A room-temperature preservation solution for bacterial RNA from sputum samples of tuberculosis patients.

Abstract

This invention relates to the field of biological sample preservation technology, specifically to a room-temperature preservation solution for bacterial RNA from sputum samples of tuberculosis patients. Each 100 mL of the RNA preservation solution comprises the following ingredients: 2-5 g tris(hydroxymethyl)aminomethane, 0.5-1.3 g mucin digester, 20-30 g guanidine isothiocyanate, 0.1-0.3 g hexadecylpyridine chloride, 0.5-1.2 g ethylhexene dicysteine, 20-40 mL fixative, 1-5 g stabilizer, 0.1-0.5 g inorganic salt, 0.3-0.6 g surfactant, and 0.1-0.5 g preservative. The preservation solution provided by this invention can effectively liquefy sputum samples at room temperature, meeting the requirements for long-distance sample transportation. The RNA quality of sputum samples treated with the preservation solution of this invention is suitable for various subsequent molecular detection methods, possessing significant application value and promising prospects for widespread application.

Description

Technical Field

[0001] This invention relates to the field of biological sample preservation technology, specifically to a room-temperature preservation solution for bacterial RNA in sputum samples from tuberculosis patients. Background Technology

[0002] The detection of Mycobacterium tuberculosis RNA in the sputum of patients with pulmonary tuberculosis is strong evidence of the presence of live bacteria and the patient's potential infectious period. Compared with traditional detection methods, RNA detection has unique advantages in assessing infection activity and the risk of transmission. Bacterial ribosomal RNA (rRNA) is a direct product of life activities, with an extremely short half-life (minutes to hours), and is continuously synthesized only when bacteria are alive and undergoing metabolism. Traditional sputum smear microscopy and DNA detection cannot distinguish between dead and live Mycobacterium tuberculosis, while a positive RNA detection, especially rRNA detection, strongly suggests the presence of metabolically active Mycobacterium tuberculosis in the sample. Studies have shown that within 4 days after effective treatment, the abundance of Mycobacterium tuberculosis mRNA decreases by more than 98%, which can dynamically reflect the treatment effect to some extent. In addition, persistently positive sputum RNA is a key indicator for diagnosing active pulmonary tuberculosis (rather than latent infection), and a positive result also suggests that the patient may be in the infectious period. However, the widespread adoption of RNA detection in clinical practice faces two major obstacles: First, Mycobacterium tuberculosis differs from ordinary viruses, possessing an extremely complex and resilient cell wall structure that makes it difficult to inactivate using conventional methods. The cell wall of Mycobacterium tuberculosis consists of a peptidoglycan layer, an arabinogalactan layer, and a mycolic acid layer, arranged from the inside out. This densely packed, lipid-rich, and highly hydrophobic envelope exhibits extremely poor fluidity and permeability, forming a natural barrier against external chemical substances and physical pressures. This unique cell wall structure not only gives Mycobacterium tuberculosis strong resistance to the external environment but also makes it difficult for conventional chemical inactivating agents to effectively penetrate and kill the bacteria. Second, RNA molecules themselves are highly degradable, requiring extremely stringent sample processing and preservation conditions, and are inconvenient to transport and deliver, affecting test results. In microbiological operations, to ensure biosafety, samples containing Mycobacterium tuberculosis typically need to be processed in a biosafety level 2 (BSL-2) or higher laboratory. Traditional physical inactivation methods, such as high-temperature autoclaving (121°C), can completely kill Mycobacterium tuberculosis. However, this process causes bacterial cell rupture, releasing large amounts of ribonuclease (RNase). Simultaneously, the high temperature itself rapidly degrades RNA. Mycobacterium tuberculosis encodes various ribonucleases (such as RNase AS and RNase J), ​​which are released after bacterial lysis and can rapidly degrade RNA in the sample. Therefore, while the DNA of samples treated with high temperature and autoclave may remain intact for subsequent PCR detection, the RNA is almost completely degraded and cannot be used to determine viability.

[0003] Currently, in clinical practice, RNA extraction and detection from sputum samples of tuberculosis patients is often performed directly in the laboratory without inactivation to avoid RNA degradation. This limitation significantly hinders the clinical adoption of RNA detection technology. Unlike the widely implemented DNA testing, RNA-based detection of Mycobacterium tuberculosis in sputum involves complex and stringent operational risks, making it difficult to routinely perform in most medical institutions in my country. This has led to a clinical dilemma: although RNA detection has important value in identifying viable bacteria, assessing infectivity, and monitoring treatment efficacy, due to biosafety concerns, this technology has long remained at the research level and has failed to truly serve clinical diagnosis and treatment.

