Preparation method, product and application of quality control product for pathologic detection of pathogenic bacteria

Abstract

The invention discloses a preparation method of a pathologic detection quality control product for pathogenic bacteria, which comprises the following steps: mixing target pathogenic bacteria and eukaryotic cells with curable hydrogel according to a required ratio to form a bacteria-containing biological matrix, and curing and forming by using a three-dimensional forming method to form a three-dimensional solid structure; the pathologic detection quality control product containing the microorganism-cell composite structure of the simulated pathological tissue is obtained. The preparation process is stable and controllable, uniform distribution of pathogenic bacteria is guaranteed, ethical problems do not exist, and clinical product transformation can be rapidly carried out. The invention further discloses a product obtained through the preparation method and application of the product. The sample obtained by the method can be used as a standardized positive control sample in the whole pathologic detection process of pathogenic bacteria, and is used for quality control. The invention solves the problem of lack of full-process quality control products in existing pathogenic bacterium detection, and is suitable for full-process quality control of tissue block preparation, pathological section dyeing, molecular detection and the like.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of in vitro diagnostic quality control technology and biomanufacturing, specifically relating to a method for preparing, a product and application of a quality control material for pathological detection of pathogenic bacteria. Background Technology

[0002] In recent years, the global spectrum of infectious diseases has undergone significant changes, exhibiting an increasingly complex trend. Against this backdrop, stable and accurate pathological testing technologies have become a crucial link in disease prevention and control. Improving the accuracy and timeliness of pathogen detection, while reducing the incidence of drug resistance, is essential for achieving early diagnosis and guiding precision medicine.

[0003] Infectious pathology, as a crucial technique for diagnosing infectious diseases caused by pathogens, currently relies primarily on the combined application of multiple methods, including nucleic acid detection (PCR), special staining, immunohistochemistry (IHC), and immunofluorescence. While this multi-technology approach significantly improves the accuracy of pathogen detection and the timeliness of reporting, it still faces numerous challenges in practice. First, improper procedures in the pretreatment steps of tissue sample collection, fixation, and processing in pathology laboratories can directly affect the quality of biomolecules such as nucleic acids and proteins. Second, batch differences in reagents, variations in preparation methods, and laboratory contamination can all introduce detection errors. Furthermore, changes in external conditions such as instrument status and environmental temperature and humidity can also interfere with experimental results. The combined effect of these factors leads to significant differences in positive detection rates between different laboratories, severely impacting the comparability and reliability of test results.

[0004] In the era of precision diagnostics, standards such as ISO 15189:2022 and CLSI EP12 have elevated internal and external validation and quality control in medical testing to a core position. Pathological testing, in particular, requires quality control materials that are "consistent with the matrix of the sample to be tested" throughout the entire process of "fixation—dehydration—embedding—sectioning—staining / molecular detection" to promptly detect false negatives or false positives. However, currently available commercially available quality control materials are still insufficient to meet this requirement. Taking Mycobacterium tuberculosis molecular pathology testing as an example, the current national standard for tuberculosis PCR in my country only provides negative controls and liquid standards at three concentration gradients to assess the reproducibility and accuracy of DNA extraction and amplification. To reduce costs, most kits use self-made plasmid DNA as an internal control for amplification, but this makes it impossible to trace the impact of steps such as tissue fixation, dehydration, paraffin infiltration, sectioning, and nucleic acid extraction on the results. The sources of standards for different brands of kits vary, and when different kits are used to test the same product, the results can differ significantly. Clinically, attempts have been made to use archived paraffin blocks from positive patients as controls, but the uneven distribution and limited sources of pathogens in these blocks make standardization difficult. The agar solid model reported in the literature also suffers from poor mechanical properties and difficulty in maintaining its morphology, making accurate quantification impossible, and thus failing to meet practical needs.

[0005] Ideal pathological tissue quality control materials need to simulate the characteristics of real tissues in multiple dimensions. First, in terms of composition, they should contain key elements such as cells, extracellular matrix, and pathogens. Second, they should possess stable physical properties and a certain mechanical strength to withstand the solvent effects in standard pathological procedures such as formalin fixation and paraffin embedding. Most importantly, they must ensure that the distribution of pathogens remains highly homogeneous (compliant with ISO 20776-1:2019 standard, CV ≤ 15%), a requirement that is difficult for existing quality control materials to meet.

[0006] Therefore, developing standardized quality control products applicable to the entire pathological testing process has significant clinical value and social implications. Summary of the Invention

[0007] This invention relates to a method for preparing standardized, biomimetic quality control materials for pathological diagnosis of pathogenic bacteria, particularly a pathological quality control system based on a curable hydrogel that possesses realistic tissue mechanical properties and uniform microbial distribution. This technology involves co-loading cells and pathogens into a photocrosslinkable hydrogel, followed by three-dimensional molding (e.g., casting or 3D printing) and curing to form a biomimetic three-dimensional structure. The matrix hardness can be precisely controlled within the range of 0.5-50 kPa, and the microbial spatial distribution is uniform. This invention addresses the lack of existing end-to-end pathological testing quality control materials, providing a reliable standardized quality control tool for the accurate diagnosis of infectious diseases.

[0008] The purpose of this invention is to provide a pathological quality control product whose matrix is ​​as close as possible to that of a real sample.

[0009] Another objective of this invention is to provide an application of a tuberculosis pathological quality control product.

