.

[0009] Furthermore, in application, the spore component DPA reacts with lanthanide compounds to generate fluorescence, thereby quantitatively detecting the spore content or fecal spore population content.

[0010] In a preferred embodiment of this application, the lanthanide compound is terbium chloride.

[0011] In a preferred embodiment of this application, in the application described above: during detection, the excitation wavelength is 275 nm, the excitation wavelength is 543 nm, the integration time is 1.25 ms, the delay is 0.1 ms, and the fluorescence intensity is detected.

[0012] This invention also provides a rapid method for detecting fecal spore count, comprising the following steps: 1) Establishment of the DPA standard curve; 2) Establishment of the spore standard curve; 3) Pre-treatment of feces; 4) Prepare a reagent for detecting fecal spore content and test the fecal spore content of the feces that have undergone pretreatment in step 3).

[0013] As a preferred embodiment of this application, in step 1) of the rapid detection method for fecal spore count, the specific steps for establishing the DPA standard curve are as follows: a1. Use Tris–HCl buffer to gradient dilute the standard DPA stock solution to a minimum concentration of 0 mM to obtain the standard DPA solution; b1. Dilute the terbium chloride mother liquor to 10 mM using sodium acetate solution to obtain a terbium chloride reagent with a concentration of 10 mM; c1. Add the 10 mM terbium chloride reagent from b1 to an equal volume of diluted standard DPA solution; d1. Time-resolved fluorescence was used to detect the fluorescence intensity of the substance obtained from c1, with an excitation wavelength of 275 nm, an integration time of 1.25 ms, and a delay of 0.1 ms. e1. Plot the DPA standard curve based on the fluorescence intensity in d1.

[0014] As a preferred embodiment of this application, in step 2) of the rapid detection method for fecal spore population content, the specific steps for establishing the spore standard curve are as follows: a2. Use Tris–HCl buffer to gradient dilute the standard spore stock solution to a minimum of 100 spores / mL to obtain a standard spore suspension; b2. The diluted standard spore suspension from a2 was autoclaved at 121°C for 30 minutes to obtain a sterilized spore suspension; c2. Dilute the terbium chloride mother liquor to 10 mM using sodium acetate solution to obtain a terbium chloride reagent with a concentration of 10 mM; d2. Add the 10 mM terbium chloride reagent from c2 to an equal volume of the sterilized spore suspension obtained in b2. e2. Time-resolved fluorescence was used to detect the fluorescence intensity of the substance obtained from d2, with an excitation wavelength of 275 nm, an integration time of 1.25 ms, and a delay of 0.1 ms. f. Plot the standard curve of spores based on the e2 fluorescence intensity.

[0015] As a preferred embodiment of this application, in step 3) of the rapid detection method for fecal spore count, the specific steps for pretreatment of the feces are as follows: a3. Collect a fecal sample of not less than 0.1g; b3. The fecal sample in a3 was thoroughly suspended in 10 times its weight of RO water to obtain a fecal suspension; c3. Centrifuge the fecal suspension (preferably 300 g) obtained in b3 for 5 min and collect the supernatant; d3. Place the supernatant obtained in c3 into a macroporous resin that has been pre-removed of ethanol, shake for 20 min, centrifuge for 5 min (preferably 300 g) and collect the supernatant; e3. Centrifuge the supernatant obtained in d3 (preferably 8800g) for 5min, and resuspend it in Tris–HCl buffer (1M, pH 8.0); f3. The resuspended fecal spore suspension obtained in e3 was subjected to high pressure treatment at 121℃ for 30 min to obtain sterilized fecal spore suspension; g3. Centrifuge the sterilized fecal spore suspension (preferably 10000 g) obtained in f3 for 5 min, collect the fecal spore supernatant, and complete the pretreatment of feces.

[0016] As a preferred embodiment of this application, in step 4) of the rapid detection method for fecal spore content, the specific steps for preparing a fecal spore content detection reagent and detecting the fecal spore content in the feces after pretreatment in step 3) are as follows: a4. Dilute the terbium chloride stock solution to 10 mM using sodium acetate solution; b4. Add the supernatant of fecal spores to an equal volume of 10 mM terbium chloride solution; c4. Time-resolved fluorescence was used, with an excitation wavelength of 275 nm, an integration time of 1.25 ms, and a delay of 0.1 ms to detect fluorescence intensity.

[0017] As a preferred embodiment of this application, the above-mentioned rapid detection method for fecal spore count further includes step 5), which sorts the fluorescence intensity of each sample, with the top 70% of fluorescence intensity being qualified donor feces and the bottom 30% being unqualified donor feces. This step is effectively used for donor screening.

