Cryptosporidium parvum sporozoite activity detection method and device and application
The ATP bioluminescence detection method solves the problems of long detection cycle, high cost and low throughput of Cryptosporidium microsporidium sporozoite activity detection in existing technologies, and realizes rapid, sensitive and reproducible sporozoite activity assessment and high-throughput drug screening, which is suitable for high-throughput drug screening of Cryptosporidium microsporidium sporozoites.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-30
- Publication Date
- 2026-03-10
AI Technical Summary
Existing technologies cannot rapidly, sensitively, and reproducibly detect the activity of Cryptosporidium microsporidium sporozoites, and are not suitable for high-throughput drug screening, resulting in long detection cycles, high costs, poor repeatability, and an inability to directly assess the activity of sporozoite stage.
The ATP bioluminescence detection method was used to lyse Cryptosporidium microsporidium sporozoites by freeze-thaw treatment and shaking treatment to release ATP. The ATP bioluminescence detection reagent was used to generate a luminescence signal, and the survival rate or activity of sporozoites was collected and quantitatively evaluated. Combined with 96-well culture plates, high-throughput screening was achieved.
It enables rapid, direct, and quantitative assessment of the sporozoite stage, reducing detection time to within ten minutes. It has high signal sensitivity, is suitable for high-throughput screening, and can detect sporozoite numbers down to the hundreds. It forms a standardized and scalable kit product, improving drug discovery efficiency.
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Figure CN121629010A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological detection and drug screening, and particularly relates to a method and device for detecting activity of micro-cryptosporidium sporozoite and application. BACKGROUND
[0002] Cryptosporidium parvum is an important intestinal protozoan parasite, and its infection can cause severe diarrhea, dehydration, electrolyte imbalance and even death, especially in immunocompromised individuals and infants. As an opportunistic pathogen, Cryptosporidium has the following biological characteristics: its life cycle includes egg capsules that are transmitted through the external environment, sporozoites that are released in the host intestinal cavity, and subsequent multiple rounds of asexual and sexual multiplication stages in host cells. However, there are few effective drugs for Cryptosporidium in clinical practice, and there is a lack of efficient, low-cost, and high-throughput screening in vitro evaluation system. Since sporozoite is the earliest stage of infection and an important target for drug action, the detection of its activity is the core of the drug screening system.
[0003] The existing in vitro activity detection and drug evaluation methods for Cryptosporidium parvum mainly include qRT-PCR counting method, high-content imaging method (HCS), host cell infection model, and metabolite-based fluorescence method. These methods have certain effects, but they generally have the following structural and functional defects, which seriously limit their use in high-throughput drug screening. Specifically, the main shortcomings of the existing technology include: 1) long detection period, which cannot achieve rapid evaluation of parasite activity: the most commonly used qRT-PCR method and host cell infection model both require 24-72h of infection incubation time, followed by cell lysis, RNA extraction, reverse transcription, and qPCR amplification, resulting in a complex and lengthy process that is not suitable for determining the direct killing effect of drugs on parasites in a short period of time; 2) high experimental cost, difficult to standardize and subject to experimental condition fluctuations: the qRT-PCR method requires high-cost consumables such as fluorescent dyes, high-content imaging requires expensive automated imaging systems and antibody labeling, and the host cell growth state (density, passage number) has a significant impact on the results, with poor reproducibility and difficulty in forming a unified, commercialized, and generalizable standard system. These shortcomings make the above methods unsuitable for industrialized screening processes; 3) cannot directly evaluate sporozoite stage activity: most methods rely on host cells as an infection medium to evaluate the proliferation of parasites in host cells, rather than the activity of sporozoites themselves. However, sporozoites, as the earliest stage of infection, are the most sensitive and suitable targets for initial screening, and the existing technology cannot achieve rapid and direct activity determination. In addition, some reagents that can detect cell viability, such as resazurin, rely on metabolic reduction, but Cryptosporidium sporozoites have very low metabolic levels, so their sensitivity is not high and they are not suitable for understanding short-term dynamic changes in parasites.
[0004] ATP bioluminescence detection is based on the reaction of luciferase catalyzing luciferin to produce photons with the participation of ATP, and its signal can theoretically reflect the activity of the organism. This method has been used for the activity detection of some parasites (such as Giardia and Amoeba), but there is no stable, sensitive and applicable ATP bioluminescence system for Cryptosporidium oocysts for drug screening.