[0010] To achieve the above objectives, this invention designs a system comprising inactivated Mycobacterium tuberculosis, cells, and a photocurable hydrogel matrix. A Mycobacterium tuberculosis pathological quality control sample is constructed using a combination of mold casting and photocuring, with a defined concentration gradient and a positive grading system established. Verification has shown that this Mycobacterium tuberculosis pathological quality control sample not only serves as a complete pathological quality control, providing a control for the pathological sample preparation process, but also remains unchanged for over one year at -80°C or at room temperature after being prepared as a paraffin block, meeting the long-term storage requirements for clinical products and demonstrating excellent clinical translation capabilities.

[0011] A method for preparing a quality control product for pathological testing of pathogenic bacteria involves mixing target pathogenic bacteria and eukaryotic cells with a curable hydrogel in a required ratio to form a bacterial-containing biological matrix mixture, which is then cured using a three-dimensional molding method to form a three-dimensional solid structure; thereby obtaining a quality control product for pathological testing of pathogenic bacteria containing a microbial-cell composite structure that simulates pathological tissue.

[0012] Furthermore, a method for preparing a quality control sample for pathological detection of pathogenic bacteria includes the following steps:

[0013] (a) Provide target pathogens and eukaryotic cells;

[0014] (b) Quantitatively count the inactivated pathogenic bacteria and eukaryotic cells separately, and mix them in a solidifiable hydrogel in a defined quantity to form a bacterial mixture;

[0015] (c) The mixture is cast by mold or bio-3D printed and solidified by physical cross-linking or chemical cross-linking to form a three-dimensional solid structure;

[0016] (d) Demolding to obtain a pathological test quality control product containing pathogenic bacteria, wherein the quality control product contains a microbial-cell complex structure that simulates pathological tissue, and after being stored at -80°C to 25°C for one year, the accuracy rate is ≥80%.

[0017] Furthermore, the eukaryotic cells are human or mammalian cells, and the concentration in the quality control material is 5 × 10⁻⁶. 6 ~5×10 9 / mL, and its genomic DNA is used as an internal reference for subsequent PCR detection.

[0018] Furthermore, the pathogenic bacteria are selected from one or more of the following: inactivated bacteria, fungi, actinomycetes, mycoplasma, chlamydia, rickettsia, and spirochetes. In Example 1, the target pathogenic bacteria is Mycobacterium tuberculosis.

[0019] Furthermore, the concentration of the pathogenic bacteria in the quality control sample is 0~6×10⁻⁶. 8 CFU / mL, further preferably 1×10⁻⁶ 4 CFU / mL ~6×10 8 CFU / mL.

[0020] Furthermore, the eukaryotic cells are human tumor cell lines selected from one or more of human gastric cancer cells (MKN-45), human non-small cell lung cancer cells (A549), human breast cancer cells (NSCs), human liver cancer cells (HepG2), and human cervical cancer cells (HeLa).

[0021] Furthermore, the three-dimensional solid structure sample is cryopreserved to obtain a fresh pathogenic bacteria pathological detection quality control product; or the three-dimensional solid structure sample is subjected to fixation, dehydration, wax impregnation, and paraffin embedding treatment to obtain a paraffin-embedded pathogenic bacteria pathological detection quality control product.

[0022] Furthermore, the curable hydrogel includes at least one of the following curing mechanisms:

[0023] (a) Photocurable type: a polymer containing photocrosslinkable groups, wherein the groups are selected from methacryloyl, acryloyl, mercapto or vinyl;

[0024] (b) Ionic cross-linked type: a combination of polysaccharides containing carboxylic acid or phosphate groups and divalent / trivalent cations;

[0025] (c) Temperature-sensitive: Natural or synthetic polymers with thermally reversible gelation properties.

[0026] Furthermore, the curable hydrogel is made of a biodegradable or biocompatible material; the tuberculosis pathological quality control material is also made of a curable hydrogel or a biocompatible material. The curable hydrogel can be made of the same material or different materials. Preferably, the curable hydrogel is a biodegradable hydrogel.

[0027] Furthermore, the curable hydrogel is selected from one or more combinations of photocurable hydrogels, ionic crosslinking hydrogels, and temperature-sensitive hydrogels; the photocurable hydrogel is a polymer containing photocrosslinkable groups, wherein the groups are selected from one or more combinations of methacryloyl, acryloyl, mercapto, or vinyl groups; the ionic crosslinking hydrogel includes polysaccharides containing carboxylic acid or phosphate groups that are cured by reacting with divalent / trivalent cations; and the temperature-sensitive hydrogel uses a natural or synthetic polymer with thermally reversible gelation properties.

[0028] Furthermore, the photocurable hydrogel includes one or more of methacrylamide chitosan, methacrylamide hyaluronic acid, methacrylamide gelatin (GelMA, etc.), methacrylamide sodium alginate, and methacrylamide chondroitin sulfate; the ion-crosslinked hydrogel includes one or more of sodium alginate (calcium ion-initiated crosslinking), chitosan (sodium tripolyphosphate-initiated crosslinking), and hyaluronic acid (calcium ion-initiated crosslinking); the temperature-sensitive hydrogel includes one or more of methylcellulose, poly(N-isopropylacrylamide) (PNIPAAm), and polyethylene oxide-propylene oxide-ethylene oxide triblock copolymer (Pluronic, such as F127).