[0018] The present invention also provides a kit for rapid detection of fecal spore content, the kit comprising reagents for preparing DPA standard curve, reagents for preparing spore standard curve, fecal pretreatment (purification) reagents, and reagents for detecting fecal spore content.

[0019] As mentioned in the kit, the reagents for preparing the DPA standard curve are soluble standard DPA reagents and reagents for detecting DPA content.

[0020] As mentioned in the aforementioned kit, the reagents for preparing the spore standard curve are reagents that can resuspend standard spores and reagents for detecting the content of standard spores.

[0021] As described in the aforementioned kit, the fecal pretreatment (purification) reagent is a reagent that can dissolve and remove impurities from feces.

[0022] As described in the aforementioned kit, the fecal spore count detection reagent is a fecal bacterial spore resuspension reagent and a reagent for detecting fecal bacterial spore count.

[0023] As described in the aforementioned kit, the detected fecal spore count is the content of bacterial spores in the feces of healthy individuals.

[0024] Furthermore, the composition of the reagents used to prepare the DPA standard curve is as follows: Tris–HCl buffer, 50mM, pH 8.0, 100ml; Terbium chloride stock solution, 30 mM, 1 ml; Standard DPA stock solution, 10mM, 10ml; Sodium acetate trihydrate, 40 mM, 100 ml.

[0025] The reagents used to prepare the spore standard curve consist of the following components: Tris–HCl buffer, 50mM, pH 8.0, 100ml; Terbium chloride stock solution, 30 mM, 1 ml; Sodium acetate trihydrate, 40 mM, 100 ml; Standard spore S1-GT1, 1,000,000,000 spores / ml, 1ml; The fecal pretreatment (purification) reagent is: RO water, 100ml; 100 ml of macroporous resin stored in 95% ethanol; Tris–HCl buffer, 50mM, pH 8.0, 100ml; The reagent for detecting fecal spore count is: Tris–HCl buffer, 50mM, pH 8.0, 100ml; Terbium chloride stock solution, 30 mM, 1 ml; Sodium acetate trihydrate, 40 mM, 100 ml.

[0026] Another aspect of the present invention provides a testing standard system, which is a standard system for screening and removing unqualified donor feces.

[0027] The use of reagents for dissolving feces, removing impurities from feces, dissolving spores, and plotting standard curves in kits for rapid detection of fecal bacterial spore content and establishment of standard systems; The fluorescence produced by the reaction of DPA (pyridine dicarboxylic acid) with terbium chloride indicates that the spores have fully released fluorescence, which can be used for quantitative detection of the spores.

[0028] As described above, the kit also includes optional fecal spores from healthy individuals that have undergone special treatment to fully release DPA, which reacts with terbium chloride to produce fluorescence, enabling quantitative detection of fecal spores from healthy individuals.

[0029] As described above, the reagents used to dissolve standard DPA and to detect DPA content are reagents for establishing a DPA standard curve.

[0030] As described above, the reagents for resuspending standard spores and for detecting the content of standard spores are reagents for establishing a spore standard curve.

[0031] As described above, the fecal dissolving reagent and the fecal impurity removal reagent are reagents for the preliminary treatment of feces.

[0032] As described above, the reagent for resuspending fecal bacterial spores and the reagent for detecting fecal bacterial spore content are reagents for measuring the content of fecal bacterial spores.

[0033] As described above, the spore content to be detected is the bacterial spore content in the feces of healthy individuals.

[0034] As described above, this is a standard system for screening and removing unqualified donor feces.

[0035] The specific steps are as follows: use the above method to perform fluorescence detection on the samples, and then sort the fluorescence intensity of each sample. The top 70% of the fluorescence intensity is qualified donor feces, and the bottom 30% of the fluorescence intensity is unqualified donor feces. This step is effective for donor screening.

[0036] Compared with existing technologies, the beneficial effects of this invention are: (1) The special component DPA in bacterial spores can react rapidly with terbium ions of lanthanide compounds to produce fluorescence. The fluorescence intensity can be detected to quantify bacterial spores, thereby quickly screening out donor feces with high spore content.

[0037] (2) The kit contains purification reagents to remove interference from feces, which can remove a large number of impurities in feces that interfere with subsequent spectral absorption, thereby making the detection more accurate.