[0005] In addition, the application limitations of ATP detection in the study of Cryptosporidium activity in the prior art are: 1) There is no ATP activity detection system for Cryptosporidium oocysts: the number of Cryptosporidium oocysts is small, the metabolism is low, the life cycle is short, and the structure is special, which makes the requirement for ATP detection sensitivity higher; 2) Most of the existing kits are designed for bacteria or mammalian cells: they are not optimized for the physical properties (such as membrane structure and intracellular ATP release efficiency) of parasitic oocysts; 3) The time of fusion, lysis and incubation, background ATP and other factors have not been optimized: which leads to unstable signal and poor repeatability, and is not suitable for direct large-scale drug evaluation.
[0006] Therefore, it is a major demand in the field of parasitic drug development to develop a rapid, sensitive, repeatable, simple and convenient Cryptosporidium oocyst activity detection system suitable for high-throughput drug screening. SUMMARY
[0007] The purpose of the present application is to provide a method and device for detecting the activity of Cryptosporidium parvum oocysts and its application, in order to solve the problems raised in the background art.
[0008] To achieve the above-mentioned purpose, the present application provides the following technical solutions:
[0009] A method for detecting the activity of Cryptosporidium parvum oocysts, comprising the following steps:
[0010] Providing a test sample containing dead or live Cryptosporidium parvum oocysts, and diluting and / or freeze-thawing the test sample by gradient, and then adding it to a detection container;
[0011] Adding ATP bioluminescence detection reagent to the detection container for shaking treatment, so as to lyse and release ATP from Cryptosporidium parvum oocysts, and generate a luminescence signal by reaction;
[0012] Collecting the luminescence signal, and quantitatively evaluating the survival rate or activity of Cryptosporidium parvum oocysts according to the intensity of the luminescence signal.
[0013] Further, the detection container is a 96-well culture plate.
[0014] Further, the freeze-thaw treatment procedure is liquid nitrogen-room temperature cycling, and the number of cycles is 1-10.
[0015] Further, the shock treatment time is 0.5-10 min.
[0016] Further, the shock treatment uses a 731 cpm shock plate procedure.
[0017] Further, the reaction time is 5-15 min.
[0018] Another object of the present application is to provide an application of the above-mentioned Cryptosporidium parvum oocyst activity detection method in high-throughput drug screening of Cryptosporidium parvum oocysts.
[0019] Further, the method for high-throughput drug screening of Cryptosporidium parvum oocysts comprises the following steps:
[0020] Prepare Cryptosporidium parvum oocysts, resuspend them in sterile PBS to obtain an oocyst sample, and add them to a detection container;
[0021] Add the drug to be screened and dimethyl sulfoxide to the detection container for incubation to obtain an incubation system;
[0022] Add an ATP bioluminescence detection reagent to the incubation system for shock treatment to lyse Cryptosporidium parvum oocysts and release ATP, and perform a reaction to generate a luminescence signal;
[0023] Collect the luminescence signal, and quantitatively evaluate the survival rate or activity of Cryptosporidium parvum oocysts according to the intensity of the luminescence signal;
[0024] According to the evaluation results of the survival rate or activity of Cryptosporidium parvum oocysts, candidate drugs are screened from the drugs to be screened;
[0025] Verify the effect of the candidate drugs to determine drugs with anti-Cryptosporidium activity.
[0026] Further, the final concentration of dimethyl sulfoxide is not more than 2%, and the incubation time is 0.5-4 h.
[0027] Another object of the present application is to provide a device for high-throughput drug screening of Cryptosporidium parvum oocysts, which is used to realize the above-mentioned application, and comprises:
[0028] A detection container;
[0029] A sample preparation module for preparing Cryptosporidium parvum oocysts, resuspending them in sterile PBS to obtain an oocyst sample, and adding them to the detection container;
[0030] an incubation module for incubating the oocyst sample, the drug to be screened, and dimethyl sulfoxide to obtain an incubation system;
[0031] an ATP bioluminescence detection module for adding ATP bioluminescence detection reagents to the incubation system and performing shock treatment, so that the Cryptosporidium parvum oocysts are lysed to release ATP, and a luminescence signal is generated through reaction;
[0032] a luminescence signal analysis module for collecting the luminescence signal and quantitatively evaluating the survival rate or activity of the Cryptosporidium parvum oocysts according to the intensity of the luminescence signal;
[0033] a drug screening module for screening candidate drugs from the drug to be screened according to the evaluation result of the survival rate or activity of the Cryptosporidium parvum oocysts;
[0034] a candidate drug verification module for verifying the effect of the candidate drugs and determining the drug with anti-Cryptosporidium activity.
[0035] The Cryptosporidium parvum oocyst activity detection method provided by the application can realize direct, rapid, and quantitative evaluation of the activity of the oocyst stage in a short time based on the ATP bioluminescence principle, and can be used for high-throughput screening of large-scale compound drug libraries. The method has the following advantages: high signal sensitivity, wide linear range, short reaction time (about ten minutes), simple principle, no dependence on cell infection, expensive equipment, or complex staining, ability to detect as low as hundreds of oocysts, ability to be used in a primary screening system of thousands of compounds, and standardization and expansion into a kit product. BRIEF DESCRIPTION OF DRAWINGS
[0036] Figure 1 A method flowchart for high-throughput drug screening of Cryptosporidium parvum oocysts provided by an embodiment of the application.
