A method for evaluating the infectivity of Haemophilus influenzae

Abstract

The present invention belongs to the technical field of bacterial infection assessment, and specifically provides a method for assessing the infectivity of Haemophilus influenzae, comprising the following steps: (1) constructing nasal epithelial organoids; (2) incubating nasal epithelial organoids with Haemophilus influenzae to be tested, obtaining infected nasal epithelial organoids, and conducting subsequent infection status assessment; (3) infection status assessment: comparing the mRNA expression levels of TNFa, IL6, IL8, DNAH5, RSPH1, and GAS8 in nasal epithelial organoid cells before and after Haemophilus influenzae infection; when the expression of TNFa and IL8 in the infected cells increases, and the expression of IL6, DNAH5, RSPH1, and GAS8 decreases, the Haemophilus influenzae to be tested has respiratory tract infectivity, thereby quickly assessing the infectivity and destructiveness of Haemophilus influenzae from patient samples, and providing an important research and application platform for the high-throughput screening and development of Haemophilus influenzae pathogenicity and antibacterial drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of bacterial infection assessment, and in particular relates to a method for assessing the infectivity of Haemophilus influenzae. Background Art

[0002] Respiratory diseases are common and frequently occurring diseases, with the main lesions occurring in the trachea, bronchi, lungs, and chest cavity. Mild lesions often cause coughing, sputum purulent sputum, chest pain, and respiratory impairment, while severe lesions cause dyspnea, hypoxia, and even respiratory failure leading to death. Chronic respiratory diseases (CRDs) are long-term diseases of the airways and other structures of the lungs, including asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, and pulmonary fibrosis, which are characterized by high recruitment of inflammatory cells (neutrophils) and / or destructive cycles of infection. Early intervention for chronic respiratory diseases is extremely necessary. On the one hand, the early symptoms of CRD patients are easily ignored, and the chronic progression of CRD will seriously affect the patient's lung function and may lead to respiratory failure, requiring lobectomy or lung transplantation. On the other hand, the current standardization of the prevention, diagnosis, and treatment of chronic respiratory diseases is far from enough, and the accessibility of medical services needs to be improved urgently, and primary medical services have a heavy responsibility.

[0003] Bacterial infection is considered to be the main cause of acute exacerbation of chronic respiratory diseases. However, because many patients with chronic respiratory diseases are often colonized by bacteria even in the clinically stable period, no exact mechanism has been found to explain how bacterial infection leads to worsening of the disease. Haemophilus influenzae (Hi) is the most common bacterial species isolated from respiratory samples of patients with chronic respiratory diseases. It is aerobic or facultative anaerobic, with an optimal growth temperature of 35-37°C. It has special growth requirements and requires the addition of fresh blood components (mainly containing X and V factors) to grow. It is 0.3-0.4um wide and 1.0-1.5um long. It has no flagella or spores, and most have pili. Some strains have capsules. The Hi capsule polysaccharide antigen is type-specific and is divided into af 6 serotypes based on this. Among them, Haemophilus influenzae type b (Hib) is the most pathogenic. Hi has a "symbiotic" relationship with Streptococcus pneumoniae, and mainly plays a synergistic role in the pathogenesis of respiratory infections. Hi can cause strong airway inflammation and affect the host humoral immune response. Although treatable with antibiotics, the condition may recur or even worsen due to an increase in bacterial numbers, a change in the location of colonizing bacteria in the airways, or the acquisition of a new, more virulent, and more pro-inflammatory bacterial species.

[0004] At present, traditional in vitro cell experiments and animal models are still widely used, but these models cannot accurately and truly reproduce the interaction between the human respiratory environment and cells. To this end, the present invention uses an in vitro nasal epithelial organoid model to study the effect of Haemophilus influenzae on the respiratory epithelial barrier, evaluate its infectivity and destructiveness, and provide a possible evaluation method for the high-throughput screening and development of Haemophilus influenzae pathogenicity and antibacterial drugs. Summary of the invention

[0005] The purpose of the present invention is to overcome the problem that traditional in vitro cell experiments and animal models cannot accurately reproduce the interaction between the human respiratory environment and cells, and cannot quickly and accurately evaluate the infectivity and destructiveness of respiratory bacteria.