[0038] (3) The reagent kit developed based on this principle can quickly screen qualified donor feces, which is helpful for large-scale clinical production of spore preparations and has good application prospects. At the same time, it can also detect various types of spores and be applied in various fields. Attached Figure Description

[0039] Figure 1 The standard curves show the fluorescence intensities corresponding to the 0 μM-3000 μM DPA solutions and the 0 μM-5000 μM DPA solutions in Example 1. Figure 2 The image shows a microscopic image of Paraclostridium Bifermentans S1-GT1 spores from Example 2. Figure 3 The standard curve of fluorescence intensity for the standard spore suspension with concentrations of 0 spores / ml to 2000000000 spores / ml in Example 2; Figure 4 Images of fecal spore suspensions after treatment with two different methods in Example 3; Figure 5 This illustrates the impact of two different methods on the standard DPA in Example 3. Detailed Implementation

[0040] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0042] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations according to this application. As used herein, the singular form includes the plural form unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this description, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0043] In this invention, some conventional operating equipment, devices and components have been omitted or only briefly described.

[0044] Unless otherwise specified in the examples, the conditions shall be performed according to the standard conditions or the conditions recommended by the manufacturer.

[0045] In this application, all percentages not explicitly stated represent weight percentages, and all ratios are mass ratios.

[0046] Implementing the kit of this invention (rapid detection kit for fecal spore count). 1. Kit Components The kit of this invention includes reagents for preparing DPA standard curves, reagents for preparing spore standard curves, reagents for fecal pretreatment (purification), and reagents for detecting fecal spore population content; it also includes a donor fecal screening standard system.

[0047] 1.1 DPA Standard Curve Reagent The reagents for preparing the DPA standard curve are used to dissolve standard DPA, detect DPA content, and establish a DPA standard curve. Their composition is shown in Table 1.

[0048] Table 1. DPA Standard Curve Reagents

[0049] 1.2 Spore Standard Curve Reagent The spore standard curve reagent is used to resuspend standard spores, detect spore content, and establish a spore standard curve. Its composition is shown in Table 2.

[0050] Table 2. Spore Standard Curve Reagents

[0051] 1.3 Fecal pretreatment reagents The fecal pretreatment reagent is used to dissolve and remove impurities from feces. Its composition is shown in Table 3.

[0052] Table 3. Reagents for fecal pretreatment

[0053] Table 3 lists HPD-100 type macroporous adsorption resin with a particle size of (60-16 mesh) 0.3-1.25 mm >90%, water content of 65-75%, and specific surface area of ​​650-700 m². 2 / g, relative adsorption capacity mg / g (phenol / dry basis) >40, bulk density: 0.65-0.7g / ml (wet), wet apparent density: 0.68-0.75g / ml, wet true density: 1.03-1.07g / ml, average pore size: 85-90Å.

[0054] 1.4 Reagents for detecting fecal microorganism spore content The fecal bacterial spore content detection reagent is used to resuspend fecal bacterial spores and detect the content of fecal bacterial spores. Its composition is shown in Table 4.

[0055] Table 4. Reagents for detecting fecal microbial spore content

[0056] 2. How to use 2.1 Establishment of DPA Standard Curve a. Use Tris–HCl buffer to gradient dilute the standard DPA stock solution to a minimum concentration of 0 mM; b. Dilute the terbium chloride mother liquor to 10 mM using sodium acetate solution; c. Add the 10 mM terbium chloride reagent sequentially to an equal volume of diluted standard DPA solution; d. Time-resolved fluorescence was used, with an excitation wavelength of 275 nm, an integration time of 1.25 ms, and a delay of 0.1 ms to detect fluorescence intensity; e. Plot the DPA standard curve based on fluorescence intensity.

[0057] 2.2 Establishment of Spore Standard Curve a. Gradually dilute the standard spore stock solution to a minimum concentration of 0 mM using Tris–HCl buffer; b. Autoclave the diluted standard spore suspension at 121℃ for 30 min; c. Dilute the terbium chloride mother liquor to 10 mM using sodium acetate solution; d. Add the 10 mM terbium chloride reagent sequentially to an equal volume of sterilized spore suspension; e. Time-resolved fluorescence was used, with an excitation wavelength of 275 nm, an integration time of 1.25 ms, and a delay of 0.1 ms to detect fluorescence intensity; f. Plot a standard curve for spores based on fluorescence intensity.