[0004] With the development of molecular biology techniques, molecular detection methods such as targeted next-generation sequencing (tNGS), metagenomic sequencing (mNGS), and polymerase chain reaction (PCR) have become effective in qualitatively diagnosing tuberculosis infection due to their speed, sensitivity, and high specificity. These methods require nucleic acid extraction from sputum for testing. However, sputum contains a large amount of mucus, making cellular components difficult to separate, which poses a significant challenge to nucleic acid extraction. More importantly, if sputum samples are not tested immediately after collection, prolonged storage can lead to contamination, bacterial growth, or microbial death, resulting in nucleic acid degradation and inaccurate test results. The microbial composition and abundance in the original sample may also change, failing to reflect the true situation. In many parts of my country, especially in some grassroots units with poor medical conditions, samples often need to be transported long distances to large hospitals with testing capabilities. During long-term transportation in complex terrain conditions such as rugged mountain roads, bacteria in the sputum sample may rupture, causing RNA release, which is then rapidly degraded by RNase. Therefore, developing a preservation solution that can stably preserve bacterial RNA in sputum samples at room temperature while completely inactivating Mycobacterium tuberculosis is of great significance for overcoming the clinical translation bottleneck of RNA detection and promoting the widespread application of live bacteria detection technology.

[0005] Currently, sputum pretreatment products on the market are mainly divided into two categories: one is a sputum dissolving agent with sodium hypochlorite and surfactants as the main components; the other is a sputum digestion solution with acetylcysteine, sodium citrate, and phosphate buffer as the main components; and there are also liquefaction agents with ammonia-based aqueous solutions or dithiothreitol as the main components. Patent CN106967778A discloses a rapid sputum dissolving and preservation solution containing ethanol, glycerol, vegetable oil, cladonic acid, disodium EDTA, SDS, and thioglycolate, which can shorten the dissolution rate from 2 minutes to 30 seconds. However, the main uses of the above products are: homogenization of sputum before smears or culture; thin-layer cell preparation of human cell specimens before pathological analysis; and digestion of sputum samples. Although these digestion products can be used for nucleic acid extraction, they do not have the function of protecting the stability of nucleic acids (especially RNA) during transportation, nor can they inactivate or inhibit the activity of pathogenic microorganisms (especially Mycobacterium tuberculosis), which brings significant biosafety risks to subsequent experimental operations.

[0006] The inventor's previous patent CN119061004A disclosed a sputum sample microbial community nucleic acid (DNA) preservation solution. By combining N-octylpyridine bromide and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and with the aid of ethanol as a fixative, it can effectively preserve the microbial community and DNA in sputum, maintaining DNA integrity for up to 14 days at room temperature without disrupting the diversity of the microbial community structure. However, this formulation is primarily designed for DNA preservation; its protective effect is insufficient for RNA, which has a very short half-life and is highly sensitive to RNases, to meet the requirements of RNA detection. Studies have shown that 70% ethanol can effectively preserve mycobacterial RNA under suitable conditions, while also possessing bactericidal properties. However, a single component cannot simultaneously achieve the multiple objectives of sputum liquefaction, RNase inhibition, RNA stabilization, and long-term preservation.

[0007] Therefore, there is an urgent need in this field to develop an RNA preservation solution specifically for sputum samples from tuberculosis patients. This solution should possess the following characteristics: 1. It should be able to efficiently lyse the mucus components in the sputum, homogenizing the sample; 2. It should be able to completely penetrate the thick cell wall of Mycobacterium tuberculosis, achieving complete bacterial inactivation, eliminating biosafety risks, and without affecting the extraction of Mycobacterium tuberculosis RNA; 3. It should be able to effectively inhibit endogenous and exogenous RNase activity, protecting bacterial RNA molecules from degradation; 4. It should be able to stably preserve RNA at room temperature, meeting the needs of long-distance sample transportation; 5. The preserved RNA should be suitable for subsequent molecular detection (such as RT-qPCR, RNA sequencing, etc.), accurately reflecting the viability of live bacteria in the original sample. Such a multifunctional RNA preservation solution integrating inactivation, preservation, and liquefaction is expected to completely solve the biosafety risks of Mycobacterium tuberculosis RNA detection, promoting this clinically valuable technology from the laboratory to the clinic and enabling its widespread application. Summary of the Invention