[0037] Figure 2 A quantitative result graph of Cryptosporidium oocyst activity based on ATP bioluminescence; in the graph, A is a linear relationship graph between Cryptosporidium oocysts and ATP bioluminescence signal intensity; B is an ATP bioluminescence signal and 18S rRNA-based qPCR Ct value correlation graph; and C is a correlation analysis result of ATP bioluminescence signal intensity and qPCR Ct value.
[0038] Figure 3 An ATP bioluminescence detection system parameter optimization experiment result graph; in the graph, A is the influence of freeze-thaw cycle times on oocyst ATP signal; B is the influence of shock mixing time on ATP oocyst ATP release; and C is a curve graph of ATP luminescence signal stability changing with time.
[0039] Figure 4Line graph showing changes in ATP signal in sporozoite samples with different survival ratios (mixture of live and dead spores).
[0040] Figure 5 The figure shows the results of the optimization experiment on incubation time and solvent tolerance in the detection system; in the figure, A is the change of ATP signal of sporozoites under different incubation times; B is the effect of different final concentrations of DMSO on the ATP signal of sporozoites.
[0041] Figure 6 This diagram illustrates the establishment of a high-throughput insecticide screening system (HTS) targeting Cryptosporidium sporozoites. In the diagram, A represents the Z′-factor value and S / B value in the HTS system; B represents the preliminary screening results of 5000 compounds, with 14 candidate compounds obtained from the preliminary screening marked in red; C represents the summary of the preliminary and secondary screening results of the 14 compounds, with candidate drugs from the secondary screening marked in red; and D is a schematic diagram of the screening method.
[0042] Figure 7 Figure 1 shows the results of the in vitro insecticidal efficacy verification of the candidate compounds; in the figure, A is the effect-concentration curve of the in vitro insecticidal efficacy of the candidate compounds; B is a summary of the insecticidal efficacy and cytotoxicity of the candidate compounds; C is the inhibitory effect of the candidate compounds on the ability of sporozoites to invade host cells.
[0043] Figure 8 The figure shows the in vivo efficacy verification results of the candidate compound in an infected mouse model. In the figure, A is a schematic diagram of the in vivo drug efficacy evaluation experiment in the Cryptosporidium mouse infection model; B is the survival rate curve of mice in different treatment groups; C is the weight change curve of infected mice; D is the graph of the change in the amount of parasite excreted in feces after infection; E is a schematic diagram of the histopathological (H&E) results of mouse small intestinal tissue. Detailed Implementation
[0044] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0045] The present invention aims to address the problems of high cost, long cycle, low throughput, and difficulty in standardization in existing in vitro drug screening methods for Cryptosporidium, as well as the lack of a suitable method for directly evaluating the activity changes of sporozoites over a short period of time.
[0046] Specifically, in one embodiment of the present invention, a method for detecting the activity of Cryptosporidium microsporidium sporozoites is provided, comprising the following steps: providing a test sample containing dead or live Cryptosporidium microsporidium sporozoites, and adding the test sample to a detection container after serial dilution and / or freeze-thaw treatment; adding ATP bioluminescent detection reagent to the detection container and shaking it to cause Cryptosporidium microsporidium sporozoites to lyse and release ATP, and react to generate a luminescent signal; collecting the luminescent signal and quantitatively assessing the survival rate or activity of Cryptosporidium microsporidium sporozoites based on the intensity of the luminescent signal.
[0047] Specifically, the testing container was a 96-well culture plate; the freeze-thaw treatment procedure was liquid nitrogen-room temperature cycling, with 1-10 cycles; the shaking treatment time was 0.5-10 min; the shaking plate program used for the shaking treatment was 731 cpm (2 mm); and the reaction time was 5-15 min.
[0048] like Figure 1 As shown, in another embodiment of the present invention, a method for high-throughput drug screening of Cryptosporidium microsporidium sporozoites is also provided, specifically including the following steps:
[0049] S1. Prepare decospores of Cryptosporidium microsporum, resuspend them in sterile PBS to obtain sporospore samples, and add them to the detection container;
[0050] S2. Add the drug to be screened and dimethyl sulfoxide (DMSO) to the detection container and incubate to obtain the incubation system; wherein, the final concentration of dimethyl sulfoxide is not greater than 2%; the incubation time is 0.5-4h;
[0051] S3. Add ATP bioluminescent detection reagent to the incubation system and shake to cause Cryptosporidium microsporidium sporozoites to lyse and release ATP, and then react to generate a luminescent signal.
