A staining agent, staining method and use for morphological detection of pathogens in blood

Abstract

The application belongs to the technical field of medical detection, and is a staining agent, a staining method and application for morphological detection of pathogens in blood. The staining agent comprises fluorescent dyes, osmotic pressure regulators, pH buffers and auxiliary agents. The fluorescent dyes contain acridine orange or its acceptable salt with a mass concentration of 1 μg / mL to 100 μg / mL. The osmotic pressure regulator has a volume concentration of 0.01% to 1.0%. The pH buffer is used to adjust and maintain the pH value to be 7.0 to 7.4. The auxiliary agent is selected from at least one of a surfactant, a fixing agent and glycerol, and has a mass concentration of 10 mg / L to 10 g / L. The pathogens in blood are stained by using the staining agent, which can induce characteristic morphological changes of the pathogens, including DNA / RNA content, organelle morphology and distribution, color difference and the like, significantly improves the identification efficiency and accuracy of microscopic examination, and provides a better morphological solution for blood pathogen detection.

Description

Technical Field

[0001] This invention relates to a staining agent, staining method, and application for the morphological detection of pathogens in blood. Specifically, it relates to a staining agent that can specifically induce characteristic morphological changes in pathogens in blood for their detection, identification, and diagnosis; a staining method based on the staining agent; and its application in the preparation of related in vitro diagnostic reagents, belonging to the field of medical testing technology. Background Technology

[0002] Blood parasites are parasites that live in the blood or blood cells of animals. Common types include Plasmodium, Trypanosoma, Babesia, and microfilariae, which can cause a variety of diseases. As pathogens, parasites can directly cause parasitic diseases and also act as vectors to spread other diseases, posing a continuous threat to public health and socioeconomic security. The spread of parasitic diseases follows three key links: the source of infection (including patients, carriers, and reservoir hosts), the route of transmission (such as via water, food, and arthropods), and susceptible populations. Therefore, accurate diagnosis of blood parasites is not only a core measure for identifying the source of infection and blocking transmission routes, but also has significant value in disease prevention and control.

[0003] Taking malaria as an example, the source of infection is patients or asymptomatic carriers who carry Plasmodium gametophytes in their peripheral blood. The main vector is Anopheles mosquitoes, including common species such as Anopheles sinensis and Anopheles minimus. While the general population is susceptible to malaria, and infection can produce some immune protection, timely diagnosis and standardized management are still crucial to effectively interrupt the transmission chain. Therefore, rapid and accurate detection of Plasmodium in the blood and subsequent intervention are key to controlling the spread of malaria.

[0004] Currently, the clinical diagnosis of blood parasites (such as malaria parasites) mainly relies on the following methods: (1) Pathogenic (morphological) microscopic examination (gold standard) The method primarily utilizes thick and thin blood film smears, stained with Giemsa or Wright's stain, and observed under an oil immersion microscope. In thin blood films, the malaria parasites are morphologically intact and typical, making identification and species differentiation easy; however, low parasite density can lead to missed detections. Thick blood films, due to the higher concentration of parasites, are easier to detect, but the lysis of red blood cells during staining alters the parasite morphology, making species identification more difficult. While this method boasts high specificity and sensitivity, it is highly dependent on the experience of the microscopist, time-consuming, and has low throughput, making it unsuitable for large-scale screening.

[0005] (2) Rapid immunological detection method Rapid diagnostic test strips based on Plasmodium-specific antigens are simple and quick to use, making them suitable for on-site initial screening. However, this method suffers from insufficient sensitivity and specificity, which may lead to missed diagnoses or false positives, especially with decreased reliability in cases of low parasite density or atypical infections.

[0006] (3) Molecular biological detection method For example, quantitative real-time PCR (qPCR) amplifies Plasmodium-specific gene fragments (such as 18S rRNA and the gene encoding HRP-2), exhibiting extremely high sensitivity and the ability to identify parasite species and mixed infections. However, this method has high requirements for laboratory equipment, personnel, and costs, and also carries the risk of false positives due to contamination, making it difficult to popularize in resource-limited areas.

[0007] (4) Serological antibody detection method Tests such as ELISA and indirect fluorescent antibody assays are mainly used to detect host antibodies and are suitable for epidemiological surveys and assessment of past infections. However, because antibodies can persist for a long time after recovery, they cannot effectively distinguish between current and past infections, thus limiting their clinical diagnostic value.

[0008] It is evident that existing mainstream diagnostic techniques for blood parasites all have significant limitations, making it difficult to achieve a good balance between ease of operation, detection speed, sensitivity, specificity, and cost-effectiveness. Therefore, there is an urgent need to develop a new method for detecting blood parasites that combines high sensitivity and specificity with ease of operation and rapid detection.