[0008] Existing sputum preservation solutions have significant shortcomings in practical applications, mainly in the following aspects: long-term preservation of sputum samples at room temperature is difficult; they cannot simultaneously achieve nuclease activity inhibition, pathogen inactivation, and sputum liquefaction; their inactivation effect on pathogens such as Mycobacterium tuberculosis with thick cell walls is poor, posing biosafety risks; and their short preservation time cannot meet the needs of long-distance sample transportation, leading to inaccurate subsequent RNA detection results. Based on the above background, to overcome the technical deficiencies of existing sputum preservation solutions in simultaneously achieving effective inactivation of infectious pathogens and stable RNA preservation, this invention provides a room-temperature sputum sample RNA preservation solution.

[0009] The RNA preservation solution provided by this invention can effectively liquefy sputum samples at room temperature and possesses the following core functions: 1. It completely penetrates the thick cell wall structure of pathogenic microorganisms such as Mycobacterium tuberculosis, achieving complete inactivation and eliminating biosafety risks; 2. The inactivation process does not affect the integrity of bacterial RNA or subsequent extraction efficiency; 3. It efficiently inhibits the activity of endogenous and exogenous ribonucleases (RNases), protecting bacterial RNA molecules from degradation; 4. It can achieve long-term stable preservation of RNA at room temperature, meeting the needs of long-distance sample transportation. Sputum samples treated with this invention have RNA quality suitable for various subsequent molecular detection methods (such as RT-qPCR, RNA sequencing, etc.), accurately reflecting the viability of live bacteria in the original sample. This invention's multifunctional design, integrating liquefaction, inactivation, and preservation, effectively solves the biosafety challenges in Mycobacterium tuberculosis RNA detection, breaks through existing technological bottlenecks, and promotes the widespread clinical application of RNA-based Mycobacterium tuberculosis live bacteria detection technology from the laboratory, demonstrating significant application value and promising prospects.

[0010] The technical solution adopted in this invention is as follows:

[0011] A room-temperature preservation solution for bacterial RNA from sputum samples of tuberculosis patients, comprising the following ingredients per 100 mL: 2-5 g tris(hydroxymethyl)aminomethane, 0.5-1.3 g mucin digester, 20-30 g guanidine isothiocyanate, 0.1-0.3 g cetylpyridine chloride, 0.5-1.2 g ethylhexylcysteine, 20-40 mL fixative, 1-5 g stabilizer, 0.1-0.5 g inorganic salt, 0.3-0.6 g surfactant, and 0.1-0.5 g preservative.

[0012] The inventors unexpectedly discovered that guanidine isothiocyanate, hexadecyl pyridine chloride, and ethylenedicysteine ​​in the above-mentioned RNA preservation solution formulation exert a synergistic effect, enabling the RNA preservation solution of the present invention to maintain stable storage at room temperature. Guanidine isothiocyanate is a nuclease inhibitor; hexadecyl pyridine chloride is a quaternary ammonium salt with a long-chain alkyl group and has certain antibacterial activity; ethylenedicysteine ​​is a chelating agent commonly used in MRI contrast agents. Although the reason is unknown, the combination of the above three components in a certain proportion can significantly improve the room temperature preservation performance of the preservation solution for RNA, providing good preservation of RNA from sputum samples even after 7 days at room temperature. If guanidine isothiocyanate is replaced with other nuclease inhibitors, or hexadecyl pyridine chloride is replaced with other quaternary ammonium salts, or its dosage is not within the above-mentioned range, or ethylenedicysteine ​​is replaced with other chelating agents, the preservation solution cannot achieve the effect of room temperature preservation of RNA samples.

[0013] Furthermore, the RNA room temperature preservation solution comprises the following ingredients per 100 mL: 2-3 g tris(hydroxymethyl)aminomethane, 0.7-1.0 g mucin digester, 20-25 g guanidine isothiocyanate, 0.1-0.2 g cetylpyridine chloride, 0.6-0.9 g ethylhexene dicysteine, 30-35 mL fixative, 2-3 g stabilizer, 0.2-0.3 g inorganic salt, 0.4-0.5 g surfactant, and 0.1-0.2 g preservative.