[0058] 2.3 Fecal pretreatment a. Collect a fecal sample of no less than 0.1g; b. 10 times its weight of RO water fully suspends the feces; c. Centrifuge 300 g of fecal suspension for 5 min and collect the supernatant; d. Place the supernatant in a pre-treated macroporous resin, shake for 20 min, centrifuge at 300g for 5 min, and collect the supernatant; e. Centrifuge the supernatant at 8800g for 5 min, then resuspend in Tris–HCl buffer (1M, pH 8.0); f. High-pressure treatment of the resuspended fecal microbial spore suspension at 121℃ for 30 min; g. Centrifuge 10,000 g of the sterilized fecal spore suspension for 5 min and collect the fecal spore supernatant.

[0059] 2.4 Detection of fecal spore count (fecal bacterial spore content) a. Dilute the terbium chloride stock solution to 10 mM using sodium acetate solution; b. Add the supernatant of fecal spores to an equal volume of 10 mM terbium chloride solution; c. Time-resolved fluorescence was used, with an excitation wavelength of 275 nm, an integration time of 1.25 ms, and a delay of 0.1 ms to detect fluorescence intensity.

[0060] 2.5 Screening for qualified donor feces The fluorescence intensity of each sample was ranked, with the top 70% of fluorescence intensity considered as qualified donor feces and the bottom 30% as unqualified donor feces.

[0061] It should be understood that this embodiment is a spore quantitative detection kit, the purpose of which is to quantitatively detect spore content by utilizing the reaction of a special component DPA in spores with lanthanide compounds to generate fluorescence. According to common knowledge in the art, any method that can utilize DPA to detect spore content can also be used for this detection.

[0062] The beneficial effects of the present invention will be further illustrated below using experimental examples.

[0063] The technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0064] The reagent preparation methods used in the following examples are as follows: Preparation of Tris–HCl buffer (1) Preparation of Tris–HCl buffer (1M, pH 8.0) Weigh 12.1g of Tris, add 80ml of RO water, dissolve thoroughly, add an appropriate amount of concentrated hydrochloric acid (30%) until the solution pH is 8.0, and bring the RO water to a final volume of 100ml.

[0065] (2) Preparation of Tris–HCl buffer (50 mM, pH 8.0) Add 5 ml of Tris–HCl buffer (1M, pH 8.0) to 80 ml of RO water, mix thoroughly, add an appropriate amount of concentrated hydrochloric acid (30%) until the solution pH is 8.0, and bring the RO water to a final volume of 100 ml.

[0066] DPA (10µm) solution preparation Weigh 16.836 mg of 2,6-pyridinedicarboxylic acid (DPA) and dissolve it in 10 ml of Tris–HCl buffer (50 mM, pH 8.0) until fully dissolved.

[0067] Preparation of terbium chloride solution Preparation of sodium acetate (40 mM) solution Weigh out 54.432 mg of sodium acetate trihydrate and dissolve it in 10 ml of RO water until fully dissolved.

[0068] (2) Preparation of terbium chloride (30mM) solution Weigh 11.1984 mg of terbium chloride hexahydrate and dissolve it in 10 ml of sodium acetate (40 mM) solution until fully dissolved.

[0069] (3) Preparation of terbium chloride (10mM) solution Add an appropriate amount of terbium chloride (10 mM) solution to two volumes of sodium acetate (40 mM) solution and mix thoroughly.

[0070] The standard spores used in this invention are the *Paraclostridium bifermentans* S1-GT1 spore strain, which was independently isolated by our team. It is of high purity and free of special impurities, such as... Figure 2 As shown.

[0071] The preservation information for strain S1-GT1 is as follows: Strain name: Paraclostridium bifermentans S1-GT1 Preservation Institution: Guangdong Microbial Culture Collection Center (GDMCC), Address: Institute of Microbiology, Guangdong Academy of Sciences, Postcode: 510075, Tel: 020-87137633.

[0072] Accession number: GDMCC NO: 64683.

[0073] Standard spores (double-fermented Clostridium paraflavum) Paraclostridium bifermentans The preparation of the mother liquor (S1-GT1) includes the following steps: The preserved S1-GT1 strain was streaked onto RCM plates using the three-zone streak method for revival. After anaerobic incubation at 37°C for 24 h, single colonies were picked and inoculated into RCM liquid medium, and anaerobic incubated at 37°C for 24 h to obtain the proliferated S1-GT1 bacterial suspension. The bacterial suspension was gradually expanded to 100 mL and inoculated into 2 L of sporulation medium at a 5% inoculation ratio, and anaerobic incubated at 37°C for 14 days.