[0009] In the prior art, Chinese patent CN104749144A discloses a blood cell detection reagent and a blood cell processing and identification method. This method achieves detection by combining a red light-excited fluorescent dye with a spheroidizing component and an organic alcohol. The spheroidizing component keeps red blood cells intact and does not damage the internal structure of white blood cells; the organic alcohol enhances cell membrane permeability and facilitates dye entry into the cell. This method aims to achieve rapid differentiation of various blood cells, especially immature white blood cells and reticulocytes, through a single detection. Its identification mainly relies on the forward scattered light (FSC) and side scattered light (SSC) signals from flow cytometry to analyze the cell's physical morphology and internal particle complexity, while fluorescent staining is mainly used to distinguish whether the cell contains DNA and RNA (i.e., the presence or absence and approximate type of nucleic acids). Therefore, it is evident that the technical solution disclosed in this patent primarily targets the differentiation and counting of routine blood cells. While its methods, including spheroidization, single nucleic acid signal discrimination, and physical scattering light analysis, enable rapid screening of blood cells, they fail to provide the ability to identify specific morphological characteristics of pathogens such as parasites in the blood. Consequently, they are insufficient to meet the clinical needs for accurate pathogen identification and classification. In the field of detecting heterologous pathogens such as parasites, the effectiveness and reliability of its method still have significant room for improvement.

[0010] In addition, Chinese patents CN104169719B and CN106840812B disclose methods and systems for detecting pathogens in biological samples. These methods employ at least two dyes: the first dye primarily stains DNA (e.g., Hoechst series dyes, which stain cell nuclei / pathogen nucleic acids), and the second dye stains other cellular components different from DNA (e.g., acridine orange AO, which can stain RNA, cytoplasm, etc.). By using different excitation / emission wavelengths, the DNA region and other component regions exhibit different colors or fluorescent signals, thus forming easily distinguishable "stained areas." The spatial and morphological relationship between these two regions is then extracted as a basis for judgment. Through comprehensive judgment using multiple parameters (size, location, shape, etc.), the detection of pathogens in the sample is achieved.

[0011] However, this method requires at least two dyes to stain DNA and other cellular components, and this staining technique fails to induce morphological changes in blood cells and has limited ability to distinguish minute morphological features within cells. Therefore, it is foreseeable that this method will be less effective in identifying low concentrations of parasites or microorganisms and may lack sufficient sensitivity.

[0012] Furthermore, in cell staining applications, the traditional Wright staining method, through the experimental steps of "fixation-staining-color separation," can achieve differentiation staining of different structures such as the cell nucleus, cytoplasmic organelles, and granules, presenting different color contrasts. In contrast, existing live cell staining methods are limited by dye selection and staining conditions, making it difficult to achieve differentiation staining of different components within the cell. This results in limited morphological information acquisition capabilities and makes it difficult to meet the needs of high-precision pathogen detection. Summary of the Invention

[0013] The purpose of this invention is to provide a staining agent, staining method, and application for the morphological detection of pathogens in blood. The invention utilizes a specifically formulated staining agent to stain parasites, microorganisms, and / or cells in the blood. Under the action of the staining agent, osmotic pressure regulator, surfactant, and buffer, characteristic staining effects can be produced on the intracellular structural components of parasites, microorganisms, and / or cells. These effects include, but are not limited to, changes in DNA content, RNA content, morphology, distribution, and intracellular structures (such as the nucleus, organelles, and cytoskeleton), as well as color changes in the structural components of parasites. This improves the efficiency and accuracy of microscopic examination, providing a superior morphological solution for the diagnosis of blood parasitic diseases.

[0014] This invention is achieved through the following technical solution: a staining agent for morphological detection of pathogens in blood, the staining agent comprising: (1) A fluorescent dye containing acridine orange or an acceptable salt thereof, wherein the mass concentration of acridine orange or an acceptable salt thereof in the dye is from 1 μg / mL to 100 μg / mL; (2) An osmotic pressure regulator, wherein the volume concentration in the dye is 0.01% to 10.0%; (3) pH buffer, used to adjust and maintain the pH of the staining agent to 7 to 7.4; (4) An auxiliary agent selected from surfactants, fixatives, glycerol or combinations thereof, wherein the mass concentration of the auxiliary agent in the dyeing agent is from 10 mg / L to 10 g / L.

[0015] The fluorescent dye further includes at least one nucleic acid dye selected from the group consisting of: 4',6-diimide-2-phenylindole (DAPI), ethidium bromide, Hoechst series dyes, SYBR series dyes, or acceptable salts or combinations thereof.

[0016] The osmotic pressure regulator is selected from one or more of sodium chloride, potassium chloride, glucose, sucrose, and trehalose.

[0017] The pH buffer is selected from one or more combinations of sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, PBS, PB, glycine, MES, and Tris.

