Method for evaluating disinfection effectiveness of air purifier on virus aerosol

Abstract

The invention relates to a method for evaluating the disinfection effectiveness of an air purifier on virus aerosol, and relates to the technical field of disinfection effect evaluation. Comprising the following steps: (1) arranging an aerosol infection cabin of a four-stage biosafety laboratory; (2) starting a Collison aerosol generator to generate virus aerosol, and injecting the virus aerosol into the aerosol infection cabin until a set time; (3) starting fan circulation of the air purifier, under the condition that the disinfection mode is started or not started, enabling the air purifier to act for a set time, then sampling the virus aerosol, and taking out the gel filter membrane after sampling is finished to carry out infectious titer detection; (4) carrying out disinfection and air filtration and purification on the experiment infection cabin; and (5) calculating the virus killing rate according to the initial virus concentration detected under the condition that the disinfection mode is started and the virus concentration detected under the condition that the action of the disinfection mode is started for set time. The method is simple and convenient to operate and high in safety performance.

Description

Technical Field

[0001] This application relates to the field of disinfection effect evaluation technology, and in particular to a method for evaluating the effectiveness of air purifiers in disinfecting viral aerosols. Background Technology

[0002] Viruses, bacteria, and other microorganisms in the air can easily enter the human body through the respiratory tract, especially pathogens transmitted through aerosols, which seriously threaten human health. Therefore, air purification and disinfection are crucial.

[0003] Currently, the market is flooded with air purification products based on various principles, and their disinfection effects are widely debated. An effective method for evaluating the effectiveness of air disinfection is essential for both new product development and the supervision and inspection of product quality. CN103805499A discloses an air disinfection effectiveness evaluation system, which includes a microbial aerosol generator, an airtight cabinet, two samplers, and one suction pump. The main unit of the microbial Collison aerosol generator is placed outside the airtight cabinet, and an atomizing cup containing the microbial solution is placed inside the airtight cabinet. The main unit of the microbial Collison aerosol generator and the atomizing cup are connected by a flexible hose. The suction pump of one sampler is pre-placed inside the airtight cabinet, and the sampling head is placed outside the airtight cabinet. The inlet and outlet of the sampling head are independently connected to the airtight cabinet, forming a closed loop. An air purifier is placed outside the airtight cabinet, with its inlet connected to the airtight cabinet and its outlet connected to the inlet of another sampler. The outlet of the other sampler is connected to the inside of the airtight cabinet, forming a closed loop. An air filter is installed on the airtight cabinet. This invention effectively evaluates the one-time disinfection effect of air purifiers on aerosol microorganisms passing through them, while also addressing biosafety issues in microbial aerosol experiments.

[0004] The drawbacks of the aforementioned patent documents are: 1. The safety of biosafety in microbial aerosol experiments depends on two factors: the airtightness of the airtight cabinet and the effectiveness of disinfection of the air and materials within it. If the airtightness and disinfection effectiveness cannot be guaranteed, the biosafety issues raised in the aforementioned patent documents cannot be addressed. Furthermore, the aforementioned documents do not specifically disclose the product model of the airtight cabinet, making it unclear to those skilled in the art how its airtightness is ensured. Therefore, there are significant safety risks when verifying the disinfection of highly pathogenic microorganisms, such as the novel coronavirus and Ebola virus.

[0005] 2. In the above literature, the disinfection verification process involves two samplers, which makes sampling cumbersome and the post-sampling calculations complex.

[0006] There are no existing reports on the disinfection validation of highly pathogenic microorganisms such as the novel coronavirus. How to provide a simple, safe, and reproducible method for evaluating the effectiveness of air disinfection in air purifiers is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0007] To address the problems existing in related technologies, this application provides a method for evaluating the effectiveness of air purifiers in disinfecting viral aerosols. This method is simple to operate and has high safety performance.

[0008] The method for evaluating the effectiveness of air purifiers in disinfecting viral aerosols in this application includes the following steps: (1) Layout of the aerosol infection chamber in a biosafety level 4 laboratory: The benchtop airborne microbial sampler, air purifier, and Collison aerosol generator were placed sequentially in the center of the aerosol infection chamber of the biosafety level 4 laboratory, with the airflow direction from the Collison aerosol generator to the benchtop airborne microbial sampler. The supply and exhaust airtight valves and airtight doors of the aerosol infection chamber were closed at the beginning of each experiment to ensure the airtightness of the aerosol infection chamber. (2) Turn on the Collison aerosol generator to generate viral aerosols and inject them into the aerosol infection chamber for the set time. (3) Turn on the air purifier fan circulation. With the disinfection mode on, let the air purifier work for a set time as the experimental group; at the same time, without the disinfection mode on, let the air purifier work for the same time as the control group. Both the control group and the experimental group were sampled using a desktop airborne microbial sampler to collect viral aerosols. After sampling, the gel filter membrane was removed and immediately 10 ml of DMEM solution was added. The solution was then incubated at 37°C for 10 min to completely dissolve the gel filter membrane before the infectious titer was detected. (4) Disinfect and purify the air in the laboratory aerosol infection chamber; (5) The virus killing rate can be calculated based on the initial virus concentration detected when the disinfection mode is turned on and the virus concentration detected after the disinfection mode has been turned on for a set time.