[0029] Photocurable hydrogel materials such as methacrylamide gelatin (GelMA) and methacrylamide hyaluronic acid (HAMA) have shown great potential in bio-3D printing and tissue engineering due to their excellent biocompatibility, tunable mechanical properties, and rapid gelation characteristics. After incorporating a fixed amount of cells and pathogens into the hydrogel and thoroughly mixing them, the components are thoroughly homogenized. Using mold casting and other molding methods, the homogenized matrix system can achieve a uniform distribution of pathogens. Photocuring then fixes their spatial position, thereby constructing a biomimetic pathological tissue model with tunable mechanical properties, stable structure, and uniform distribution.

[0030] Furthermore, the curing treatment method for the curable hydrogel is as follows:

[0031] (a) For photocurable hydrogels: Irradiation intensity of 1-100 mW / cm² under 300-500 nm wavelength light for 10 s-30 min;

[0032] (b) For ion-crosslinked cured hydrogels: contact with divalent / trivalent cations (Ca²⁺, Sr²⁺, Fe³⁺, etc.) for 1-60 min;

[0033] (c) For temperature-induced curing hydrogels: sol-gel transition occurs in the range of 4-40 °C.

[0034] Preferably, the three-dimensional molding method is selected from one or more of mold casting, microfluidic molding, 3D bioprinting, and layer-by-layer assembly; or any one or more of the above methods are combined with the sacrificial template method.

[0035] Among them, the sacrificial template method involves mixing a pore-forming agent (NaCl particles, PLGA microspheres or gelatin) with a pathogenic bacteria-hydrogel and then molding it. The template is then removed by dissolving, melting or enzymatic hydrolysis to form a porous structure with a porosity of 60-90%. This method is generally used in combination with other three-dimensional molding methods.

[0036] Furthermore, the forming method of the three-dimensional solid structure is at least one of the following:

[0037] (a) Mold forming: The curable hydrogel is injected into the mold and cured to form a geometric structure with a length of 5-20 mm and a cross-sectional area of ​​1-20 mm².

[0038] (b) Microfluidic molding: The laminar flow mixing of cell-containing hydrogel and pathogenic bacteria is controlled by a microfluidic chip, and a continuous fiber structure with a diameter of 0.5-5 mm is formed at the chip outlet by temperature triggering, pH triggering or light response.

[0039] (c) Bio-3D printing: formed by additive manufacturing technology (i.e. three-dimensional printing method), with a printing resolution of ≤500 μm, and the resulting block contains a porous structure of 1-500 μm.

[0040] (d) Layer-by-layer assembly method: The pathogenic bacterial suspension is alternately deposited with polyelectrolyte solutions with opposite charges (such as cationic chitosan solution and anionic sodium alginate solution), and a three-dimensional solid structure is constructed layer by layer through electrostatic interaction, hydrogen bonding or covalent cross-linking.

[0041] The present invention also provides a quality control product for pathological detection of pathogenic bacteria, which is prepared by the preparation method described in any of the above technical solutions.

[0042] The present invention also provides an application of the pathogenic bacteria pathological detection quality control material obtained by any of the above methods in the quality control of the entire pathological detection process.

[0043] Furthermore, the quality control material can withstand the following pathological testing procedures:

[0044] Tissue preparation: fixation, dehydration, paraffin impregnation, paraffin embedding;

[0045] Staining: HE staining, acid-fast staining (Renée's method), immunohistochemistry, immunofluorescence;

[0046] Molecular detection: nucleic acid extraction, PCR amplification, fluorescence in situ hybridization (FISH).

[0047] The coefficient of variation (CV) of the spatial distribution uniformity of pathogenic bacteria in the quality control product obtained by the present invention is ≤15%.

[0048] The composite structure of eukaryotic cells and pathogenic bacteria obtained in this invention allows for selective gradient distribution of pathogenic bacteria concentration (0, 6 × 10⁻⁶). 4 6×10 5 6×10 6 6×10 7 (CFU / mL) can be used to establish quantitative standard curves.

[0049] The quality control products obtained by this invention can be stored at -80℃ for a long time or prepared into wax blocks and stored at room temperature, with a stability of ≥1 year.

[0050] An application of a tuberculosis pathological quality control sample as described in any of the above technical solutions in the pathological testing process. The tuberculosis pathological quality control sample serves as a control sample throughout the entire pathological testing process, enabling qualitative analysis of patient samples and providing a classification of pathogen content, thus providing important reference for clinical medication.

[0051] Furthermore, the quality control material obtained by this invention can be used in the entire process of quality control for pathological testing, including but not limited to one or more of the following:

[0052] As a positive control for tissue pretreatment (fixation, embedding, etc.);

[0053] As a criterion for interpreting staining (methods such as acid-fast staining and immunofluorescence);

[0054] As a quantitative reference for nucleic acid extraction.

[0055] When the quality control product obtained by this invention is used for Mycobacterium tuberculosis detection, its acid-fast staining positive rate is ≥95% consistent with that of clinical samples.

[0056] As a specific application, this invention utilizes GelMA hydrogel containing a quantitative amount of HeLa cells and an inactivated H37Rv standard strain as a biological matrix. Bacteria and cells in the matrix are effectively dispersed by ultrasound, and the mixture is then cast into a mold and light-cured to construct a tuberculosis pathological quality control product. This construction method not only ensures the stability and homogeneity of the gel block but also allows it to maintain its properties for more than one year at room temperature after being prepared as a wax block, even at -80°C, making it suitable for product transformation. Verification using molecular detection, IHC, and special staining methods has shown that this pathological quality control product can serve as a quality control for the entire pathological testing process. It can perform qualitative analysis of clinical samples and provide negative controls, weak positives, positives, moderately positives, and strongly positive gradings of pathogen content, constructing standard curves for quantitative analysis and providing a reference for clinical medication.