[0052] S4. Collect the luminescence signal and quantitatively assess the survival rate or activity of Cryptosporidium microlucrificum sporozoites based on the intensity of the luminescence signal;
[0053] S5. Based on the assessment results of the survival rate or activity of Cryptosporidium microsporidium sporozoites, candidate drugs are screened from the drugs to be screened.
[0054] S6. Verify the efficacy of candidate drugs to identify those with anti-cryptosporidiosis activity.
[0055] In another embodiment of the present invention, a device for high-throughput drug screening of Cryptosporidium microsporidium sporozoites is also provided for implementing the above method, comprising:
[0056] Container for testing;
[0057] The sample preparation module is used to prepare decospores of Cryptosporidium microphyllum, which are resuspended in sterile PBS to obtain sporospore samples and added to the detection container;
[0058] The incubation module is used to incubate sporozoite samples, the drug to be screened, and dimethyl sulfoxide to obtain an incubation system;
[0059] The ATP bioluminescence detection module is used to add ATP bioluminescence detection reagent to the incubation system and shake it to cause Cryptosporidium microsporidium sporozoites to lyse and release ATP, and then react to generate a luminescent signal.
[0060] A luminescence signal analysis module is used to collect the luminescence signal and quantitatively assess the survival rate or activity of Cryptosporidium microlucgiformes sporozoites based on the intensity of the luminescence signal.
[0061] The drug screening module is used to screen candidate drugs from the drugs to be screened based on the assessment results of the survival rate or activity of Cryptosporidium microsporidium sporozoites.
[0062] The candidate drug validation module is used to validate the efficacy of candidate drugs and identify drugs with anti-cryptosporidosis activity.
[0063] In this invention, an ATP-based bioluminescence method for detecting Cryptosporidium microsporidium sporozoite activity is established, capable of directly reflecting sporozoite survival in a very short time (approximately ten minutes). A detection system with stable signal, high sensitivity, strong repeatability, and suitability for standardized operation is provided. By optimizing key steps such as sporozoite quantity, oscillatory lysis conditions, incubation time, and DMSO tolerance range, accurate and controllable bioluminescence readings are achieved. This forms a high-throughput screening (HTS) method suitable for 96-well or even higher throughput platforms, enabling rapid screening of large-scale drug compounds. Using this system to screen Cryptosporidium inhibitors improves drug discovery efficiency and establishes a cross-platform, low-cost, and highly consistent early drug screening technology for parasites.
[0064] Example 1: Validation of the correlation between sporozoite number and ATP quantitative signal
[0065] This embodiment provides a rapid and sensitive quantitative method for detecting the activity of Cryptosporidium microsporidium sporozoites. This method uses a commercially available ATP bioluminescent assay reagent, which quantitatively reflects the number of viable sporozoites in a sample by detecting the relative luminescent units (RLU) of the reaction system. The selected cell viability assay kit differs from previous kits targeting mammalian cells; this kit possesses ultra-high sensitivity and an ultra-wide linear quantitative range, and can be widely used to detect the activity of microbial cells such as bacteria and fungi, and can be applied to the screening of antimicrobial compounds. Procedure: Sporozoites are serially diluted and added to a 96-well black culture plate, 100 μL per well; an equal volume of ATP bioluminescent assay reagent is added; after vortexing and mixing, the relative luminescent units (RLU) are immediately detected using a fluorescence microplate reader.
[0066] The results are as follows Figure 2 As shown: There is a linear relationship between different numbers of sporozoites and the corresponding ATP luminescence signals. Figure 2 The A value indicates that the ATP signal can serve as a quantitative basis for activity analysis. The ATP signal shows a high degree of consistency with the Ct value of traditional 18S rRNA qPCR (…). Figure 2 B and Figure 2 The C value indicates that ATP readings can replace molecular biological indicators for evaluating parasite activity. The system provided in this invention avoids the cumbersome steps of RNA extraction and reverse transcription required by traditional qPCR, significantly improving detection speed and throughput.
[0067] Example 2: Optimization of key parameters of ATP bioluminescence detection system
[0068] To make this ATP detection reagent and detection process suitable for the special structure and small size of Cryptosporidium microsporidium sporozoites, the embodiments of this invention have systematically optimized multiple influencing factors to ensure the best detection signal.