[0018] The surfactant is selected from one or more of anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants.

[0019] The fixative is selected from one or a combination of two or more of paraformaldehyde, glutaraldehyde, methanol, ethanol, and acetone.

[0020] The present invention also provides a staining method for morphological detection of pathogens in blood, wherein the above-mentioned staining agent is mixed with the blood sample to be tested for nucleic acid staining, so that parasites, microorganisms and / or cells in the blood sample produce characteristic morphological changes and fluorescence signal changes, which can be used for identification analysis based on DNA content, RNA content, morphology, distribution and color characteristics.

[0021] The volume ratio of the staining agent to the blood sample is from 9:1 to 99:1.

[0022] The present invention also provides the use of the above-mentioned staining agent in the preparation of in vitro diagnostic reagents, wherein the in vitro diagnostic reagents are used to detect pathogens parasitizing in animal blood or blood cells.

[0023] Furthermore, the pathogen is selected from parasites and / or microorganisms.

[0024] The parasite is selected from at least one of the following genera: Plasmodium, Trypanosoma, Babesia, Theileria, Aplasticum, Leishmania, Filarial parasites, and Toxoplasma gondii.

[0025] Unless otherwise stated, the parasites and / or microorganisms described in this invention include, but are not limited to, the following types: Plasmodium genus: Plasmodium falciparum ( Plasmodium falciparum ), Plasmodium vivax ( Plasmodium vivax ), Plasmodium malariae ( Plasmodium malariae ), Plasmodium ovale ( Plasmodium ovale ), Plasmodium norotri ( Plasmodium knowlesi Plasmodium berghei ( Plasmodium berghei ), Plasmodium yoelii ( Plasmodium yoelii ), chicken malaria parasite ( Plasmodium gallinaceum )wait; Trypanosoma genus: Trypanosoma brücken ( Trypanosoma brucei Trypanosoma cruzi (), Trypanosoma cruzi () Trypanosoma cruzi Trypanosoma eiri ( ), Trypanosoma eiri ( Trypanosoma evansi Trypanosoma japonicum ( Trypanosoma equiperdum Trypanosoma japonicum ( ), Trypanosoma japonicum ( ) Trypanosoma vivax Congo trypanosome ( Trypanosoma congolense )wait; Babesia genus: Babesia vole ( Babesia microti ), Babesia divaricata ( Babesia divergens ), Bogobabesi ( Babesia bovis ), Babesia dibuds ( Babesia bigemina Mababesi ( Babesia equi Babesia canis ( Babesia canis ), feline babesi ( Babesia felis )wait; Theileria genus: Theileria ring-shaped Theileria annulata Theileria serrulata ( Theileria sergeants ), Little Taylor worm ( Theileria parva ), Sheep Theylodes ( Theileria ovis Theileria roxburghii ( Theileria lawrencei )wait; Non-slurry type: Edge non-slurry ( Anaplasma marginale ), Central non-slurry ( Anaplasma central ), phagocytic cells without plasma bodies ( Anaplasma phagocytophilum ), flat non-slurry ( Anaplasma platys ), sheep without sebaceous body ( Anaplasma ovis )wait; Leishmania genus: Leishmania donovani ( Leishmania donovani ), Leishmania infantis ( Leishmania infantum ), giant Leishmania ( Leishmania major ), Tropical Leishmania ( Leishmania tropica ), Leishmania brasiliensis ( Leishmania braziliensis )wait; Filariaceae superfamily parasites: *Wuceta bancrocarpa* ( Wuchereria bancrofti ), Malayan brurus ( Bruges Malay Timorbrunneus ( Brugia timori Loa lo ... Loa loa ), Onchocerca filaria ( Onchocerca volvulus ), commonly found filarial worms ( Mansonella perstans ), Erwinia filarialis ( Mansonella ozzardi ), filariasis ( Mansonella streptocerca ), canine filarial worm ( Dirofilaria immitis ), creeping filarial worms ( Dirofilaria repens (etc.), and the microfilariae stage of the aforementioned filarial worms in the host's blood or tissues; Other blood parasites or microorganisms: Toxoplasma gondii ( Toxoplasma gondii Histoplasma ( ) Histoplasma capsulatum ), horsetail beetle ( Trypanosoma theileri Trypanosomes ( Trypanosoma rangeli ), regressive heat spirochetes ( Borrelia recurrentis )wait.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The staining method of the present invention can achieve accurate identification based on multicolor fluorescence and image analysis. Specifically, the staining agent can induce characteristic morphological and fluorescence changes in parasites and host cells, resulting in DNA, RNA and other components exhibiting rich color gradients such as green, red, yellow, orange, and yellow-green. By using AI algorithms to assist in the identification of the size and brightness of different color regions in the stained image, a comprehensive analysis of multidimensional features such as nucleic acid content, cell morphology, size, color distribution and relative position can be performed, significantly improving the identification accuracy of different cell types (including mature and immature blood cells) and parasites (including parasites at different developmental stages) or microorganisms.