[0014] Furthermore, the mucus digesting agent is selected from one or more of DTT (dithiothreitol), N-acetyl-L-cysteine, and TCEP (tris(2-carboxyethyl)phosphine hydrochloride); preferably TCEP, which, compared to other mucus digesting agents, can destroy the intermolecular disulfide bonds and intercellular plasmodesmata of mucin, thereby achieving the purpose of digesting mucus.

[0015] Furthermore, the inorganic salt is selected from at least one of sodium chloride, potassium chloride, lithium chloride, sodium sulfate, and sodium bicarbonate; preferably potassium chloride, to provide a salt ion environment.

[0016] Further, the fixative is selected from one or more of glycerol, ethanol, and isopropanol. Preferably, the fixative is a mixed alcohol solvent of glycerol, ethanol, and isopropanol in a volume ratio of 3-5:10-20:5-10.

[0017] Furthermore, the surfactant is selected from at least one of sodium dodecyl sarcosinate, sodium dodecyl sulfate, and Triton X-100.

[0018] Furthermore, the stabilizer is selected from at least one of sodium citrate, sodium tripolyphosphate, and sodium ascorbate.

[0019] Furthermore, the preservative is selected from at least one of proclin 300, sodium azide, imidazolidinyl urea, and methylparaben.

[0020] In this invention, the combination of the nuclease inhibitor guanidine isothiocyanate and hexadecylpyridine chloride enhances the inhibitory effect on RNase; furthermore, the combination of hexadecylpyridine chloride and ethanol better inactivates infectious pathogens; ethylenedicysteine ​​is used to chelate metal ions, sodium dodecyl sarcosinate is a surfactant with biosolubility; potassium chloride provides a salt ion environment, glycerol is a stable solution, and ethanol and isopropanol are used to precipitate nucleic acids and reduce foaming by the surfactant. The inventors have creatively discovered that the combination of guanidine isothiocyanate, hexadecylpyridine chloride, and ethylenedicysteine ​​produces a synergistic effect, improving the long-term preservation performance of sputum RNA samples at room temperature. The preservation solution of this invention can maintain the stability of sputum samples, inactivate infectious pathogens, rapidly liquefy sputum, inhibit RNase, obtain high-purity RNA, and improve detection accuracy. RNA extraction from liquefied sputum samples yields high purity. Samples preserved with the preservation solution of this invention can be used in various methods such as magnetic bead extraction, column extraction, and lysis without interference. Attached Figure Description

[0021] Figure 1 These are the results of bacterial culture using the preservation solutions from Examples 1, 2, and 3.

[0022] Figure 2 The results of bacterial culture in the preservation solutions of Comparative Examples 1 and 2 are shown.

[0023] Figure 3 The results were obtained through the RAA-cas12 test strip. Detailed Implementation

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0025] Unless otherwise specified, the experimental materials and reagents used in the following examples are all commercially available products.

[0026] Example 1

[0027] Weigh out 25g guanidine isothiocyanate, 0.2g hexadecylpyridine chloride, 0.4g sodium dodecyl sarcosinate, 0.9g ethylidene dicysteine, 0.3g potassium chloride, 3g tris(hydroxymethyl)aminomethane, 2g sodium citrate, 1g TCEP, 5mL glycerol, 15mL isopropanol, 10mL ethanol, and 0.1g proclin 300. Make up the volume to 100mL with sterile deionized water. Filter the solution through a 0.22μm sterile filter membrane to obtain the sputum RNA preservation solution.

[0028] Example 2

[0029] Weigh out 20g guanidine isothiocyanate, 0.1g hexadecylpyridine chloride, 0.5g sodium dodecyl sarcosinate, 0.6g ethylidene dicysteine, 0.2g potassium chloride, 2g tris(hydroxymethyl)aminomethane, 3g sodium ascorbate, 0.7g TCEP, 5mL glycerol, 15mL isopropanol, 10mL ethanol, and 0.2g proclin 300. Make up the volume to 100mL with sterile deionized water. Filter the solution through a 0.22μm sterile filter membrane to obtain the sputum RNA preservation solution.