[0069] The experimental procedure for this part is as follows: 1) First, the effect of freeze-thaw treatment on ATP release from Cryptosporidium sporozoites was investigated: Because the sporozoite membrane is different from that of mammalian cell membranes, the lysis buffer in the kit may have limited lysis. Therefore, in this embodiment of the invention, freeze-thaw treatment was added before detection to maximize ATP release. The freeze-thaw program set in this embodiment of the invention was liquid nitrogen-room temperature cycling, and 0, 2, 4, 6, 8, and 10 freeze-thaw cycles were set respectively. The detection was then performed immediately, and the differences in ATP signal intensity among the groups were compared. 2) Standardized setting of shaking time after mixing sample and detection reagent: Shaking and mixing the sample and detection time is a key factor in the effective lysis of the sample and the release of ATP. Therefore, finding the optimal mixing time is the key to obtaining a true ATP fluorescence detection signal. Therefore, this embodiment of the invention compares the effect of shaking time of 0-10 minutes on the final fluorescence signal intensity. This embodiment of the invention uses the shaking program built into the Biotek multi-functional fluorescence microplate reader - 731 cpm (2 mm). After adding an equal volume of detection reagent to the sample, shaking is performed immediately at different time ranges, and the fluorescence value is directly read and compared after the shaking program ends. 3) Engineering optimization of luminescence signal stability: In order to ensure that the maximum ATP fluorescence signal can be detected, this embodiment of the invention further systematically evaluates the time-dependent intensity change of the ATP luminescence system. This embodiment of the invention performs fluorescence signal detection within a period of 0-120 minutes after the sample and detection time are fully shaken and mixed, and interprets the signal stability results.
[0070] The results are as follows Figure 3 As shown: The results of the freeze-thaw cycle test indicate that, compared to the non-freeze-thaw group, two freeze-thaw cycles significantly enhanced ATP release, but further increases in the number of freeze-thaw cycles did not increase the fluorescence signal. This may be related to the degradation of ATP during freeze-thaw cycles. Figure 3 (A). Furthermore, although the ATP fluorescence detection signal of untreated sporozoites is lower than that of frozen-thawed sporozoites, the generated fluorescence signal is sufficient for subsequent viability determination. Moreover, to adapt to high-throughput operations, reduce processing steps, and avoid additional data fluctuations, this embodiment of the invention omits the freeze-thaw step in the later drug screening process. This significantly improves the stability, reproducibility, and industrial operability of the method. The results of the shaking time study indicate that shaking for 2 minutes is sufficient to fully rupture the sporozoite structure and release ATP; shaking for less than 1 minute or more than 5 minutes leads to a decrease or instability in signal levels. Figure 3 (B). Therefore, the optimal oscillation time was determined to be 2 minutes in this embodiment of the invention. The time stability results of the ATP luminescence system in this embodiment of the invention show that the signal increases slightly from 0 to 10 minutes after the reaction, and the signal continuously decreases from 10 to 120 minutes. Figure 3(C). Therefore, the optimal detection time is determined in this embodiment of the invention to be within 10 minutes after the addition of the detection reagent.
[0071] Example 3: Quantitative calibration of the activity of mixed live and dead sporozoites
[0072] To ensure that the ATP signal accurately reflects the proportion of viable sporozoites, this invention constructs a mixed live / dead sporozoite sample. Cryptosporidium sporozoite inactivation samples are obtained by resuspending sporozoites in PBS, incubating them in an 80°C water bath for 20 minutes, and then allowing them to stand at room temperature for 30 minutes, ultimately obtaining a dead sporozoite sample. Live sporozoite samples refer to freshly decapitated sporozoite samples, with a default viability of 100%. This invention mixes live and dead sporozoites in ratios of 0:10, 2:8, 4:6, 6:4, 8:2, and 10:0 to ensure a consistent total number of sporozoites, and performs ATP fluorescence signal detection under the aforementioned optimal detection conditions, obtaining a linear graph of the live / dead sporozoite ratio versus signal intensity. The results are as follows: Figure 4 As shown, the results indicate that the ATP signal decreases linearly with the decrease in the survival rate. Although trace amounts of residual ATP are retained in dead sporozoites, this does not affect the overall linear trend. The results in this section demonstrate that the embodiments of the present invention are sensitive in detecting sporozoite viability and can serve as an important calibration method for determining drug inhibition rates, laying the foundation for further drug screening.
[0073] Example 4: Optimization of DMSO tolerance and drug incubation time
[0074] To ensure the detection method established in this invention is applicable to subsequent drug screening processes, this invention first detects the activity-time curve of sporozoites under room temperature incubation. Because Cryptosporidium sporozoites cannot survive long-term in vitro in cell-free systems, determining the optimal incubation time for the drug is crucial to ensure the compound exerts its full effect. In this invention, freshly decapitated sporozoites were resuspended in PBS, and equal amounts of sporozoites were placed at room temperature. Incubation was stopped at 0, 0.5, 1, 2, 3, 4, 5, 6, 9, 12, 24, and 48 hours, and ATP signal was detected. Next, this invention conducted a tolerance study on the DMSO concentration in the system. DMSO is a common solvent for most compounds, and sporozoite activity is affected by DMSO. Therefore, during the incubation of sporozoites with the drug, it is essential to ensure that the DMSO concentration does not affect sporozoite activity to accurately reflect the drug's effect. Therefore, this invention investigated the changes in ATP signal intensity of sporozoites after incubation at room temperature for 3 hours with different concentrations of DMSO (0.25%-8%).