[0074] After culturing the above bacterial culture for 14 days, a portion of the liquid was taken out for microscopic examination. The remaining liquid was aliquoted and centrifuged at 4000 g for 10 min. After removing the supernatant, the precipitate was resuspended in a small amount of sterile water and slowly poured into twice the volume of 50% sucrose solution. The solution was then centrifuged at 3200 g for 20 min, the supernatant was removed, and the precipitate was washed twice with sterile water to obtain spores.

[0075] Resuspend the spores in an appropriate volume of sterile water, take a small amount of the liquid and examine it under a microscope according to the above steps, then take a small amount of the liquid for serial dilution and spread it on an RCM plate for plate counting. Dispense the remaining liquid into 1.5 mL EP tubes to obtain the standard spore stock solution for later use.

[0076] Sporulation medium Weigh the culture medium components according to the table below, add 1 L of RO water, mix well, and then sterilize at 121℃ for 15 minutes to obtain the sporulation culture medium.

[0077] Table 1 Components of Sporulation Culture Medium

[0078] Example 1: Optimization of buffer solution preparation and establishment of terbium chloride standard curve. 1. Dipyridinic acid (DPA) is an important component of bacterial spores and exhibits significant fluorescence properties. When DPA reacts with terbium chloride (TbCl3), it forms a fluorescent complex. Under ultraviolet light excitation, this complex emits fluorescence at a specific wavelength. A 10 mM standard DPA stock solution was diluted with Tris–HCl buffer (50 mM, pH 8.0) to prepare final concentrations of 0 μM, 1.46484375 μM, 2.9296875 μM, and 5.859375 μM. The concentrations of DPA, terbium chloride (TCH) were 11.71875 μM, 23.4375 μM, 46.875 μM, 93.75 μM, 187.5 μM, 375.750 μM, 1500 μM, and 3000 μM. The TCH stock solution was diluted to 10 mM with sodium acetate (40 mM). 50 μL of each of the diluted DPA solution and terbium chloride (10 mM) solution were added sequentially to black 96-well fluorescent plates with duplicate wells. The multi-mode microplate reader was set to excitation wavelengths of 275 nm and 543 nm, with a time-resolved fluorescence integration time of 1.25 ms and a delay of 0.1 ms. Fluorescence intensity was measured. The results are shown in Table 5. The fluorescence intensity increased linearly with increasing DPA solution concentration (R² = 0.9938).

[0079] Table 5. Fluorescence Intensities Corresponding to DPA Solutions with Concentrations of 0 μM to 3000 μM

[0080]

[0081] 2. Dilute the 10 mM standard DPA stock solution with Tris–HCl buffer (50 mM, pH 8.0) to prepare the following concentrations: 0 uM, 1.22070312 uM, 2.4414062 uM, 4.882812 uM, 9.765625 uM, 19.53125 uM, 39.0625 uM, 78.125 uM, 156.25 uM, 312.5 uM, 625 uM, and 1250 uM. The terbium chloride stock solution was diluted to 10 mM with sodium acetate (40 mM) at concentrations of 2500 μM, 5000 μM, and 10 mM. 50 μL of each of the diluted DPA solution and terbium chloride (10 mM) solution were added sequentially to black 96-well fluorescent plates with duplicate wells. The multi-mode microplate reader was set to excitation wavelength of 275 nm, excitation wavelength of 543 nm, time-resolved fluorescence integration time of 1.25 ms, and delay of 0.1 ms. Fluorescence intensity was measured. The results are shown in Table 6. The fluorescence intensity increased linearly with increasing DPA solution concentration (R² = 0.9893).

[0082] Table 6. Fluorescence Intensities Corresponding to DPA Solutions with Concentrations of 0 μM-3000 μM

[0083]

[0084] Standard curves were plotted based on the fluorescence intensity of the reactions between DPA solutions of different concentrations and 10 mM terbium chloride solution, as shown in Tables 5 and 6. The results are as follows: Figure 1 As shown. Figure 1 The standard curves show the fluorescence intensity corresponding to 0 μM-3000 μM DPA solutions and the fluorescence intensity corresponding to 0 μM-5000 μM DPA solutions.