[0009] Furthermore, the viral aerosol mentioned in step (2) is a novel coronavirus aerosol and the volume of the novel coronavirus solution is 5 ml, and the air compressor is turned off to stop aerosol generation after 3 minutes of injection.

[0010] Further, in step (3), the MD8 airscan desktop airborne microbial sampler and the specially matched gel filter membrane 17528-080ACD are used for sampling. The sampling flow rate is 30 L / min and the sampling volume is 100 L.

[0011] The beneficial effects of this application are: The evaluation method in this application is based on a biosafety level 4 (BSL-4) laboratory environment. Viral aerosol diffusion occurs within the BSL-4 aerosol infection chamber, where an air purifier's fan circulates and carries some air through the purifier. With disinfection mode activated, the air purifier is operated for a set time (experimental group); simultaneously, without disinfection mode activated, the air purifier is operated for the same duration (control group). A benchtop airborne microbial sampler is used to sample viral aerosols from both the control and experimental groups. After sampling, the gel filter membrane is removed for infectivity titer testing, allowing the disinfection kill rate to be calculated. This method is simple to operate and highly safe. Attached Figure Description

[0012] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments thereof.

[0013] Figure 1 This is a schematic diagram of the layout of the aerosol infection chamber in the Level 4 biosafety laboratory in this application; Figure 2 This is a schematic diagram showing the results of SARS-CoV-2 Omicron titer detection at a wind speed of 1. Figure 3 This is a schematic diagram showing the results of SARS-CoV-2 Omicron titer detection at a wind speed of 2. Detailed Implementation

[0014] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art. Example 1

[0015] The method for evaluating the effectiveness of air purifiers in disinfecting viral aerosols in this application includes the following: 1. Experimental equipment and materials 1.1 Air purifier The Chenxing brand SKAiR DF-1900 electrostatic air sterilizer (air purifier), manufactured by a company in Zhejiang, uses an integrated, round-hole electrostatic module as its core component, installed at the air intake of the air conditioner. It employs high-voltage electrostatic technology, achieving a voltage intensity of 4000V-7000V. The technical principle is as follows: 1. The electric field breaks down the outer structure of pathogens such as bacteria or viruses (e.g., bacterial cell membrane or viral envelope). 2. Reactive oxygen species disrupt the biofield of microorganisms (including bacteria); 3. Ozone breaks down the outer structure of pathogens (such as bacterial cell membranes or viral envelopes). 4. High-pressure instantaneous carbonization of protein bodies.

[0016] 1.2 Collison Aerosol Generator The MRE-type Collison 6 aerosol generator is a flat-bottomed aerosol generator designed by the Microbiological Research Establishment in Bolton, UK. All metal parts are machined from 316 stainless steel. The "T"-shaped rod on top of the BGI aerosol generator connects the air inlet and pressure gauge for easy pressure testing. Optional accessories include a glass precious liquid bottle and a telescopic sleeve for expensive or hard-to-find materials; the glass precious liquid bottle has a small well with a volume of 5 ml.

[0017] 1.3 MD8 Airscan Desktop Airborne Microbial Sampler The Sartorius MD8 airscan benchtop airborne microbial sampler has a flow rate of 2.0 m / s. 3 / h - 8.0 m 3 / h, 0.1 m per gear 3 / h; Timer: 1-99 min adjustable, 1 min increments; Temperature range: 15 ℃-35℃, maximum error ±5%; Noise level ≤62 dB(A); Weight 6.5 kg; Dimensions (L × W × H): 375×242 × 228 mm. The specially designed gel filter membrane is water-soluble, with a nominal pore size of 3 μm and a diameter of 80 mm. The gel filter membrane is 250 μm thick, belonging to deep filtration, with a retention rate of 99.9995% for Bac. sub.niger. spores and 99.94% for bacteriophage T3.

[0018] 1.4 Experimental strains The novel coronavirus (SARS-CoV-2) Omicron (BA.5.1.16) strain was isolated from the National High-Level Biosafety Laboratory in Kunming, Institute of Medical Biology, Chinese Academy of Medical Sciences. The main experimental reagents involved are shown in Table 1, and the main instruments and equipment are shown in Table 2.