[0075] The results are as follows Figure 5As shown: During the 0-4 hour incubation period at room temperature, the ATP signal remained stable, but after 5 hours of incubation, natural death occurred, resulting in a significant decrease in the ATP signal. Figure 5 (A). To ensure optimal stability of signal detection and authenticity of results, the incubation time for the drug was ultimately determined to be 3 hours in this embodiment of the invention, minimizing false positives caused by the natural death of parasites. The effect of DMSO concentration on sporozoite activity results showed that when the DMSO concentration was ≤2%, it had minimal impact on sporozoites, did not produce significant fluctuations in ATP signals, and maintained a baseline activity level (…). Figure 5 (B) When the DMSO concentration rises above 2%, the ATP signal is significantly reduced, indicating that the high concentration of DMSO has caused the death of sporozoites. Therefore, in the later stages of drug screening, the DMSO concentration can be controlled within 2%. Since the initial concentration of the compound library drugs in this embodiment of the invention is 10mM, the required amount of drug stock solution is very low. Therefore, in the screening process of this embodiment of the invention, DMSO and drugs are used at concentrations below 0.5%.
[0076] Example 5: Establishment of a high-throughput drug screening (HTS) system based on the system of the present invention.
[0077] Based on the optimal conditions for detecting Cryptosporidium sporozoite activity using the ATP fluorescence method, this invention establishes a rapid sporozoite activity screening platform applicable to 96-well plates and 5000 compounds. In this invention, fresh decospores are first prepared, resuspended in sterile PBS, and added to 96-well black cell culture plates, with 4*10⁴ spores added to each well. 5 Ascospores, 100 μL in volume. Then, the compound to be screened was added to each well to a final concentration of 40 μM, ensuring a final DMSO concentration of 0.5%. The plates were shaken at medium speed for 1 minute on a plate shaker, sealed with a micropermeable sealing membrane, and incubated at room temperature for 3 hours. Subsequently, 100 μL of ATP luminescent assay reagent was added, shaken for 2 minutes, and allowed to stand at room temperature for 10 minutes before measuring the fluorescence signal using a fluorescence microplate reader.
[0078] The results are as follows Figure 6 As shown: In this embodiment of the invention, the stability of the screening system was first evaluated. The calculated Z′-factor was 0.73, and the signal-to-background ratio (S / B) was 9.67, indicating that the screening system of this embodiment has good stability and significant signal differences. Figure 6 (A). In the preliminary screening at high concentrations in this embodiment of the invention, among 5,000 compounds, 14 drugs showed a sporozoite activity inhibition rate of over 60% ( Figure 6 B). In a secondary screening at low concentrations (4 μM), five highly active inhibitors were ultimately identified. Figure 6(C). Finally, based on the optimized ATP fluorescence activity method, this embodiment of the invention determined the Cryptosporidium sporozoites as the target for drug screening in the anti-Cryptospora drug screening system (C). Figure 6 (D).
[0079] Example 6: Evaluation of the in vitro insecticidal effect of candidate compounds
[0080] In this embodiment of the invention, five candidate compounds obtained through two rounds of HTS were used to further evaluate their in vitro anti-Cryptospora efficacy. In vitro drug efficacy was assessed by a 44-hour infection test combined with qRT-PCR to determine parasite load. Briefly, human ileocecal adenocarcinoma cells (HCT-8; ATCC CCL244) were seeded into 96-well plates and cultured to 80% confluence in RPMI-1640 medium supplemented with 10% fetal bovine serum. Then, bleached and washed Cryptosporidium oocysts (2 × 10⁻⁶) were used to detect the parasite load. 4 HCT-8 cells were infected with oocysts (per well). To optimize decapsulation and infection, oocysts were added to complete medium supplemented with 0.15% taurine. After incubation at 37°C for 3 hours, uninfected parasites were removed by changing the medium. Infected cells were then incubated with the selected compound at 37°C for another 41 hours. After incubation, 96-well plates were centrifuged, and 100 μL of 1 mg / mL bovine serum albumin (BSA) solution was added to each well. Cell lysis was then performed using a multiplex vortex mixer. The relative levels of parasite 18S rRNA and human 18S rRNA were detected using the HiScript II one-step qRT-PCR SYBR Green kit. For the detection of Cryptosporidium microsporidium in vitro invasion inhibition, HCT-8 cells with confluence >90% were seeded in 96-well plates, pretreated with the compound for 1 hour, and then infected with the parasite. Then, the oocysts were added to a monolayer of HCT-8 cells containing the compound or DMSO and incubated at 37°C for 3 hours. Afterward, the plates were washed three times with sterile PBS and analyzed according to the same protocol described above.