[0085] Example 2: Establishing a standard curve for spores and determining the lower limit of spore detection. The standard spore stock solution was diluted with Tris-HCl buffer (50 mM, pH 8.0) to prepare final concentrations of 0, 1024, 5120, 25600, 128000, 640000, 3200000, 16000000, 80000000, and 4000000000 spores / ml. 2,000,000,000 spores / ml were used to dilute the standard spore suspension. The diluted suspension was autoclaved at 121℃ for 30 min. The terbium chloride stock solution was diluted to 10 mM using sodium acetate (40 mM) solution. 50 μL of each of the sterilized spore suspension and the 10 mM terbium chloride solution were added sequentially to black 96-well fluorescent plates with duplicate wells. The multi-mode microplate reader was set to excitation wavelength 275 nm, excitation wavelength 543 nm, time-resolved fluorescence integration time 1.25 ms, and delay 0.1 ms. Fluorescence intensity was measured. The results are shown in Table 7. The fluorescence intensity increased linearly with increasing spore suspension concentration. Table 7. Fluorescence Intensities Corresponding to Concentrations of 0-2000000000 Standard Spore Suspensions / ml

[0086]

[0087] Standard curves were plotted based on the fluorescence intensity of the reaction between the standard spore suspensions of different concentrations shown in Table 7 and 10 mM terbium chloride solution. The results are as follows: Figure 3 As shown; Figure 3 The standard curve of fluorescence intensity for standard spore suspensions with concentrations ranging from 0 spores / ml to 2000000000 spores / ml is shown.

[0088] Example 3: Screening of the most reasonable method for removing fecal impurities 1. Collect 8 stool samples from healthy individuals, suspend them thoroughly in 10 times their weight of RO water, and then 300 ml of water was used. g Centrifuge for 5 minutes for preliminary treatment, collect the supernatant and divide it into three equal parts. The supernatant is then processed using three different methods: Method A: Place the pre-treated supernatant in a small amount of deethanolified macroporous resin, vortex for 20 minutes, centrifuge at 300g for 5 minutes, collect the supernatant, and then process the collected supernatant at 8800... gCentrifuge for 5 min, collect the precipitate, and resuspend the precipitate in Tris-HCl buffer (50 mM, pH 8.0). Autoclave the resuspended fecal microorganism suspension at 121℃ for 30 min to obtain a clear liquid. For Method B, place the pre-treated fecal supernatant in a small amount of activated carbon. Due to the special properties of activated carbon, subsequent centrifugation is omitted; the fecal microorganism suspension is directly resuspended in Tris-HCl buffer (50 mM, pH 8.0). Autoclave the resuspended fecal microorganism suspension at 121℃ for 30 min to obtain a clear liquid. Centrifuge the autoclaved suspension at 10000g for 5 min, collect the supernatant, and obtain a clear liquid. The untreated fecal supernatant is turbid. Compare the impurity removal effects with Methods A and B. The results are as follows: Figure 4 As shown.

[0089] 2. Remove impurities and pigments to prevent adsorption of the target analyte DPA, as this would interfere with detection. Adsorption experiments were conducted using different impurity removal methods: Method A: Standard DPA at concentrations of 0.5 mM and 0.05 mM was placed in macroporous resin (after ethanol removal) and vortexed for 20 min. 50 μL of the macroporous resin-treated standard DPA solution and terbium chloride (10 mM) solution were added sequentially to black 96-well fluorescent plates in duplicate. The multi-mode microplate reader was set to excitation wavelength 275 nm, excitation wavelength 543 nm, time-resolved fluorescence integration time 1.25 ms, and delay 0.1 ms. Fluorescence intensity was measured. Method A: Standard DPA at concentrations of 0.5 mM and 0.05 mM was added to macroporous resin (after ethanol removal) and excitation wavelength 0.05 mM. Method B involves preparing 0.5 mM and 0.05 mM DPA solutions in activated carbon and vortexing for 20 min. Then, 50 μL of the DPA solution and 10 mM terbium chloride solution are added sequentially to a black 96-well fluorescent plate in duplicate. The detection conditions for the multi-mode microplate reader are set to excitation wavelength 275 nm, excitation wavelength 543 nm, time-resolved fluorescence integration time 1.25 ms, and delay 0.1 ms. Fluorescence intensity is then measured. Method B involves preparing 0.5 mM and 0.05 mM DPA solutions in activated carbon and vortexing for 20 min. Then, 50 μL of the DPA solution and 10 mM terbium chloride solution are added sequentially to a black 96-well fluorescent plate in duplicate. The detection conditions for the multi-mode microplate reader are set to excitation wavelength 275 nm, excitation wavelength 543 nm, time-resolved fluorescence integration time 1.25 ms, and delay 0.1 ms. Fluorescence intensity is then measured. Figure 5As shown: In Method A, the impurity removal process does not affect DPA concentrations of 0.5 and 0.05 mM. In Method B, the DPA content is greatly affected in a short period of time, and its concentration will decrease rapidly. Therefore, we chose the A technical route for subsequent validation of the rapid screening methodology for spores in feces.