[0006] To this end, the present invention provides a method for evaluating the infectivity of Haemophilus influenzae, comprising the following steps:

[0007] (1) Construction of nasal epithelial organoids;

[0008] (2) Incubate nasal epithelial organoids with the tested Haemophilus influenzae to obtain infected nasal epithelial organoids for subsequent infection status assessment;

[0009] (3) Assessment of infection status: Compare the mRNA expression levels of TNFa, IL6, IL8, DNAH5, RSPH1, and GAS8 in nasal epithelial organoid cells before and after Haemophilus influenzae infection; when the expression of TNFa and IL8 in the cells increases and the expression of IL6, DNAH5, RSPH1, and GAS8 decreases after infection, the Haemophilus influenzae to be tested has respiratory infectious properties.

[0010] Specifically, in the above step (3), real-time fluorescence quantitative PCR is used to detect the mRNA expression levels of TNFa, IL6, IL8, DNAH5, RSPH1, and GAS8 in nasal epithelial organoid cells.

[0011] Specifically, the infection status assessment in the above step (3) also includes: preparing tissue sections of nasal epithelial organoids after Haemophilus influenzae infection, performing HE staining, and observing epithelial morphological characteristics. When the cells are obviously arranged loosely after infection, the Haemophilus influenzae to be tested has respiratory infectiousness.

[0012] Specifically, the infection status assessment in the above step (3) also includes: comparing the transepithelial resistivity of the nasal epithelial organoid cells before and after Haemophilus influenzae infection; when the transepithelial resistivity of the nasal epithelial organoid cells decreases after infection, the Haemophilus influenzae to be tested is respiratory infectious.

[0013] Specifically, the above-mentioned method for measuring transepithelial resistivity is to move the infected nasal epithelial organoid cells into a transwell chamber, add ALI basal culture medium, and set up a blank chamber without cells. The resistance value of each chamber is measured using a transepithelial cell resistor meter. The chamber with cells is counted as R1, and the blank chamber without cells is counted as R2. The area of ​​the chamber is S, and the resistivity of the epithelial cells in the chamber TEER = (R1-R2) / S.

[0014] Specifically, the infection status assessment in the above step (3) also includes: performing a FITC-Dextran permeability test on nasal epithelial organoid cells before and after Haemophilus influenzae infection; when the FITC-Dextran permeability of the cells after infection is higher than the FITC-Dextran permeability of the cells before infection, the Haemophilus influenzae to be tested has respiratory tract infectiousness.

[0015] Specifically, the infection status assessment in the above step (3) also includes: comparing the ciliary beat frequency values ​​of the nasal epithelial organoids before and after Haemophilus influenzae infection. When the ciliary beat frequency of the nasal epithelial organoids decreases after infection, the Haemophilus influenzae to be tested is respiratory infectious.

[0016] Specifically, the method for constructing nasal epithelial organoids in the above step (1) is as follows: collect normal nasal cartilage tissue, chop it into pieces, add neutral protease solution, digest it on a shaking table, discard the supernatant after centrifugation, add trypsin, water bath, add DMEM to neutralize the trypsin, pass it through a cell strainer after blowing, discard the supernatant after centrifugation, wash the cells with buffer, inoculate them into a culture bottle, expand nasal epithelial cells, and obtain nasal epithelial organoids.

[0017] Specifically, the above step (2) is specifically as follows: incubating the nasal epithelial organoids with the Haemophilus influenzae to be tested, and shaking the culture plate evenly and gently every 24 hours to facilitate the adsorption of bacteria to host cells; after 48 hours, discarding the bacterial solution, washing the cells with PBS buffer, obtaining infected nasal epithelial organoids, and conducting subsequent infection status assessment.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] The method for evaluating the infectivity of Haemophilus influenzae provided by the present invention is based on a nasal epithelial organoid platform, which can realistically simulate the epithelial structure of the human respiratory mucosa and directly reflect the impact of Haemophilus influenzae infection on the nasal epithelial tissue. The virulence of Haemophilus influenzae is evaluated by differences in multiple indicators such as host-responsive gene expression, tissue integrity, permeability, and ciliary movement, solving the problem that the current traditional in vitro cell experiments and animal models cannot accurately reproduce the interaction between the human respiratory environment and cells. The infectivity and destructiveness of Haemophilus influenzae from patient samples can be quickly evaluated, and an important research and application platform is provided for the high-throughput screening and development of Haemophilus influenzae pathogenicity and antibacterial drugs.