[0027] (2) The present invention can achieve automated identification and counting by moderately changing the permeability and morphology of cell membranes under near-physiological conditions, in conjunction with imaging systems and AI algorithms, effectively reducing the reliance on manual microscopic examination experience and improving the consistency and reliability of detection.

[0028] (3) The application of the present invention can significantly improve detection efficiency, increase the detection rate of samples with low protozoan density, shorten the detection time of laboratory diagnostic microscopy of parasites, reduce the influence of the technical level of microscopists on the detection results, and realize high-throughput detection of parasite morphology. Attached Figure Description

[0029] Figure 1Typical images of Plasmodium parasites at different developmental stages under a microscope for laboratory diagnostic examination.

[0030] Figure 2 This is an example of the detection method of the present invention for the infection status of Plasmodium at different developmental stages (I).

[0031] Figure 3 This is the second part of the detection scheme of the present invention to show the infection status of Plasmodium at different developmental stages.

[0032] Figure 4 This invention presents the detection scheme for malaria parasite infection at different developmental stages (Part III).

[0033] Figure 5 This is the fourth part of the detection scheme of the present invention to show the infection status of Plasmodium at different developmental stages.

[0034] Figure 6 This is a fluorescence detection image of a blood sample containing malaria parasites in Example 5.

[0035] Figure 7 This is a fluorescence detection image of a blood sample containing malaria parasites from Example 6.

[0036] Figure 8 The image shows the fluorescence detection of the blood sample containing malaria parasites in Comparative Example 1.

[0037] Figure 9 The image shows the fluorescence detection of the blood sample containing malaria parasites in Comparative Example 2. Detailed Implementation

[0038] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0039] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the claimed invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] To address the shortcomings of existing diagnostic methods for blood parasites: traditional morphological microscopy relies on experience and is inefficient; immunological methods suffer from sensitivity and specificity deficiencies, easily resulting in false positives / negatives; while molecular detection is sensitive, it requires advanced equipment and is costly, making it difficult to implement at the grassroots level. Furthermore, while existing multicolor fluorescence staining techniques (such as CN104169719B and CN106840812B) can distinguish between DNA and RNA, they lack differentiation staining effects on subtle structural features in samples, thus limiting their accuracy in identifying minute morphological changes within cells.

[0041] To address this, the present invention provides a novel staining reagent composed of a fluorescent dye, an osmotic pressure regulator, a pH buffer, and an auxiliary agent. These components work synergistically at specific concentrations to achieve differentiated, multi-level fluorescent staining of morphological components in blood samples under near-physiological conditions. Specifically, it can clearly stain the morphology of reticulocytes at different maturity levels. For example, reticulocytes can be stained and classified into developmental stages based on their morphology after staining, such as broken-reticulocyte, granular, and dotted-granular types. Furthermore, it can also identify parasites or microorganisms within red blood cells based on differences in their nucleic acid (DNA or RNA) content. For example, DNA stains green, cytoplasm and RNA stain red, and platelets stain from green to yellow. Based on the different gradients of fluorescent colors (yellow, green, orange, red, etc.) observed in the captured images, multi-dimensional morphological characteristics, including nucleic acid (DNA and RNA) content, cell morphology, size, and distribution, can be extracted, ultimately achieving accurate identification and counting of parasites or microorganisms.

[0042] The following is a detailed overview of the technical solution of this invention: This invention provides a staining agent, staining method, and application for the morphological detection of parasites in blood. The staining agent comprises the following components: Fluorescent dye: Contains acridine orange or an acceptable salt thereof, in a concentration ranging from 1 μg / mL to 100 μg / mL, and may further contain at least one of DAPI, ethidium bromide, Hoechst series dyes or SYBR series dyes to enhance the diversity and specificity of nucleic acid staining; Osmotic regulators, such as sodium chloride, potassium chloride, glucose, sucrose, trehalose, etc., with a concentration range of 0.01% to 10.0% (w / v), are used to maintain the morphological integrity of cells under near-physiological osmotic pressure. pH buffer: Selected from sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, phosphate buffer (PBS, PB), glycine, (2-(N-morpholino)ethanesulfonic acid (MES), tris(hydroxymethyl)aminomethane (Tris), etc., to maintain the pH of the staining system within a stable range of 7.0 to 7.4; Auxiliary agents include one or more of surfactants (such as anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants), fixatives (such as paraformaldehyde and glutaraldehyde), or glycerol. Their concentrations can be adjusted within a reasonable range (10 mg / L to 10 g / L) according to the specific application to optimize cell membrane permeability and stabilize staining morphology. Specifically, as anionic surfactants, options include organic carboxylates, organic sulfonates, and organic phosphates, such as dodecyl carboxylic acid and its salts, dodecyl sulfuric acid and its salts, and dodecyl sulfonic acid and its salts; as cationic surfactants, options include quaternary ammonium salts and pyridinium salts, such as hexadecyltrimethylammonium bromide, dodecyltrimethylammonium bromide, and tetradecyltrimethylammonium chloride; as amphoteric surfactants, options include sulfobetaine, alkyl betaine, and cocamidopropyl betaine, such as cocamidopropyl betaine; and as nonionic surfactants, options include fatty acid glycerides and polyols, such as fatty acid monoglycerides and fatty acid diglycerides.