[0090] Example 4 verifies the accuracy of the rapid detection kit for fecal microorganisms spores. Thirteen fecal samples were collected from healthy individuals. After being fully suspended in RO water, the samples were centrifuged at 300g for 5 minutes for preliminary treatment. The supernatant was collected and divided into two equal portions. The supernatant was then processed using two different methods.

[0091] 1. Fecal microbiota spores were detected using a rapid fecal microbiota spore detection kit. The pre-treated supernatant was placed in a pre-ethanol-free macroporous resin, vortexed for 20 min, centrifuged at 300 g for 5 min, and the supernatant was collected. The collected supernatant was then centrifuged at 8800 g for 5 min, and the precipitate was collected. The collected precipitate was resuspended in Tris-HCl buffer (1M, pH 8.0). The resuspended fecal microbiota spore suspension was autoclaved at 121℃ for 30 min. The sterilized fecal microbiota spore suspension was centrifuged at 10000 g for 5 min, and the supernatant was collected. The terbium chloride stock solution was diluted to 10 mM using sodium acetate solution. The fecal microbiota spore supernatant was added to an equal volume of 10... 50 μL of sterilized spore suspension and 10 mM terbium chloride solution were added sequentially to a black 96-well fluorescent plate with duplicate wells. The detection conditions of the multi-mode microplate reader were 275 nm excitation wavelength, 543 nm excitation wavelength, time-resolved fluorescence integration time of 1.25 ms, and delay of 0.1 ms. The fluorescence intensity was then detected.

[0092] 2. Fecal microorganism spores were counted using a conventional dilution plating method. The supernatant of the initial supernatant was centrifuged at 8800g for 5 min to collect the precipitate, resuspended in RO water, and washed twice. The collected precipitate was resuspended in 50% ethanol and incubated at 37℃ for one hour. After centrifugation at 8800g for 5 min to remove the ethanol, the precipitate was collected and diluted with sterile RO water. The diluted fecal microorganism spore suspension was then thermally activated at 65℃ for 30 min. Sterile bovine bile powder was added to bring the final concentration to 0.5%, and the mixture was thoroughly mixed. 200 μL of the suspension was evenly spread on RCM solid medium and anaerobically cultured for 48 h. Single colonies formed after spore germination were counted.

[0093] 3. The results of the rapid fecal microbiota spore detection kit were compared with those of the spore germination using the dilution plating method. The specific results are shown in the table below:

[0094] As shown in the table above, when the DPA content in feces is detected using this rapid fecal spore detection kit, samples with a fluorescence value below 30,000 have a spore germination rate below 100. This kit can complete the initial screening of feces and, according to the screening criteria, specify the rejection ratio for each batch of samples to guide donor screening.

[0095] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

[0096] This background section is provided to generally present the context of the invention. The work of the currently named inventors, the work to the extent described in this background section, and aspects of this section that did not constitute prior art at the time of application are neither expressly nor impliedly acknowledged as prior art to the invention.

Claims

1. A strain of double-fermented Clostridium parahaemolyticus ( Paraclostridium bifermentans Application of S1-GT1 in quantitative detection of spore content or quantitative detection of fecal spore population content.

2. The application according to claim 1, characterized in that: In application, the spore component DPA reacts with lanthanide compounds to generate fluorescence, thereby quantitatively detecting the spore content or fecal spore population content; during detection, the excitation wavelength is 275 nm, the excitation wavelength is 543 nm, the integration time is 1.25 ms, the delay is 0.1 ms, and the fluorescence intensity is detected; the lanthanide compound is terbium chloride.

3. A rapid method for detecting fecal spore count, characterized in that, Includes the following steps: 1) Establishment of the DPA standard curve; 2) Establishment of the spore standard curve; 3) Pre-treatment of feces; 4) Prepare a reagent for detecting fecal spore content and test the fecal spore content of the feces after pretreatment in step 3).

4. The rapid detection method for fecal spore count according to claim 3, characterized in that: In step 1), the specific steps for establishing the DPA standard curve are as follows: a1. Use Tris–HCl buffer to gradient dilute the standard DPA stock solution to a minimum concentration of 0 mM to obtain the standard DPA solution; b1. Dilute the terbium chloride mother liquor to 10 mM using sodium acetate solution to obtain a 10 mM terbium chloride reagent; c1. Add the 10 mM terbium chloride reagent from b1 to an equal volume of diluted standard DPA solution; d1. Time-resolved fluorescence was used to detect the fluorescence intensity of the substance obtained in c1, with an excitation wavelength of 275 nm, an integration time of 1.25 ms, and a delay of 0.1 ms. e1. Plot the DPA standard curve based on the fluorescence intensity in d1.