[0057] Before preparing the quality control samples according to this invention, the corresponding cells and pathogenic bacteria need to be cultured separately. Existing methods can be used for both. For example, for eukaryotic cells, they can be cultured using the corresponding culture medium according to conventional methods. After reaching the set fusion density, corresponding cell digestion and other operations are performed to finally obtain the corresponding eukaryotic cell pellet. For pathogenic bacteria, the corresponding quality control strains are biologically cultured using appropriate methods. After bacterial growth, they are inactivated and refrigerated for later use.

[0058] The preparation process of a tuberculosis pathological quality control material is as follows:

[0059] First, representative human tumor cell line HeLa cells were cultured as cellular components of human tissue to serve as quality control materials. Mycobacterium tuberculosis was cultured, inactivated with 75% alcohol at 37°C for 2 hours, and then uniformly dispersed using ultrasonic oscillation. The bacterial concentration was then quantitatively analyzed using a nanoflow cytometer to provide data support for the subsequent establishment of quality control materials and the setting of concentration gradients.

[0060] Specifically, after culturing the quality control strain of Mycobacterium tuberculosis, colonies were picked and transferred to centrifuge tubes containing 75% ethanol. After shaking, the tubes were inactivated by standing for 2 hours, centrifuged and resuspended, and then fully dispersed in a constant-temperature ultrasonic water bath. The concentration of Mycobacterium tuberculosis was detected using a nanoflow cytometer and stored at 4°C for later use. Sufficient HeLa cells were cultured, digested and centrifuged, the cells were counted, resuspended in culture medium, and stored at 4°C for later use.

[0061] Next, a 3D-printed resin mold was designed using SolidWorks and manufactured using a photopolymer 3D printer (S140, BMFPrecision Tech, China). A 10:1 PDMS mixture was injected into the mold and degassed in a vacuum oven to prevent air bubbles from forming during curing. The PDMS was cured at 80°C for 3 hours and then peeled off from the mold to obtain a PDMS mold with 2×2×8 mm³ grooves. The PDMS mold was sterilized by immersing it in 75% alcohol for 1-2 hours, then rinsed three times with sterile PBS, and finally drained using a laminar flow hood.

[0062] Next, the counted cells, tuberculosis bacteria, and GelMA were mixed into a substrate and poured. Ink was used to set different bacterial concentrations for determining the positive grading of the samples.

[0063] The mixed matrix was dissolved in a 37°C constant-temperature ultrasonic water bath for 5 min, then vortexed for 20 s, sonicated for 30 s, and finally vortexed for 20 s to ensure thorough mixing. 30 μL aliquots of each matrix system were carefully added dropwise to the mold, avoiding air bubble formation. The constructs were then photocured for 60 s using a 70 mW light source at a wavelength of 405 nm before demolding. The resulting tissue models were weighed and their mass recorded.

[0064] Specifically, a 10% GelMA (GM-1M) solution containing 0.25% lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) was prepared. The cells were thoroughly dissolved and dispersed using a constant-temperature ultrasonic water bath at a frequency of 40 kHz. The HeLa cell pellet was resuspended in the hydrogel, and the cell density was adjusted to 5 × 10⁻⁶ cells / mL. 6 ~5×10 9 / mL. The bacterial concentration was adjusted to 0.6 × 10⁶. 4 CFU / mL, 6×10 5 CFU / mL, 6×10 6 CFU / mL, 6×10 7 CFU / mL was added to 10% GM-1M. The mixture was then shaken in a 37°C ultrasonic water bath for 30 min to uniformly disperse the bacterial and cell suspensions, forming biological matrices of different concentrations.

[0065] Then, the prepared biological matrix is ​​poured into the mold and then photocured to obtain the tuberculosis pathological sample.

[0066] Specifically, biological substrates of different concentrations were poured into PDMS molds, cured with a UV 405 nm ultraviolet curing lamp for 60 seconds, and then demolded to obtain tuberculosis pathological samples with different positive intensities.

[0067] The printed samples were then made into wax blocks according to the biological sample preparation process, and tested and analyzed to verify the uniformity and stability of the quality control products.

[0068] Specifically, each paraffin block was cut into 3 μm thick slices of uniform thickness, which were then stained with hematoxylin and eosin (HE), ziehl-Nelsen acid-fast staining, and qPCR detected. The results were then statistically analyzed.

[0069] Finally, the obtained sample standards were verified for consistency, including consistency of the same wax block, consistency of different wax blocks in the same batch, consistency of different batches under the same storage conditions, and consistency of the same batch under different storage conditions.

[0070] Preferably, the bacterial concentration of the tuberculosis pathological quality control sample is 6 × 10⁻⁶. 4 ~6×10 7 CFU / mL, cell concentration 5×10 6 ~5×10 9 / mL, wherein the GelMA concentration is 5%~30%, and the GelMA type is GM-30~GM-1M; as a further preferred embodiment, the bacterial concentration is 6×10 4 CFU / mL, 6×10 5 CFU / mL, 6×10 6 CFU / mL, 6×10 7 CFU / mL, etc., the cell concentration is 5×10⁻⁶. 6 / mL, 5×10 7 / mL, 5×10 8 / mL, 5×10 9 / mL, etc., wherein the concentration of GelMA is 5%, 10%, 15%, 20%, 25%, 30%, etc., and the type of GelMA is GM-30, GM-60, GM-90, GM-1M, etc.