[0020] The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the nasal epithelial organoid culture process of Example 1 of the present invention.

[0022] Figure 2 This is a diagram of HE staining results of nasal epithelial organoids in Example 1 of the present invention.

[0023] Figure 3 This is the histopathological evaluation of the Haemophilus influenzae organoid infection model in Example 3 of the present invention; Scalebar = 20 μm.

[0024] Figure 4 This is the transepithelial resistivity measurement result of the Haemophilus influenzae organoid infection model in Example 3 of the present invention.

[0025] Figure 5 It is the result of FITC-Dextran permeability test of Haemophilus influenzae organoid infection model in Example 3 of the present invention; * compared with NT group, P < 0.0332; ** compared with NT group, P < 0.0021; *** compared with NT group, P < 0.0002; **** compared with NT group, P < 0.0001.

[0026] Figure 6 It is the analysis result of the SAVA video analysis system of the Haemophilus influenzae organoid infection model in Example 3 of the present invention; A is the measurement of ciliary beat frequency after nasal epithelial organoid infection, * compared with the NT group, P < 0.0332; ** compared with the NT group, P < 0.0021; *** compared with the NT group, P < 0.0002; **** compared with the NT group, P < 0.0001; B is a partial digital image of the whole field analysis (WFA) after nasal epithelial organoid infection.

[0027] Figure 7is the relative mRNA expression level of each gene in the model cells infected with Haemophilus influenzae organoids in Example 3 of the present invention; A is the mRNA expression level of TNFa / 18S rRNA in cells infected with nasal epithelial organoids; B is the mRNA expression level of IL6 / 18S rRNA in cells infected with nasal epithelial organoids; C is the mRNA expression level of IL8 / 18SrRNA in cells infected with nasal epithelial organoids; D is the mRNA expression level of DNAH5 / 18S rRNA in cells infected with nasal epithelial organoids; E is the mRNA expression level of RSPH1 / 18S rRNA in cells infected with nasal epithelial organoids; F is the mRNA expression level of GAS8 / 18S rRNA in cells infected with nasal epithelial organoids; * compared with the NT group, P < 0.0332; ** compared with the NT group, P < 0.0021; *** compared with the NT group, P < 0.0002; **** compared with the NT group, P < 0.0001. DETAILED DESCRIPTION

[0028] The technical scheme in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be limited by the attached claims and their equivalents.

[0029] The effect of the method for evaluating the infectivity of Haemophilus influenzae of the present invention is studied below through specific examples.

[0030] Embodiment 1:

[0031] This embodiment refers to Figure 1 The process constructed nasal epithelial organoids, and the specific steps are as follows.

[0032] (1) Tissue collection and processing

[0033] Normal nasal cartilage tissues removed surgically from the hospital were collected, and after being rinsed with PBS, the tissues were cut into pieces and placed in 2 ml centrifuge tubes, 1 ml of neutral protease solution was added, and the tubes were placed in a shaker at 4°C for digestion overnight.

[0034] (2) Tissue digestion and cell dissociation

[0035] Centrifuge the tissue digested overnight and discard the supernatant. Add 2 ml of pancreatin and transfer the sample to a 15 ml centrifuge tube. Incubate in a 37°C water bath for 15 min. Add 2 ml of DMEM (containing 10% FBS) to neutralize the pancreatin and pipette for 20 min. Pass the tissue through a cell strainer and transfer to a new 15 ml centrifuge tube and centrifuge and discard the supernatant. Wash the cells three times with PBS.

[0036] (3) Organoid culture and preparation

[0037] Use 5 ml of culture medium to inoculate the cells dissociated in (2) into a T25 culture flask pre-coated with collagen IV. When the cell density reaches 80%-90%, inoculate the cells into the transwell of a 24-well plate and add 500 μl of culture medium to the lower chamber of each chamber.

[0038] (4) Organoid staining and identification

[0039] For the organoids to be identified, the PET membrane of the transwell chamber was scraped off with the needle of a 50 ml syringe, and immediately fixed in 4% paraformaldehyde solution for 30 min. After routine dehydration, the tissue sections were paraffin-embedded, and HE staining was performed. The results were as follows: Figure 2 shown.

[0040] Embodiment 2:

[0041] In this example, an organoid infection model was constructed, and the specific steps are as follows.