5. The rapid detection method for fecal spore count according to claim 3, characterized in that: In step 2), the specific steps for establishing the spore standard curve are as follows: a2. The standard spore stock solution was serially diluted with Tris–HCl buffer to a minimum concentration of 0 mM to obtain the standard spore suspension; b2. The standard spore suspension in a2 is autoclaved at 121°C for 30 min to obtain a sterilized standard spore suspension. c2. Dilute the terbium chloride mother liquor to 10 mM using sodium acetate solution to obtain a terbium chloride reagent with a concentration of 10 mM; d2. Add the 10 mM terbium chloride reagent from c2 to the sterilized standard spore suspension obtained in b2. e2. Time-resolved fluorescence was used to detect the fluorescence intensity of the substance in d2, with an excitation wavelength of 275 nm, an integration time of 1.25 ms, and a delay of 0.1 ms. f. Plot the standard curve of spores based on the e2 fluorescence intensity.

6. The rapid detection method for fecal spore count according to claim 3, characterized in that: In step 3), the specific steps for pre-treatment of feces are as follows: a3. Collect a fecal sample of not less than 0.1g; b3. The fecal sample in a3 was thoroughly suspended in 10 times its weight of RO water to obtain a fecal suspension; c3. Centrifuge the fecal suspension obtained in b3 for 5 minutes and collect the supernatant; d3. Place the supernatant obtained in c3 into a macroporous resin that has been pre-removed of ethanol, shake for 20 min, centrifuge for 5 min, and collect the supernatant; e3. Centrifuge the supernatant obtained in d3 for 5 min, and resuspend it in 1 M Tris-HCl buffer at pH 8.0 to obtain a resuspended fecal spore suspension. f3. The resuspended fecal spore suspension obtained in e3 was subjected to high pressure treatment at 121℃ for 30 min to obtain sterilized fecal spore suspension; g3. Centrifuge the sterilized fecal spore suspension obtained in f3 for 5 minutes, collect the fecal spore supernatant, and complete the fecal pretreatment.

7. The rapid detection method for fecal spore count according to claim 3, characterized in that: Step 4), preparing the fecal spore content detection reagent, and the specific steps for detecting the fecal spore content of the pretreated feces in Step 3) are as follows: a4. Dilute the terbium chloride stock solution to 10 mM using sodium acetate solution; b4. Add the supernatant of fecal spores to an equal volume of 10 mM terbium chloride solution; c4. Use time-resolved fluorescence with an excitation wavelength of 275 nm and an integration time of 1.25 ms, and a delay of 0.1 ms to detect fluorescence intensity and acquire the results.

8. The rapid detection method for fecal spore count according to claim 3, characterized in that: The method also includes step 5), which sorts the fluorescence intensity of each sample; the top 70% of fluorescence intensity is considered qualified donor feces, and the bottom 30% is considered unqualified donor feces; this is used for donor screening.

9. A kit for rapid detection of fecal spore count, characterized in that: The kit includes reagents for preparing DPA standard curves, reagents for preparing spore standard curves, fecal pretreatment reagents, and reagents for detecting fecal spore population content.

10. A kit for rapid detection of fecal spore count according to claim 9, characterized in that: The reagents used to prepare the DPA standard curve are: Tris–HCl buffer, 50mM, pH 8.0, 100ml; Terbium chloride stock solution, 30 mM, 1 ml; Standard DPA stock solution, 10mM, 10ml; Sodium acetate trihydrate, 40 mM, 100 ml; The reagents used to prepare the spore standard curve are: Tris–HCl buffer, 50mM, pH 8.0, 100ml; Terbium chloride stock solution, 30 mM, 1 ml; Sodium acetate trihydrate, 40 mM, 100 ml; Standard spore S1-GT1, 1,000,000,000 spores / ml, 1ml; The fecal pretreatment reagent is: RO water, 100ml; 100 ml of macroporous resin stored in 95% ethanol; Tris–HCl buffer, 50mM, pH 8.0, 100ml; The reagent for detecting fecal spore count is: Tris–HCl buffer, 50mM, pH 8.0, 100ml; Terbium chloride stock solution, 30 mM, 1 ml; Sodium acetate trihydrate, 40 mM, 100 ml.

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