[0043] The staining method of this invention involves mixing the aforementioned staining agent with a blood sample to be tested at a certain volume ratio (staining agent: blood sample = 9:1 to 99:1). While maintaining the basic physiological state of the cells, this induces observable characteristic morphological and fluorescence signal changes in the parasites and their host cells. The stained sample can then be used to acquire multi-channel fluorescence images through a microscopic imaging system. Furthermore, based on multi-dimensional features such as DNA / RNA content, morphology, size, color gradient, and spatial distribution in the images, combined with image analysis algorithms, automatic identification, classification, and counting of parasites can be achieved.

[0044] In practical applications, an integrated detection process can be adopted to achieve automated identification and counting. For example, the stained sample is placed in a dedicated detection plate or counting plate and inserted into a matching microscopic imaging system. The system automatically completes image acquisition and calls the built-in AI algorithm to perform real-time analysis of multi-dimensional features such as fluorescence intensity, morphology, size, distribution, and relative position of different color regions (such as green, red, and yellow) in the image. Finally, the system automatically completes target classification and counting according to preset recognition logic and outputs a structured detection report. This process requires no manual intervention, significantly reduces reliance on the experience of microscopists, improves the consistency and reliability of detection, and is suitable for high-throughput, standardized laboratories and rapid on-site screening scenarios.

[0045] In summary, the staining agent and staining method of the present invention can achieve the following effects in blood fluorescence staining: (1) Unlike traditional staining which only stains, the staining agent of the present invention can actively induce parasites and blood cells to undergo characteristic morphological and structural changes that are conducive to microscopic identification, enhance the visual and signal differences between the target and the background, and achieve the specificity and sensitivity of detection.

[0046] (2) The staining agent of the present invention can achieve gradient changes of multicolor fluorescence, realize the transition from single color differentiation (green / red) to continuous color gradient (yellow, orange, yellow-green, etc.), and can simultaneously extract multidimensional features such as nucleic acid content, morphology, distribution, and color, thus improving the accuracy of identification.

[0047] (3) The staining method of the present invention can be completed under near-physiological conditions. The sample pretreatment is simple and the staining is fast. It can be combined with automated imaging and analysis. It can achieve high-throughput and low-manual-dependence detection while maintaining high sensitivity. It is suitable for laboratories at different resource levels.

[0048] (4) The staining method of the present invention is not only applicable to the detection of common blood parasites (such as Plasmodium, Babesia, filaria, etc.), but can also be extended to the morphological identification of cells (such as immature leukocyte-immature leukocyte typing, immature red blood cell-reticulocyte typing, etc.), and has a wide range of applications.

[0049] More specifically, in one practical application of the present invention, targeting Plasmodium parasites at different developmental stages (see...) Figure 1 Using the staining reagent and staining method of this invention, effective identification of nucleic acid distribution characteristics after Plasmodium infection can be achieved. Specifically, such as... Figure 2 to Figure 5 As shown, under the detection scheme of this invention, the surface cell structure of Plasmodium and the distribution of its characteristic metabolite—malaria pigment—can be observed simultaneously under bright field conditions; at the same time, under dark field conditions, the staining characteristics and spatial distribution of nucleic acid substances in the host's erythrocytes after Plasmodium infection can be clearly identified. Through the above multimodal imaging analysis, the morphological characteristics and internal structure of Plasmodium at different developmental stages can be comprehensively classified and accurately counted from more dimensions and richer information levels, significantly improving the accuracy and reliability of Plasmodium identification.

[0050] in, Figure 1 The image shows the morphological changes of Plasmodium at different developmental stages under a laboratory diagnostic microscope. Figure 1 In Figure a, all are PV ring bodies, with visible punctate purplish-red chromatin and nearly ring-shaped blue cytoplasm; in Figure b, all are PV trophozoites, with visible diffuse chromatin and irregularly shaped cytoplasm; in Figure c, all are PV schizonts, with visible central or eccentric brown pigment spots and mature merozoites; in Figure d, all are PVdv gametophytes, with visible enlarged cell bodies and scattered pigment spots.