[0081] The results are as follows Figure 7 As shown: qRT-PCR results indicate that, except for kojic acid, all five candidate compounds exhibited varying degrees of in vitro insecticidal activity. Figure 7 (A), and determined the median inhibitory concentration (MIC) of these four compounds (A). Figure 7 (B). Since sporozoites represent the invasion stage of Cryptosporidium, this invention further investigated the effects of the screened compounds on Cryptosporidium invasion. The results showed that all five compounds significantly induced Cryptosporidium invasion in vitro. Figure 7 (C).
[0082] Example 7: Animal Model Validation
[0083] This invention utilizes IFN-γ gene knockout mice (B6.129S7-Ifngtm1Ts / J) (IFN-γ- / -) to establish an in vivo Cryptosporidium infection model. Six 6-8 week old female IFN-γ- / - mice were infected via oral gavage. Each group consisted of six mice, and the gavage solution was 200 μL of sterile PBS containing 5 × 10⁻⁶ mg / L of sterile PBS. 4 Cryptosporidium oocysts expressing Nluc were identified. Mice were administered the designated compound once daily via intraperitoneal injection or gavage from day 3 to day 9 post-infection. During treatment, mouse body weight and fecal samples were collected daily. After treatment, mouse body weight and fecal samples were collected every two days. Data were recorded weekly from day 21 to day 35 post-infection. Figure 8 (A) At the end of the experiment, the animals were euthanized with sodium pentobarbital (50 mg / kg). Ileal tissue samples were collected, fixed in formalin, embedded in paraffin, and cut into 5 μm thick sections. The sections were stained with hematoxylin and eosin and observed under an optical microscope (Olympus Corporation, Japan).
[0084] The results are as follows Figure 8 As shown: Mice were treated with Cryptosporidium oocysts via oral gavage for 3 days prior. The load of Cryptosporidium oocysts in mouse feces was determined by monitoring the activity of parasite-derived luciferase. Figure 8 As shown in B, infected control mice began to die early (day 8) and exhibited a high mortality rate (50%) at the end of the experiment (day 35). At the end of the experiment, the mortality rate of mice treated with ZL0420 and SIB1757 was 16.7%, while mice treated with sulbactam or abexinolta did not die throughout the study period. In the experiments of this embodiment, mice treated with the vector control group showed continuous weight loss (…). Figure 8 (C). All mice treated to reduce Cryptosporidium infection maintained relatively stable body weight throughout the experiment. As expected, all infected mice began shed detectable Cryptosporidium oocysts in their feces on day 3 post-infection, while the oocyst shedding in the vector control mice gradually increased until day 9 post-infection. Oocyst counts remained consistently lower in the vector control mice after treatment with compound ZL0420 (10 mg / kg), sulbactam (20 mg / kg), SIB1757 (20 mg / kg), and abexinolta (25 mg / kg) until the end of treatment. Figure 8Consistent with the decreased oocyst count, all treated mice exhibited relatively normal physiological parameters, including activity, posture, and appetite, until day 35 post-infection, compared to the untreated group. After treatment, H&E staining and histopathological examination were performed on distal small intestine sections from mice according to this embodiment of the invention. Small intestine sections from control mice receiving only vector treatment showed significant histopathological changes, including acute inflammation, crypt hypertrophy, blunted villi, and a decreased villi height to crypt length ratio. Figure 8 (E). In contrast, the small intestinal tissue of mice treated with the four compounds showed minimal local inflammation and moderate villus atrophy, as well as an increase in the villus crypt height ratio.