[0042] (1) Isolation, growth and storage of Haemophilus influenzae

[0043] Haemophilus influenzae strains BALF-1, BALF-2, BALF-3, and BALF-4 were isolated from bronchoalveolar lavage fluid (BALF) of patients with respiratory diseases in hospitals. The strains were propagated on chocolate agar at 37°C and 5% CO2. The colonies on the agar plates were transferred to 37.0 g / L brain heart infusion broth (BHI) (Cat. No. 10805) containing 2 μg / ml sterile coenzyme I (NAD) solution (Qingdao Rishui Biotechnology Co., Ltd., Cat. No. 22127) and 10 μg / ml hemin solution (Cat. No. 21004) for culture. The cultured bacteria were suspended in BHI broth containing 20% ​​glycerol and stored at -80°C for use.

[0044] (2) Infection of nasal epithelial organoids

[0045] All experiments related to Haemophilus influenzae infection were conducted in a biosafety level 3 laboratory (BSL-3). Haemophilus influenzae frozen at -80°C was revived, and 20 μl of Haemophilus influenzae serotypes with an OD of 0.3 were used to incubate the nasal epithelial organoids prepared in Example 1. During this period, the culture plate was shaken evenly and gently every 24 hours to facilitate bacterial adsorption to host cells. After 48 hours, the bacterial solution was discarded, and the cells were washed 3 times with 1×PBS to obtain infected nasal epithelial organoids for subsequent infection status evaluation experiments.

[0046] Embodiment 3:

[0047] In this example, nasal epithelial organoids not infected with Haemophilus influenzae were used as a control group (NT), and the following infection status evaluations were performed on the infected nasal epithelial organoids.

[0048] (1) Histopathological evaluation (HE staining)

[0049] For each group of nasal epithelial organoids 48 hours after infection, the PET membrane of the transwell chamber was scraped off with the needle of a 50ml syringe, and immediately fixed in 4% paraformaldehyde solution for 30 minutes. After routine dehydration, paraffin embedding was performed, and tissue sections were made. HE staining was used to reflect the degree of tissue damage. The epithelial morphological characteristics were observed under different magnifications using an Olympus BX53F microscope. The results are shown in Figure 2. Figure 3 As shown, 48 hours after infection with different serotypes of Haemophilus influenzae, the morphological damage of nasal epithelial organoids in each infection group was obvious, and the cells were obviously loosely arranged.

[0050] (2) Transepithelial electrical resistivity (TEER) measurement

[0051] For each group of nasal epithelial organoids 48 hours after infection, 200μl PBS was added to the upper chamber of the transwell chamber for washing three times, and the liquid was discarded. The culture medium of the transwell chamber was replaced, 500μl Ali was added to the lower chamber, and 100μl Ali was added to the upper chamber. At the same time, a blank chamber without cells was set up, 500μl Ali was added to the lower chamber of the chamber, and 100μl Ali was added to the upper chamber. According to the instructions of the Millicell Electircal Resistance System (Millicell-ERS), a transepithelial cell resistor was used to measure the resistance value of each chamber to reflect the degree of tissue integrity. The chamber with cells was counted as R1, the blank chamber without cells was counted as R2, and the area of ​​the chamber was S (0.33cm 2 ), the formula for calculating the resistivity of epithelial cells in the chamber is: TEER = (R1-R2) / S. Figure 4 As shown, TEER values ​​of nasal epithelial organoids were reduced after infection compared with the control group.

[0052] (3) Fluorescein-labeled dextran (FITC-Dextran) permeability test

[0053] For the nasal epithelial organoids in each group 48 hours after infection, add 200μl PBS to the upper chamber of the transwell chamber and wash three times, and discard the liquid. Replace the culture medium of the transwell chamber, add 500μl Ali to the lower chamber, and add 200μl fluorescein isothiocyanate-dextran (Sigma-Aldrich, 46944) diluted to 4mg / ml with sterile PBS to the upper chamber. After incubation at 37°C for 8 hours, pipette 3 equal portions of 100μl of the lower chamber liquid from each chamber into a 96-well plate, and use a multifunctional fluorescence microplate reader to detect the fluorescence emission intensity of FITC in the lower chamber liquid (excitation wavelength 480nm, emission wavelength 530nm). The ratio of the measured values ​​of each experimental group to the measured values ​​of the control group is the relative permeability of FD40. The data were statistically analyzed, and the results are as follows. Figure 5 As shown, the FITC-Dextran permeability of nasal epithelial organoids was significantly increased after infection (P < 0.05), indicating that Haemophilus influenzae can significantly destroy the epithelial barrier of nasal epithelial organoids.