[0051] Figure 2 to Figure 5 This invention describes the detection method for detecting Plasmodium infection at different developmental stages. Figure 2 Image a1 contains bright-field and fluorescent-field images of the same erythrocyte (RBC). Under bright-field conditions, only changes in cell shape are visible in the circular bodies. Under fluorescent conditions, the chromatin of the Plasmodium circular bodies appears green, and the cytoplasm appears orange-red. Image a2 shows an infection of a reticulocyte. Under bright-field conditions, the Plasmodium appears as a pale spot. Under fluorescent conditions, the green chromatin of the Plasmodium and the typical reticulated orange-red morphology of the reticulocyte are visible. This staining morphology demonstrates the ability of this invention to achieve color gradient changes, which is difficult to achieve with existing in vivo AO staining methods. Figure 3 Images b1 and b2 show the typical trophozoite morphology of Plasmodium after staining according to this invention. Clear pigment granules are visible in the bright field image, while diffuse trophozoite cytoplasm and chromatin are visible in the fluorescent field. Figure 4 C1 and C2 represent the typical morphology of Plasmodium schizonts after staining according to this invention. In bright field images, central or off-center pigment clusters and white refractive merozoites are visible, while in fluorescent field images, bright merozoites are visible with green / yellow staining. Figure 5 In the image, d1 and d2 represent the morphology of the female and male gametophytes after staining according to the present invention. In the bright field image, the cells are full and enlarged, and the pigment particles are dispersed. Under the fluorescence field, they appear orange-yellow and diffuse green fluorescence.

[0052] The specific implementation of the present invention will be described below with reference to the embodiments. Of course, the scope of protection of the present invention is not limited to the following embodiments.

[0053] Example 1: Preparation of dye one Dissolve 0.8 g of sodium chloride, 0.0296 g of sodium dihydrogen phosphate dihydrate, 0.29 g of disodium hydrogen phosphate dodecahydrate, and 0.02 g of potassium chloride in 90 mL of deionized water. After stirring and dissolving, bring the volume to 100 mL to prepare a phosphate buffer solution (PBS) with a pH of 7.4.

[0054] Take 100 mL of the above PBS buffer solution, add 0.5 mL of glycerol and 0.4 mg of acridine orange hydrochloride hydrate, and stir thoroughly to prepare staining agent one.

[0055] Example 2: Preparation of dye two Take 0.8 g of sodium chloride, 0.0296 g of sodium dihydrogen phosphate dihydrate, 0.29 g of disodium hydrogen phosphate dodecahydrate, and 0.02 g of potassium chloride, dissolve them in 90 mL of deionized water, stir to dissolve, and then make up to 100 mL to prepare a phosphate buffer (PBS) with pH 7.4.

[0056] Take 100 mL of the above PBS buffer solution, add 1 mg of sodium dodecyl sulfate, 0.3 g of trehalose and 1.5 mg of acridine orange hydrochloride hydrate, and stir thoroughly to prepare staining agent two.

[0057] Example 3: Blood Sample Staining A 10 μL sample was collected using a disposable capillary blood collection tube and thoroughly mixed with 290 μL of the staining agent prepared in Example 1. The mixture was then dropped into a cell counting chamber and allowed to stand for 5 minutes to allow the cells to settle. The cell counting chamber was then inserted into a matching microscopic imaging instrument for analysis.

[0058] Example 4: Staining of blood samples Using a pipette, 10 μL of normal human venous whole blood was drawn and thoroughly mixed with 290 μL of staining agent II prepared in Example 2. The mixture was then dropped into a cell counting chamber and allowed to stand for 5 minutes to allow the cells to settle. The cell counting chamber was then inserted into a matching microscopic imaging instrument for analysis.

[0059] Example 5: Fluorescence Image Detection and Analysis The stained samples from Example 3 were analyzed for fluorescence detection using microscopic imaging technology (the integrated blood cell and immune detection machine described in Chinese Patent CN117368172A). During detection, both the bright-field light source and the fluorescence light source were turned on simultaneously, and bright-field and fluorescence field images were acquired and fused for analysis.

[0060] Test results are shown Figure 6 ,in, Figure 6 In the image, A is the bright field image and B is the fluorescence field image.

[0061] Depend on Figure 6 As can be seen, after staining with the staining agent of this invention, the erythrocytes infected with Plasmodium exhibit clear abnormal nucleic acid staining morphology, mainly showing green and yellow fluorescence signals, which are significantly different from uninfected erythrocytes. Combined with the granular structure observed in the erythrocytes under bright field and the distribution characteristics of nucleic acid content under fluorescent field, the erythmodium can be accurately identified as being in the immature clonal development stage.