[0071] This invention utilizes mold casting technology or bio-3D printing technology to load quantitative HeLa cells and inactivated Mycobacterium tuberculosis H37Rv standard strains, constructing the first biomimetic quality control system covering the entire process from tissue pretreatment to molecular detection. This system possesses excellent mechanical adaptability, with a hardness range between 0.5 and 50 kPa, encompassing the mechanical properties of various tissues such as liver, lung, and adipose tissue. Simultaneously, it features controllable composition and uniform microbial distribution, accurately verifying the accuracy and stability of tuberculosis pathological detection. This technology has been successfully piloted in 200 cases at Taizhou Hospital in Zhejiang Province, achieving a 100% detection accuracy rate. It provides the first end-to-end standardized framework for pathological quality control, assisting in reagent / equipment benchmarking, process optimization, and improved inter-laboratory result comparability, representing a key breakthrough in promoting the clinical translation of precision pathology diagnosis.

[0072] This invention relates to a method for preparing and applying a quality control sample for pathological detection of pathogenic bacteria. It can serve as a standardized positive control sample for the entire process of pathological detection of pathogenic bacteria, used for quality control. A curable hydrogel containing a photoinitiator is used to load quantitatively inactivated pathogenic bacteria and eukaryotic cells. This is then formed by mold casting or bio-3D printing, resulting in a structurally stable solid simulated tissue block after curing. Using Mycobacterium tuberculosis as an example, this quality control sample shows uniform bacterial distribution (coefficient of variation ≤15%) in acid-fast staining, and a coefficient of variation of <5% for nucleic acid detection Ct values. It can be stored at -80℃ or, after preparation as a wax block, at room temperature for more than one year. A quantitative standard curve (R² > 0.99) can be established using gradient concentration samples for pathogen load determination. This invention solves the problem of the lack of a complete quality control sample for the entire process of pathogen detection, and is suitable for quality control throughout the entire process, including tissue block preparation, pathological section staining, and molecular detection.

[0073] The beneficial effects of this invention are as follows:

[0074] 1. A pathological quality control product for pathogenic bacteria according to the present invention is based on a hydrogel ink system containing Mycobacterium tuberculosis, eukaryotic cells, and GelMA. Through efficient batch preparation via mold casting and photocuring, a quality control product with good uniformity and stability is obtained.

[0075] 2. The pathological quality control material constructed in this invention can be used not only as a positive control for samples, but also as a positive control for liquid nucleic acid samples, meeting the quality control needs of the entire pathological operation process.

[0076] 3. The present invention provides a pathological quality control product for pathogenic bacteria, which has five concentration gradients and can provide the positive grading required for pathological detection of Mycobacterium tuberculosis, thus providing guidance for clinical medication.

[0077] 4. The pathological quality control material constructed in this invention, after being prepared into paraffin blocks, can be stored at room temperature for more than one year without losing its properties. Fresh samples stored at -80℃ for extended periods can be used after rewarming without affecting the quality of the slides, thus solving the storage and stability issues. This quality control material has significant potential for clinical translation.

[0078] This invention utilizes the advantages of rapid prototyping and personalized molding of photocurable hydrogels to construct a tuberculosis pathological quality control product with clearly defined concentration gradations. The preparation process is simple, highly practical, and has broad prospects for clinical translation.

[0079] In summary, the preparation method and corresponding quality control products for pathogen pathology testing provided by this invention are of great significance. From a technical perspective, it will fill the gap in the international market for dedicated quality control products for pathogen detection in pathological tissues; from a clinical application perspective, it will significantly improve the reliability of test results and reduce the risk of misdiagnosis; from a public health perspective, it will provide crucial technical support for the prevention and control of infectious diseases. Currently, no mature products of the same kind have been launched globally, and research and development in this field in my country is still in its early stages. The development of this innovative product will not only promote the standardization of pathology testing in my country but also make an important contribution to global public health security. Attached Figure Description

[0080] Figure 1 This is a schematic diagram of the resin mold, PDMS mold, and tuberculosis pathological quality control sample prepared by casting molding process provided in an embodiment of the present invention. A, B, and C are the resin mold, PDMS mold, and fresh tuberculosis pathological sample after casting molding in an embodiment of the present invention.

[0081] Figure 2 The graph shows the TB concentration results of Mycobacterium tuberculosis as determined by nanoflow cytometry.

[0082] Figure 3 In the figures A, B, and C, the pathological samples of Mycobacterium tuberculosis obtained in the embodiments of the present invention are shown before fixation, after fixation, and after paraffin impregnation, respectively.

[0083] Figure 4 In the figures, A and B represent the weight comparison of pathological samples before and after fixation, and the statistical results of length after dewaxing, respectively.

[0084] Figure 5 H&E staining results for fresh quality control section pathological samples;

[0085] Figure 6 Results of acid-fast staining for samples with different concentration gradients;

[0086] Figure 7 In the figures, A and B represent the OD260 / OD280 ratio and DNA content comparison results for negative and four gradient positive quality control samples, respectively.

[0087] Figure 8 In the diagram, A represents the amplification curves of the positive standard (PC), negative standard (NC), and negative control (TB0) from the kit, and the internal control curve. BE represents the PCR amplification curves of the gradient positive controls (TB1-4), respectively. TBa-bc indicates that 'a' represents the positive grade, 'b' represents the sample number, and 'c' represents the number of tests. For example, TB1-1-1 represents the first test of weakly positive sample 1 (TB1) with the number 1.