[0054] (4) SAVA video analysis system

[0055] For each group of nasal epithelial organoids 48 hours after infection, 200 μl PBS was added to the upper chamber of the transwell chamber for washing three times, and the liquid was discarded. The 24-well culture plate was opened and placed on an Olympus IMT-2 inverted phase contrast microscope and visualized using a 20x objective. The video output sampled images at 30 frames per second (fps), and the digital image sampling rate was set to 85fps. Each digital image frame consisted of 640 columns × 480 rows of pixels. The video images were analyzed using the SAVA video analysis system. For each transwell, whole field analysis (WFA) images (1×1 pixel points) of three areas were collected. Each measurement point represents one cilium, and its ciliary beat frequency (CBF) value was recorded. The data were statistically analyzed, and the results are shown in the following table. Figure 6 As shown, the ciliary beat frequency of nasal epithelial organoids decreased after infection (P < 0.05), indicating that the ciliary beat function was affected after Haemophilus influenzae infection.

[0056] (5) Real-time fluorescence quantitative PCR technology

[0057] Total RNA was isolated and extracted using Trizol reagent (Life Technologies, 15596018). RT Master Mix Perfect Real Time Reverse Transcriptase Kit (TaKaRa, Cat#RR036A-1) was used to reverse transcribe and synthesize cDNA. THUNDERBIRD The qPCR Mix kit (ToYoBo, Lot: 841600) detects the mRNA level of a single gene by real-time fluorescence quantitative PCR, and the value is compared and analyzed based on the cycle threshold (CT) and the internal reference (18S rRNA). The specific steps are as follows.

[0058] 1) Isolation and extraction of total RNA

[0059] For each group of nasal epithelial organoids infected 48 hours later, add 500μl Trizol reagent to each transwell chamber and gently blow with the pipette tip to fully lyse the cells. Add 100μl chloroform (chloroform: trizol = 1:5), invert to mix, let stand for 4-5 minutes, and centrifuge at 12000g for 15 minutes at 4°C. Add 200μl isopropanol to several 1.5ml Eppendorf tubes without RNase, cover with lid, write labels, and precool at 4°C. After centrifugation, draw 200μl of aqueous phase into the Eppendorf tube with isopropanol (isopropanol: aqueous phase = 1:1), invert and mix 5 times. Centrifuge after placing at 4°C for more than 2 hours. After centrifugation, discard the supernatant, add 1ml 75% ethanol to each tube to wash, invert to mix and centrifuge. Discard the supernatant, add 1ml 75% ethanol again to wash, invert to mix and centrifuge. Discard the supernatant, add 1 ml of anhydrous ethanol to wash, invert to mix and centrifuge. Discard the supernatant, invert the centrifuge tube and air dry. After air drying, add 20 μl of DEPC water and gently pipette to mix. Measure the RNA concentration using an ultra-micro spectrophotometer.

[0060] 2) Reverse transcription to synthesize cDNA

[0061] The RT reaction solution was prepared according to the components listed in Table 1 (the reaction solution was prepared on ice). The reverse transcription reaction conditions were shown in Table 2.

[0062] Table 1 RT reaction solution composition

[0063]

[0064] Table 2 Reverse transcription reaction conditions

[0065]

[0066]

[0067] 3) Real-time fluorescence quantitative PCR

[0068] The PCR reaction solution was prepared according to the components listed in Table 3 (the reaction solution was prepared on ice). The Real Time PCR reaction conditions (two-step method) are shown in Table 4. The primer sequences used in this example are shown in Table 5.

[0069] Table 3 PCR reaction solution components

[0070]

[0071] Table 4 Real Time PCR reaction conditions

[0072]

[0073] Table 5 Real-time fluorescence quantitative PCR primer sequences

[0074]

[0075]

[0076] The experimental results are as follows Figure 7 As shown, after infection, the mRNA expressions of TNFa and IL8 in nasal epithelial tracheal cells were significantly increased (P < 0.05), and the mRNA expression of IL6 was significantly decreased (P < 0.05), indicating that the secretion levels of TNFa and IL8 were positively correlated with the inflammatory response caused by infection, and the secretion level of IL6 was negatively correlated with the inflammatory response caused by infection; and the mRNA expressions of cilia-related genes DNAH5, RSPH1, and GAS8 were significantly decreased (P < 0.05), which was consistent with the determination of the cilia beat frequency of nasal epithelial organoids.