[0062] The results of this embodiment show that the staining agent described in this invention, combined with an automated microscopic imaging system, can effectively distinguish between infected and uninfected cells and further accurately identify the developmental stage (schizont) of Plasmodium.

[0063] Example 6: Fluorescence Image Analysis The stained samples from Example 4 above were analyzed for fluorescence detection using microscopic imaging technology (the integrated blood cell and immune detection machine described in Chinese Patent CN117368172A). During detection, both the bright-field light source and the fluorescence light source were turned on simultaneously, and bright-field and fluorescence field images were acquired and fused for analysis.

[0064] Test results are shown Figure 7 ,in, Figure 7 In the image, A is the bright field image and B is the fluorescence field image.

[0065] Depend on Figure 7 As can be seen, after staining with the second staining agent of this invention, normal erythrocytes showed no obvious fluorescent signal under a fluorescent field. However, erythrocytes infected with Plasmodium showed clear abnormal nucleic acid staining morphology due to the invasion of Plasmodium, mainly manifested as yellow, orange, and green fluorescent signals, exhibiting rich color gradient changes. By combining the morphology of erythrocytes under a bright field and the distribution and color differences of nucleic acid signals under a fluorescent field for comprehensive analysis, the trophozoite stage of the Plasmodium can be accurately identified.

[0066] The results of this embodiment show that the staining agent of the present invention, combined with an automated microscopic imaging system, can not only effectively identify infected red blood cells, but also accurately determine the developmental stage (trophozoite) of Plasmodium based on fluorescence color gradient and morphological characteristics, further verifying the feasibility of the staining agent of the present invention in parasite stage counting.

[0067] Comparative Example 1: Traditional staining method (physiological saline formulation) Using the same Plasmodium-infected blood samples as in Example 3, a comparative experiment was conducted using conventional staining methods.

[0068] Staining agent: physiological saline solution containing 2 μg / mL acridine orange.

[0069] Staining method: Take 10 μL of blood sample, mix it thoroughly with 290 μL of the above staining agent, drop it into a cell counting chamber, let it stand for 5 min until the cells settle, and then analyze it according to the same fluorescence detection method as in Example 5.

[0070] Test results are shown Figure 8 .

[0071] Depend on Figure 8 As can be seen, after staining with traditional staining methods, red blood cells maintain their original biconcave disc-shaped physiological state without significant morphological changes (see A1, A2). Although nucleic acid substances can be observed in the cells under a fluorescent field (see B1, B2, showing green fluorescence), the nucleic acid staining effect is limited and cannot effectively distinguish the content and morphological distribution of nucleic acids in the cells.

[0072] Results analysis: Acridine orange staining solution prepared with traditional physiological saline can only confirm the presence of abnormal nucleic acid substances in red blood cells, but it does not have the effect of differentiation staining, and therefore cannot be used to count the different developmental stages of Plasmodium (such as schizonts and trophozoites).

[0073] Furthermore, based on the detection results of Example 5 and Comparative Example 1, it can be seen that traditional staining methods can only indicate the presence or absence of nucleic acids, but cannot effectively classify the species or developmental stage of Plasmodium, and have obvious limitations in terms of clinical application value for accurate diagnosis and treatment of the disease.

[0074] Comparative Example 2: Traditional staining method (PBS buffer formulation) Using the same blood sample as in Example 4, staining was performed using conventional staining methods: Staining agent: pH 7.4 PBS buffer containing 2 μg / mL acridine orange.

[0075] Staining method: Take 10 μL of blood sample, mix it thoroughly with 290 μL of the above staining agent, drop it into a cell counting chamber, let it stand for 5 min until the cells settle, and then analyze it according to the same fluorescence detection method as in Example 6.

[0076] Test results are shown Figure 9 .

[0077] Depend on Figure 9 As can be seen, after staining with traditional staining methods, red blood cells maintain their original biconcave disc-shaped physiological state (see A1, A2). Although abnormal nucleic acid substances caused by scar protozoan infection can be observed under fluorescence (see B1, B2, showing green fluorescence), there is a lack of color gradient changes, making it impossible to distinguish the differences in nucleic acid content and morphological distribution characteristics.

[0078] Results Analysis: While the acridine orange staining solution prepared with traditional pH 7.4 PBS buffer provides a near-physiological pH environment, it still lacks differentiation staining effectiveness. It can only confirm the presence of abnormal nucleic acid substances within cells and cannot accurately identify the developmental stage of Plasmodium. Furthermore, in traditional staining methods, RNA in reticulocytes and DNA in nucleated erythrocytes both exhibit green fluorescence, failing to effectively distinguish cells infected by these nuclear parasites.

[0079] Furthermore, based on the detection results of Example 6 and Comparative Example 2, it can be seen that the traditional PBS staining scheme also cannot effectively classify the species or developmental stage of Plasmodium, further verifying the key role of the auxiliary agents (surfactants, glycerol, etc.) in the staining agent of the present invention in achieving "differentiation staining" and "stage counting".