[0030] Example 3

[0031] Weigh out 25g guanidine isothiocyanate, 0.08g hexadecylpyridine chloride, 0.4g sodium dodecyl sarcosinate, 1.1g ethylidene dicysteine, 0.3g potassium chloride, 3g tris(hydroxymethyl)aminomethane, 2g sodium citrate, 1g TCEP, 5mL glycerol, 15mL isopropanol, 10mL ethanol, and 0.1g proclin 300. Make up the volume to 100mL with sterile deionized water. Filter the solution through a 0.22μm sterile filter membrane to obtain the sputum RNA preservation solution.

[0032] Comparative Example 1

[0033] Everything else is the same as in Example 1, except that hexadecylpyridine chloride is replaced with an equal mass of hexadecyltrimethylammonium bromide.

[0034] Comparative Example 2

[0035] Everything else is the same as in Example 1, except that ethylidene cysteine ​​is replaced with an equal mass of ethylenediaminetetraacetic acid.

[0036] Application examples

[0037] The RNA preservation performance of the preservation solutions used in the above examples and comparative examples was tested: sputum samples from 5 patients were selected. 30 minutes before collecting saliva samples, the subjects rinsed their mouths with water and kept their oral tracts clean. After cleaning their mouths, they did not eat, smoke, chew gum, or drink water. The sputum was immediately placed in a clean and sterilized sputum container after coughing up. Samples from 5 subjects were obtained. Samples 1-5 were added to the preservation solutions of Examples 1-3 and Comparative Examples 1 and 2 at a volume ratio of 1:1.5, respectively. Each sample 1-5 was divided into 3 equal portions. The sputum samples from the examples and comparative examples with added preservation solutions were placed at room temperature (25±1℃) for 0 days, 3 days, and 7 days, respectively. The total amount and purity of nucleic acids were tested after purification at each time point.

[0038] The kit used was a column-extraction bacterial RNA extraction kit (Meiji Biotechnology R4182-02). Nucleic acid extraction and testing were performed according to the following steps:

[0039] (1) Take 1 ml of the sample containing the preservation solution into a 2.0 ml centrifuge tube, centrifuge at 12,000×g for 3 minutes to collect bacteria, discard the culture medium, and invert the tube onto absorbent paper to remove any remaining liquid.

[0040] (2) Add a spoonful of glass beads (0.1~0.6mm) to the centrifuge tube containing microbial precipitate.

[0041] (3) Add 0.45ml Buffer ATL and 0.45ml Buffer PCL, tighten the cap, and transfer to a vortex mixer at the highest speed for 10 minutes or a bead mill for rapid bead milling for 30~60 seconds.

[0042] (4) Centrifuge at 12,000 × g for 5 minutes at room temperature.

[0043] (5) Transfer 300µl of supernatant to a new centrifuge tube, add 300µl of BufferGDP, and invert and mix 6-8 times.

[0044] (6) Place the gDNAFilter Mini Column into a 2ml collection tube. Transfer the entire mixture to the gDNA filter column. Centrifuge at 12,000 xg for 30-60 seconds.

[0045] (7) Discard the gDNA filter column, add 180µl of isopropanol to the filtrate, and pipette 3-5 times.

[0046] (8) Pack the HiPureRNAMiniColumn into a 2ml collection tube. Transfer the entire mixture to the column. Centrifuge at 12,000× g for 30-60 seconds.

[0047] (9) Discard the filtrate and reassemble the column into the collection tube. Add 500µl of Buffer RW2 to the column and centrifuge at 12,000× g for 30-60 seconds.

[0048] (10) Discard the filtrate and reassemble the column into the collection tube. Add 500µl of Buffer RW2 to the column and centrifuge at 12,000 × g for 30-60 seconds.

[0049] (11) Discard the effluent and put the column back into the collection tube. Centrifuge at 12,000 × g for 2 minutes.

[0050] (12) Transfer the column to a 1.5 ml centrifuge tube and add 30-100 µl of RNase-free water to the center of the column membrane. Let stand at room temperature for 2 minutes. Centrifuge at 12,000 × g for 1 minute. Discard the column and store the RNA at -80 °C.