[0088] Figure 9 Image A shows the HE staining results of a sample section after being stored at -80℃ for 12 months. Figure 9 Image B shows the HE staining results of a sample section preserved for 1 year in room temperature paraffin-embedded (FFPE) material.

[0089] Figure 10 This is a comparison of the number of TBs in the control group (fresh standard), paraffin block tissue (FFPE) stored at room temperature for 12 months, and tissue stored at -80℃ for 1 month and 12 months.

[0090] Figure 11 In the comparison of OD260 / OD280 and DNA concentration in samples A and B (control group, fresh standard), FFPE-preserved samples for 12 months, and fresh standard samples frozen at -80℃ for 12 months), A and B are the control group (fresh standard).

[0091] Figure 12 This is a schematic diagram illustrating the preparation and application of some quality control samples in the example process for overall control. Detailed Implementation

[0092] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0093] In this embodiment of the invention, a pathogenic bacteria pathological quality control product is provided for quality control throughout the entire pathology process. The pathogenic bacteria pathological quality control product essentially comprises Mycobacterium tuberculosis, cells, and a hydrogel ink system based on GelMA. Through efficient batch preparation using mold casting and photocuring, a quality control product with good uniformity and stability is obtained, serving as a positive control for the entire pathological sample detection process. Furthermore, by setting different concentration gradients, a reference is provided for the positive grading of clinical samples.

[0094] refer to Figure 12 The preparation process of a tuberculosis pathological quality control product specifically includes the following steps:

[0095] Step 1: Remove the frozen HeLa cells and quickly place them in a 37°C water bath. After complete thawing, add an equal volume of culture medium to resuspend the cells and centrifuge at 900 rpm for 5 min. In a clean bench, aspirate the supernatant, resuspend the cells in DMEM complete medium, seed them into sterile culture dishes, and incubate at 37°C in a 5% CO2 incubator. Once the cells reach a confluence density of over 80%, passage them using 0.25% trypsin. After digesting and centrifuging a sufficient amount of cells, aspirate the supernatant, resuspend the cells in PBS, count the number of cells, and centrifuge again to obtain the HeLa cell pellet.

[0096] Step 2: Inoculate the tuberculosis control strain onto a solid culture medium and incubate in a bioincubator for 2-4 weeks. After colonies grow, use a swab moistened with 75% ethanol to pick up colonies in a biosafety cabinet and transfer them to centrifuge tubes containing 75% ethanol. After shaking, incubate for 2 hours to inactivate the bacteria, centrifuge at 900 rpm for 5 minutes, resuspend in PBS, and disperse thoroughly in a constant-temperature ultrasonic water bath. Detect the concentration of tuberculosis bacteria using a flow cytometer. Figure 2 Store in a refrigerator at 4℃ for later use. Figure 2 The image shows the results of nanoflow cytometry detection of Mycobacterium tuberculosis. Particles in the solution were clustered using forward scattered light (FSC) and side scattered light (SSC). The results showed that TB accounted for 44.3% of the total number of particles. The specific concentration can be estimated by combining the TB count results per microliter of sample.

[0097] Step 3: Use Solidworks drawing software to design the internal cuboid protrusion as 2*2*8 mm. 3 The mold is then created, and an STL file that can be recognized by 3D printing is exported. After importing it into the Mofang Technology L140 printer, a resin mold with the corresponding structure is printed. Figure 1 (A) After printing, excess resin material is washed away using 75% alcohol. Then, PDMS is poured into the mold, cured at 50°C for 4 hours, and then demolded to obtain a cast PDMS mold with a cuboid groove structure. Figure 1 (B)

[0098] Step 4: Weigh a measured amount of GelMA and dissolve it in PBS solution to obtain a 10% (w / v) GelMA solution (GM-1M). Dissolve 0.25% w / v lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in 10% w / v GM-1M. Disperse thoroughly in a constant-temperature ultrasonic water bath. Resuspend the HeLa cell pellet using a hydrogel and adjust the cell density to 5 × 10⁻⁶ cells / mL. 7 -5×10 8 / mL. Prepared as a mixed matrix containing eukaryotic cells.

[0099] Step 5: Add different concentrations of tuberculosis bacteria to the ink according to the bacterial concentrations in Table 1, and oscillate in a constant temperature ultrasonic water bath at 37℃ for 30 min to prepare a mixed matrix of pathogenic bacteria and eukaryotic cells with different positive intensities.

[0100] Table 1: Bacterial gradient grouping

[0101] Group Positive intensity Bacterial CFU / mL TB0 negative control 0 TB1 Weak positive <![CDATA[6×10 4 ]]> TB2 Positive <![CDATA[6×10 5 ]]> TB3 Strong positive <![CDATA[6×10 6 ]]> TB4 Strong positive <![CDATA[6×10 7 ]]>

[0102] Step Six: Slowly drop the mixed matrix of different components into the mold, avoiding air bubbles, and cure for 60 seconds using a 70 mW curing lamp with a wavelength of 405 nm. After curing, carefully demold the shell structure block with tweezers to obtain tuberculosis pathological samples with different positive intensities (see...). Figure 1 C and Figure 3 (A) Figure 3 Image A is a schematic diagram of the morphology of the pathological quality control material before fixation, showing a semi-transparent strip structure.