[0080] Experimental Example 1: Specificity Detection Experimental objective: To evaluate the detection specificity of the staining agent described in this invention on blood samples from uninfected individuals and to verify whether it produces nonspecific positive results.

[0081] Experimental Methods: A total of 172 blood samples were collected from healthy volunteers who were confirmed negative for Plasmodium by clinical microscopy and PCR. Staining agents one and two prepared in Example 1 of this invention were used to process each sample according to the staining methods described in Examples 3 and 4, respectively. Automated image acquisition and analysis were performed using the microscopic imaging systems described in Examples 5 and 6. The detection process was fully automated and required no manual interpretation. The system automatically output "Plasmodium positive" as the judgment criterion, and the test results were recorded.

[0082] The test results are shown in Tables 1 and 2.

[0083] Table 1. Results of staining agent specificity detection

[0084] Table 2. Results of specificity detection of the staining agent.

[0085] As shown in Tables 1 and 2, the staining agents 1 and 2 described in this invention have good specificity for blood samples from healthy individuals.

[0086] Experiment Example 2: Sensitivity Detection Experimental objective: To evaluate the detection sensitivity of the staining agent described in this invention on positive samples of Plasmodium malaria with different infection densities and developmental stages.

[0087] Experimental Methods: A total of 95 blood samples were collected from individuals clinically confirmed to be positive for Plasmodium by microscopic examination. The parasite species in the samples were identified as Plasmodium vivax. The parasite density in the samples ranged from 80 to 41,195 individuals / μL. The specific detection procedure was the same as in Experiment 1.

[0088] The test results are shown in Tables 3 and 4.

[0089] Table 3. Results of staining agent sensitivity test

[0090] Table 4. Results of staining agent sensitivity test

[0091] As shown in Tables 3 and 4, the staining agents 1 and 2 described in this invention have extremely high detection rates for Plasmodium samples of different species, different developmental stages, and different infection densities, which can meet the sensitivity requirements of clinical screening and diagnosis of Plasmodium.

[0092] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A staining agent for morphological detection of pathogens in blood, characterized in that: The staining agent comprises: (1) A fluorescent dye containing acridine orange or an acceptable salt thereof, wherein the mass concentration of acridine orange or an acceptable salt thereof in the dye is from 1 μg / mL to 100 μg / mL; (2) An osmotic pressure regulator, wherein the volume concentration in the dye is 0.01% to 10.0%; (3) pH buffer, used to adjust and maintain the pH of the staining agent at 7.0 to 7.4; (4) An auxiliary agent selected from surfactants, fixatives, glycerol or combinations thereof, wherein the mass concentration of the auxiliary agent in the dyeing agent is from 10 mg / L to 10 g / L.

2. The staining agent according to claim 1, characterized in that: The fluorescent dye also includes at least one nucleic acid dye selected from the group consisting of DAPI, ethidium bromide, Hoechst series dyes, SYBR series dyes, or acceptable salts or combinations thereof.

3. The staining agent according to claim 1, characterized in that: The osmotic pressure regulator is selected from one or more of sodium chloride, potassium chloride, glucose, sucrose, and trehalose.

4. The staining agent according to claim 1, characterized in that: The pH buffer is selected from one or more combinations of sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, PBS, PB, glycine, MES, and Tris.

5. The staining agent according to claim 1, characterized in that: The surfactant is selected from one or more of anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants.

6. The staining agent according to claim 1, characterized in that: The fixative is selected from one or a combination of two or more of paraformaldehyde, glutaraldehyde, methanol, ethanol, and acetone.

7. A staining method for morphological detection of pathogens in blood, characterized in that: The staining agent described in any one of claims 1 to 6 is mixed with the blood sample to be tested for nucleic acid staining, so that the parasites, microorganisms and / or cells in the blood sample produce characteristic morphological changes and fluorescence signal changes, which are used for identification analysis based on DNA content, RNA content, morphology, distribution and color characteristics.

8. The staining method according to claim 7, characterized in that: The volume ratio of the staining agent to the blood sample is from 9:1 to 99:

1.

9. The use of the staining agent according to any one of claims 1 to 6 in the preparation of in vitro diagnostic reagents, characterized in that: The in vitro diagnostic reagent is used to detect pathogens that parasitize in animal blood or blood cells.

10. The application according to claim 9, characterized in that: The pathogens are selected from parasites and / or microorganisms.

11. The application according to claim 10, characterized in that: The parasite is selected from at least one of the following genera: Plasmodium, Trypanosoma, Babesia, Theileria, Aplasticum, Leishmania, Filarial parasites, and Toxoplasma gondii.

Images