[0051] The extracted nucleic acids were analyzed for concentration and purity using a Nanodrop One micro-volume spectrophotometer, with concentration units expressed as ng / µl. Purity was indicated using the A260 / A280 ratio. A260 (nm) is the absorption wavelength of the highest absorption peak for nucleic acids, and A280 (nm) is the absorption wavelength of the highest absorption peak for proteins and phenolic substances. The A260 / A280 ratio is used to assess the purity of nucleic acid samples: the A260 / A280 ratio for pure RNA is 1.8-2.0. A ratio below 1.8 indicates contamination by proteins (aromatics) or phenolic substances, requiring sample purification. The results are shown in Table 1 below.

[0052] Table 1: Concentration and purity of nucleic acid RNA at different times with different preservation solutions

[0053]

[0054] As shown in Table 1, the samples preserved in Examples 1, 2, and 3 after treatment with the preservation solution of the present invention maintained a relatively stable nucleic acid concentration after 0, 3, and 7 days of storage at room temperature, without significant degradation, and the purity remained between 1.8 and 2.0 (below 1.8 indicates the presence of protein in the sample, and above 2.0 indicates RNA degradation). In contrast, the nucleic acid concentration and purity of the samples in Comparative Examples 1 and 2 decreased significantly after 7 days (…). Figure 1 , Figure 2 It is evident that the sputum sample nucleic acid preservation solution provided by this invention can effectively maintain the stability of nucleic acids in sputum, with minimal change in the total nucleic acid amount and excellent preservation of nucleic acid purity. The effect of Example 2 is superior to that of Examples 1 and 3.

[0055] Application Example 2

[0056] To verify the inactivation effect of the preservation solution on infectious pathogenic microorganisms, mycobacteria were picked and preserved according to Examples 1, 2, 3, and Comparative Examples 1 and 2, and then inoculated on Roche solid medium and cultured at 37°C for 21 days to observe the results and whether bacterial plaques appeared. No bacterial plaques appeared in Examples 1, 2, and 3, while bacterial plaques appeared in Comparative Examples 1 and 2. Figure 1 , 2 For the cultivation results.

[0057] Application Example 3

[0058] The RNA extracted from the examples and comparative examples was specifically detected using the RAA-Cas12 system. The results, presented on a test strip, showed that the RNA preserved in Comparative Example 2 was either not extracted or had degraded. Figure 3 The results, from left to right, are Examples 1, 2, 3, Comparative Examples 1, 2, and Control P and Control N.

[0059] Based on the changes in RNA extraction concentration, total amount of nucleic acid RNA, and culture results, the preservation solution formula of the present invention can be derived, which is significantly better than the preservation scheme of the comparative method for protecting nucleic acid RNA in sputum.

[0060] In summary, the results show that the examples in this invention are slightly better than the comparative examples in terms of sample extraction, post-extraction preservation, and post-sequencing phylogenetic analysis. This indicates that the combination of guanidine isothiocyanate, hexadecylpyridine chloride, and ethylenedicysteine ​​can produce a synergistic effect, improving the long-term preservation performance of sputum RNA samples at room temperature. If other reagents are replaced, the preservation effect is significantly reduced. The preservation effect of Examples 1 and 2 is better than that of Example 3, indicating that the relative amounts of hexadecylpyridine chloride and ethylenedicysteine ​​affect the preservation results. Ultimately, Examples 1 and 2 are the most preferred embodiments of this invention, but the preservation solution of Example 3 can also achieve good preservation requirements, such as stable preservation at room temperature for 3 days, and can also meet the detection requirements, and therefore is also within the scope of protection of this invention.

Claims

1. A room-temperature preservation solution for bacterial RNA from sputum samples of tuberculosis patients, characterized in that, The components and contents of each 100 mL of the preservation solution are as follows: 2-3 g tris(hydroxymethyl)aminomethane, 0.7-1.0 g mucin digester, 20-25 g guanidine isothiocyanate, 0.1-0.2 g cetylpyridine chloride, 0.6-0.9 g ethylidene cysteine, 30-35 g fixative, 2-3 g stabilizer, 0.2-0.3 g potassium chloride, 0.4-0.5 g sodium dodecyl sarcosinate, and 0.1-0.2 g proclin 300; the mucin digester is selected from tris(2-carboxyethyl)phosphonic acid hydrochloride; the stabilizer is selected from at least one of sodium citrate and sodium ascorbate; the fixative is a mixed alcohol solvent of glycerol, ethanol, and isopropanol in a volume ratio of 3-5:10-20:5-10.

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