[0077] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.

Claims

1. A method for evaluating the infectivity of Haemophilus influenzae, characterized in that: The following steps are involved: (1) Construction of nasal epithelial organoids; (2) Incubate nasal epithelial organoids with the tested Haemophilus influenzae to obtain infected nasal epithelial organoids for subsequent infection status assessment; (3) Evaluation of infection status: Comparison of mRNA expression levels of TNFa, IL6, IL8, DNAH5, RSPH1, and GAS8 in nasal epithelial organoid cells before and after infection with Haemophilus influenzae. When the expression of TNFa and IL8 in cells increases and the expression of IL6, DNAH5, RSPH1, and GAS8 decreases after infection, the Haemophilus influenzae to be tested has respiratory tract infection. The infection status assessment in step (3) further comprises: comparing the transepithelial resistivity of the nasal epithelial organoid cells before and after infection with Haemophilus influenzae, and when the transepithelial resistivity of the nasal epithelial organoid cells decreases after infection, the Haemophilus influenzae to be tested has respiratory tract infection; The transepithelial resistivity measurement method is to transfer the infected nasal epithelial organoid cells into a transwell chamber, add ALI basal medium, and set up a blank chamber without cells at the same time, and use a transepithelial cell resistance meter to measure the resistance value of each chamber. The chamber with cells is counted as R1, and the blank chamber without cells is counted as R2. The area of ​​the chamber is S, and the resistivity of the epithelial cells in the chamber TEER = (R1-R2)*S; The infection status assessment in step (3) further includes: performing a FITC-Dextran permeability test on the nasal epithelial organoid cells before and after infection with Haemophilus influenzae. When the FITC-Dextran permeability of the cells after infection is higher than the FITC-Dextran permeability of the cells before infection, the Haemophilus influenzae to be tested has respiratory tract infectiousness.

2. The method for evaluating the infectivity of Haemophilus influenzae according to claim 1, characterized in that: In the step (3), real-time fluorescence quantitative PCR is used to detect the mRNA expression levels of TNFa, IL6, IL8, DNAH5, RSPH1, and GAS8 in nasal epithelial organoid cells.

3. The method for evaluating the infectivity of Haemophilus influenzae according to claim 1, characterized in that: The infection status assessment of step (3) further includes: preparing tissue sections of nasal epithelial organoids after Haemophilus influenzae infection, performing HE staining, and observing epithelial morphological characteristics. When the cells are obviously arranged loosely after infection, the Haemophilus influenzae to be tested has respiratory tract infection.

4. The method for evaluating the infectivity of Haemophilus influenzae according to claim 1, wherein: The infection status assessment in step (3) further includes: comparing the ciliary beat frequency values ​​of the nasal epithelial organoids before and after infection with Haemophilus influenzae. When the ciliary beat frequency of the nasal epithelial organoids decreases after infection, the Haemophilus influenzae to be tested is respiratory infectious.

5. The method for evaluating the infectivity of Haemophilus influenzae according to claim 1, characterized in that: The method for constructing nasal epithelial organoids in step (1) is specifically as follows: normal nasal cartilage tissue is collected, minced, added with a neutral protease solution, digested on a shaking table, centrifuged and discarded the supernatant, added with trypsin, water bathed, added with DMEM and trypsin, pipetted, passed through a cell strainer, centrifuged and discarded the supernatant, washed the cells with a buffer solution, inoculated into a culture bottle, and expanded the nasal epithelial cells to obtain nasal epithelial organoids.

6. The method for evaluating the infectivity of Haemophilus influenzae according to claim 1, characterized in that: The step (2) specifically comprises: incubating the nasal epithelial organoids with the Haemophilus influenzae to be tested, and evenly and gently shaking the culture plate every 24 hours to facilitate the adsorption of bacteria to host cells; discarding the bacterial solution after 48 hours, washing the cells with PBS buffer, obtaining the infected nasal epithelial organoids, and conducting subsequent infection status assessment.

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