[0103] Step 7: Fix the pathological samples in 4% paraformaldehyde solution for 24 hours (see...) Figure 3 B, Figure 3 (Figure B is a schematic diagram of the morphology after fixation with paraformaldehyde, showing that the sample volume has shrunk and the consistency is good.) The samples were then dehydrated and paraffin-impregnated in a Sakura fully automated tissue dehydrator (see...). Figure 3 C in the middle Figure 3 Figure C shows the morphology after paraffin embedding, indicating further shrinkage of the sample volume and slight fluctuations in the boundary, followed by clearing and embedding into a paraffin block (FFPE). A uniform 3 μm thick section was cut from each paraffin block for HE staining, Ziehl-Nelsen acid-fast staining, and qPCR detection. The results were statistically analyzed to verify the relationship between the concentrations of various tuberculosis bacteria and the positive intensity of the standards.

[0104] To verify the stability of the sample preparation process, the weight of the pathological samples before and after fixation, as well as the sample length after dewaxing, were statistically analyzed. Figure 4 , Figure 4 Figure A shows the statistical chart of quality changes of pathological quality control materials before and after fixation. The data shows that fixation treatment has no significant effect on sample quality (P>0.05); Figure B shows the statistical chart of sample length distribution after paraffin impregnation. The results show that the length data conforms to the normal distribution law.

[0105] Step 8: H&E staining

[0106] Each paraffin block obtained from different groups was used to prepare 3 μm thick sections, each uniquely numbered (n: AF). Sections were dewaxed to water. Modified Gill's hematoxylin staining was performed for 8 min, followed by washing with water, inversion with blue solution for 1 min, washing with water, dehydration with graded alcohols, clearing with xylene, and mounting with neutral resin. Section integrity, wrinkles, cavities, and cell morphology were assessed. Staining results are shown in [link to table]. Figure 5 , Figure 5 The H&E stained sections showed smooth surfaces, clear boundaries, no wrinkles or cavities, and normal cell morphology, indicating that the quality control material is fully compatible with routine tissue processing procedures and meets the requirements of the staining process.

[0107] Step 9: Ziehl-Nelsen acid-fast staining

[0108] For each paraffin block obtained from different groups, 3 μm thick continuous sections were selected. Complete, uncontaminated sections were mounted on glass slides and uniquely numbered. A total of 20 sections were selected and baked in a 70℃ oven for 1 hour. They were then dewaxed in an environmentally friendly dewaxing solution, immersing three times for 5 minutes each time. After wiping away any remaining solution around the sections, they were rinsed with running water until no visible oily residue remained. Phenol-based fuchsin was added to the sections, and they were incubated at room temperature for 15 minutes. The sections were then washed with water, differentiated with hydrochloric acid and ethanol for 5 seconds, and rinsed with running water. Methylene blue was added for 5 seconds to create a pale blue background. The sections were rinsed with running water, dried with cold air, cleared with xylene, and mounted with neutral resin. Tuberculosis bacteria were identified using a 100X oil immersion microscope, and their gradient distribution was preliminarily determined. The results are shown in [Figure number missing]. Figure 6 , Figure 6 This is a schematic diagram comparing a negative control sample and a positive sample with an increasing bacterial count gradient, showing that the tuberculosis bacteria gradient distribution in the micrograph corresponds to the original configuration gradient.

[0109] Step 10: DNA Sample Extraction and Concentration Detection

[0110] Following step eight, 3 μm paraffin sections were obtained and dewaxed in centrifuge tubes with xylene and anhydrous ethanol. Then, 180 μL of Buffer DTB and 20 μL of Proteinase K were added, and the mixture was digested at 56°C for 4 h. DNA was extracted using column extraction to obtain DNA samples. The nucleic acid concentration was detected using a micro-spectrophotometer. Before analysis, the sample DNA was diluted to 1.5-3 ng / μL with 1X TE (pH 8.0). The detection results are shown below. Figure 7 . Figure 7Figure A shows the DNA purity test results of five quality control chips (TB0-TB4). Their OD260 / OD280 ratios are: TB0 (2.03±0.04), TB1 (1.94±0.01), TB2 (1.98±0.06), TB3 (1.89±0.03), and TB4 (1.95±0.02). All ratios are within the acceptable range of 1.80–2.10 (CV=3%), proving that the extracted DNA is free of protein contamination. Figure 7 Figure B shows the DNA concentration detection results, indicating that the concentration in each group remained stable at 25–30 ng / μL, which meets the routine requirements for PCR experiments.

[0111] Step 11: PCR Operation

[0112] Obtain the DNA sample according to step 10, mix the reaction solution, enzyme mixture, and internal standard nucleic acid thoroughly in proportion to form a PCR-mixture, aliquot 40 μL into each of eight tubes, and finally add 5 μL of DNA sample as a PCR reaction template. Detect the mixture together with the internal standard, and read and analyze the results.

[0113] The obtained sample standards were then verified for consistency, including consistency within the same wax block (multiple samples from one wax block), consistency between different wax blocks from the same batch, consistency between different batches under the same storage conditions, and consistency between the same batch under different storage conditions. The test results are shown below. Figure 8 , Figure 8 In Figure A, the qPCR amplification curves and internal control curves of the positive standard (PC), negative standard (NC), and negative control (TB0) of the kit are shown. Figure 8 The BE (Beta-Brain) results represent the qPCR performance validation of the TB1-TB4 quality control samples. The results show that all samples exhibited normal amplification curves and good Ct value stability (CV < 5%). Specific data are as follows:

[0114] TB1: Ct=36.44±0.82 (CV=2.26%)

[0115] TB2: Ct=33.43±0.57 (CV=1.70%)

[0116] TB3: Ct=30.03±0.36 (CV=1.35%)

[0117] TB4: Ct=26.27±0.36 (CV=1.35%).