[0085] In summary, the technical solution provided by the embodiments of the present invention has the characteristics of being rapid, highly sensitive, high-throughput, and standardizable, and can effectively solve the key problem that existing technologies cannot meet the needs of parasite drug screening. Specifically, the embodiments of the present invention address the problems of long detection cycles, low throughput, and insensitivity to the sporozoite stage in existing Cryptosporidium activity detection methods by providing a rapid activity evaluation method based on ATP bioluminescence detection and its high-throughput screening system. Compared with the prior art, the embodiments of the present invention have the following significant technical effects: 1) It achieves rapid, direct, and quantitative detection of the sporozoite stage: the detection time is shortened from the traditional 48-72 hours to ≤3 hours, greatly improving the detection efficiency; the ATP signal can directly reflect sporozoite activity, avoiding the delay and interference caused by host cell infection models; and quantifiable results can be obtained without complex sample processing (such as RNA extraction and fluorescence staining). 1) Significantly improves detection speed, suitable for direct activity evaluation in the early stages of parasite invasion; 2) Significantly improves detection sensitivity, with the lowest detectable number of sporozoites as low as 100; 3) Constructs a stable and reproducible standardized detection system, improving experimental repeatability and cross-laboratory consistency, making it suitable for industrialization and kit development; 4) Suitable for high-throughput screening (HTS), supporting large-scale drug library screening, greatly improving the efficiency of anti-cryptosporidosis drug discovery, and achieving rapid, large-scale screening.
[0086] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.
Claims
1. A method for detecting the activity of microsporidian spores, characterized in that, The method comprises the following steps: providing a to-be-tested sample containing dead or live microcystic cryptosporidium sporozoites, diluting the to-be-tested sample by a gradient ratio and / or performing freeze-thaw treatment, and adding the to-be-tested sample into a detection container; adding ATP bioluminescence detection reagents into the detection container to perform shaking treatment, so that the microcystic cryptosporidium sporozoites are lysed to release ATP, and a reaction is performed to generate a luminescence signal; collecting the luminescence signal, and quantitatively evaluating the survival rate or activity of the microcystic cryptosporidium sporozoites according to the intensity of the luminescence signal.
2. The method of claim 1, wherein the microsporidia spore activity is detected by the method comprising the steps of: The detection container is a 96-well culture plate.
3. The method for detecting the sporospora microsporidia sporozoan activity according to claim 1, characterized in that, The freeze-thaw treatment program is liquid nitrogen-room temperature circulation, and the number of cycles is 1-10.
4. The method of claim 1, wherein the microsporidia spore activity is detected by the method comprising the steps of: The shaking treatment time is 0.5-10 min.
5. The method for detecting the activity of microsporidia sporozoite according to claim 1 or 4, characterized in that, The shaking plate program used in the shaking treatment is 731 cpm.
6. The method for detecting the sporospora microsporidia sporozoan activity according to claim 1, characterized in that, The reaction time is 5-15 min.
7. Application of the microcystic cryptosporidium sporozoite activity detection method in any one of claims 1-6 in high-throughput drug screening of microcystic cryptosporidium sporozoites.
8. Use according to claim 7, characterized in that, The method for high-throughput drug screening of microcystic cryptosporidium sporozoites comprises the following steps: preparing microcystic cryptosporidium sporozoites, resuspending the sporozoites in sterile PBS to obtain a sporozoite sample, and adding the sporozoite sample into a detection container; adding to-be-screened drugs and dimethyl sulfoxide into the detection container to perform incubation, so as to obtain an incubation system; adding ATP bioluminescence detection reagents into the incubation system to perform shaking treatment, so that the microcystic cryptosporidium sporozoites are lysed to release ATP, and a reaction is performed to generate a luminescence signal; collecting the luminescence signal, and quantitatively evaluating the survival rate or activity of the microcystic cryptosporidium sporozoites according to the intensity of the luminescence signal; screening candidate drugs from the to-be-screened drugs according to the evaluation result of the survival rate or activity of the microcystic cryptosporidium sporozoites; verifying the effect of the candidate drugs to determine drugs with anti-cryptosporidium activity.
9. Use according to claim 7, characterized in that, The final concentration of the dimethyl sulfoxide is not more than 2%, and the incubation time is 0.5-4 h.
10. A device for high-throughput drug screening of Cryptosporidium parvum oocysts for use in the method of any one of claims 7 to 9, wherein the device comprises: comprise: a detection container; a sample preparation module for preparing microcystic cryptosporidium sporozoites, resuspending the sporozoites in sterile PBS to obtain a sporozoite sample, and adding the sporozoite sample into the detection container; an incubation module for incubating the sporozoite sample, to-be-screened drugs and dimethyl sulfoxide to obtain an incubation system; an ATP bioluminescence detection module for adding ATP bioluminescence detection reagents into the incubation system to perform shaking treatment, so that the microcystic cryptosporidium sporozoites are lysed to release ATP, and a reaction is performed to generate a luminescence signal; a luminescence signal analysis module for collecting the luminescence signal, and quantitatively evaluating the survival rate or activity of the microcystic cryptosporidium sporozoites according to the intensity of the luminescence signal; a drug screening module for screening candidate drugs from the to-be-screened drugs according to the evaluation result of the survival rate or activity of the microcystic cryptosporidium sporozoites; a candidate drug verification module for verifying the effect of the candidate drugs to determine drugs with anti-cryptosporidium activity.