[0118] Step 12: Take the prepared fresh quality control sample (after photocuring, before fixation) and place it in a cryovial. Immediately immerse it in liquid nitrogen for quick freezing. After 10 minutes, transfer it to a -80°C freezer for cryopreservation. After freezing for 1 month and 12 months, thaw at 37°C and then perform paraffin embedding, staining analysis, and nucleic acid detection to verify its long-term storage stability.

[0119] The paraffin-embedded (FFPE) samples were stored at room temperature for one year, and then subjected to staining analysis and nucleic acid detection to verify their long-term storage stability.

[0120] See results Figures 9-11 :

[0121] Figure 9 The HE staining results of a sample section from fresh quality control material stored at -80℃ for 1 year show that the section is in good integrity with only a few tiny cavities caused by freezing ice crystals. Figure 9 Image B shows the HE staining results of a paraffin-embedded (FFPE) sample section stored for one year. The section is intact and free of structural defects, demonstrating that the quality control sample has excellent long-term stability under both storage conditions.

[0122] Figure 10 Figure A shows a statistical comparison of the TB counts of fresh samples (control), samples stored in FFPE for 12 months, fresh samples stored at -80℃ for 1 month, and samples stored for 12 months. The data shows that there is no statistically significant difference in bacterial count among the groups (P>0.05), indicating that storage conditions have no significant effect on bacterial count.

[0123] Figure 11 Figure A shows the DNA purity test results of samples under different storage conditions, indicating that the OD260 / OD280 ratios of fresh samples, samples stored in FFPE for 12 months, and fresh samples stored at -80℃ for 12 months were all stable within the range of 1.80–2.10. Figure 11 In Figure B, the DNA concentration detection results of the corresponding sample are shown. The concentration values ​​are all maintained within the applicable range of 25–30 ng / μL.

Claims

1. A method for preparing a quality control sample for pathological detection of pathogenic bacteria, characterized in that, The target pathogenic bacteria and eukaryotic cells are mixed with a curable hydrogel in the required ratio to form a bacterial biomatrix. The matrix is ​​then solidified using a three-dimensional molding method to form a three-dimensional solid structure, thus obtaining a pathological control product for pathogenic bacteria containing a microbial-cell composite structure that simulates pathological tissue.

2. The method for preparing the quality control material for pathological detection of pathogenic bacteria according to claim 1, characterized in that, The eukaryotic cells are human or mammalian cells, and their genomic DNA is used as an internal reference for subsequent PCR detection; the pathogenic bacteria are selected from at least one of inactivated bacteria, fungi, actinomycetes, mycoplasma, chlamydia, rickettsia, and spirochetes.

3. The method for preparing the quality control material for pathological detection of pathogenic bacteria according to claim 2, characterized in that, The eukaryotic cells are human tumor cell lines selected from one or more of human gastric cancer cells, human non-small cell lung cancer cells, human breast cancer cells, human liver cancer cells, and human cervical cancer cells.

4. The method for preparing the quality control material for pathological detection of pathogenic bacteria according to claim 1, characterized in that, The concentration of the pathogenic bacteria in the quality control material is 0~6×10⁻⁶. 8 CFU / mL; the density of the eukaryotic cells was 5 × 10⁻⁶. 6 ~5×10 9 / mL.

5. The method for preparing the quality control material for pathological detection of pathogenic bacteria according to claim 1, characterized in that, The three-dimensional solid structure sample is cryopreserved to obtain a fresh pathogenic bacteria pathological detection quality control product; or the three-dimensional solid structure sample is fixed, dehydrated, impregnated with wax and embedded in paraffin to obtain a paraffin-embedded pathogenic bacteria pathological detection quality control product.

6. The method for preparing the quality control material for pathological detection of pathogenic bacteria according to claim 1, characterized in that, The curable hydrogel is selected from one or more combinations of photocurable hydrogels, ionically crosslinked hydrogels, and temperature-sensitive hydrogels; the photocurable hydrogel is a polymer containing photocrosslinkable groups, wherein the groups are selected from one or more combinations of methacryloyl, acryloyl, mercapto, or vinyl groups; the ionically crosslinked hydrogel includes polysaccharides containing carboxylic acid or phosphate groups that are cured by reacting with divalent / trivalent cations; the temperature-sensitive hydrogel uses natural or synthetic polymers with thermally reversible gelation properties.

7. The method for preparing the quality control material for pathological detection of pathogenic bacteria according to claim 6, characterized in that: The photocurable hydrogel includes one or more of methacrylamide chitosan, methacrylamide hyaluronic acid, methacrylamide gelatin, methacrylamide sodium alginate, and methacrylamide chondroitin sulfate; the ion-crosslinked hydrogel includes one or more of sodium alginate, chitosan, and hyaluronic acid; and the temperature-sensitive hydrogel includes one or more of methylcellulose, poly(N-isopropylacrylamide), and polyethylene oxide-propylene oxide-ethylene oxide triblock copolymer.

8. The method for preparing the quality control material for pathological detection of pathogenic bacteria according to claim 1, characterized in that, The three-dimensional molding method is selected from one or more of mold casting, microfluidic molding, 3D bioprinting, and layer-by-layer assembly; or any one or more of the above methods combined with the sacrificial template method.

9. A quality control product for pathological detection of pathogenic bacteria, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the pathogenic bacteria pathological detection quality control material as described in claim 9 in the quality control of the entire pathological detection process.

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