[0019] Table 1 Main experimental reagents

[0020] Table 2 Main Instruments and Equipment

[0021] 2. Experimental Methods 2.1 Layout of the Experimental Infection Chamber The air purifier, Collison aerosol generator, and benchtop airborne microbial sampler were placed in the center of the aerosol infection chamber (approximately 12 m³) in the biosafety level 4 laboratory. Figure 1 Each time, the laboratory closes the supply and exhaust air valves and airtight doors of the aerosol infection chamber to ensure its airtightness.

[0022] 2.2 Experimental Design Immediately after aerosol injection into the aerosol infection chamber, a benchtop airborne microbial sampler was used to collect samples as a positive control group; cell maintenance solution was used as a negative control to observe for contamination and cell growth. The experimental group had its fan circulation and disinfection modes activated, and the aerosol injection was followed by 60 minutes of exposure; the control group had its fan circulation activated but disinfection mode disabled, and the aerosol injection was followed by 60 minutes of exposure (see table below).

[0023] Experimental protocol table for disinfection of novel coronavirus aerosols by air purifiers

[0024] Experimental steps: 1) First, the new gel filter membrane should be installed on the benchtop airborne microbial sampler; 2) Set the air purifier's speed, ionization state, and action time before turning it on and then turn off the aerosol infection chamber; 3) Turn on the Collison aerosol generator to inject novel coronavirus aerosols for the set time; 4) After the procedure is completed, turn on the airborne bacteria sampler to collect samples. After sampling, remove the gel filter membrane. 5) Disinfect and purify the air in the experimental infection chamber.

[0025] 2.3 Aerosol Generation Before each experiment, 5 ml of SARS-CoV-2 Omicron (BA.5.1.16) was injected into the small well of a glass precious liquid bottle. The air compressor flow rate was set to 20 L / min. After 3 minutes of injection, the air compressor was turned off to stop aerosol generation.

[0026] 2.4 Aerosol Sampling Sampling was performed using an MD8 airscan benchtop airborne microbial sampler and a dedicated gel filter membrane 17528-080ACD. The sampling flow rate was 30 L / min, and the sampling volume was 100 L.

[0027] 2.5 Gel Filtration Membrane Treatment After sampling, the gel filter membrane was removed, and 10 ml of DMEM solution was immediately added and incubated at 37 ℃ for 10 min to completely dissolve the gel filter membrane before the infectious titer was detected.

[0028] 2.6 Infectivity titer detection 2.6.1 Preparing Cells Take out a 96-well cell culture plate, add 8,000 to 10,000 Vero cells to each well, and after two days of culture, when the cell abundance reaches 60%-90%, it can be used for experiments.

[0029] 2.6.2 Diluting the virus sample to be tested The virus was diluted according to the number of wells to be inoculated. DMEM was used as the basal medium, with antibiotic-free and serum-free DMEM as the diluent. Eight wells were inoculated for each dilution, and 1000 µl of virus dilution was prepared for each dilution. For example, in a 10... -1 Add 900 µl of diluent to 100 µl of virus solution, and so on, to serially dilute to 1010. -3 .

[0030] 2.6.3 Vaccination Take a 96-well cell culture plate and use a multi-channel pipette to add 100 µl of diluted virus solution to each well. The order of addition is from right to left, from top to bottom, and from high dilution to low dilution (10 µl per well). -3 10 -2 10 -1 The original solution was added. Then, the cells were incubated in a 37 ℃ CO2 incubator for 5 days, and the cytopathic effects were observed.

[0031] 2.6.4 Results statistics and analysis.

[0032] The cytopathic effects of the control and experimental groups were observed and recorded. Then, the disinfection kill rate was calculated according to "2.1.3 Air Disinfection Efficacy Identification Test" in the "Disinfection Technical Specifications" (2002 edition).

[0033] 3 Experimental Results 3.1 Omicron (BA.5.1.16) stock solution titer of novel coronavirus (SARS-CoV-2) This evaluation experiment used the SARS-CoV-2 Omicron (BA.5.1.16) strain. The infectivity titer of the original SARS-CoV-2 Omicron (BA.5.1.16) strain was determined to be 3.46 × 10⁻⁶. 7 TCID 50 / ml.

[0034] 3.2 Disinfection Experiment Results Air samples were collected in the laboratory aerosol infection chamber at an average temperature of 21-23.5 ℃ and a relative humidity of 45%-55%, with the air purifier disinfection mode turned on or off. Infectivity titers for SARS-CoV-2 virus were tested. Each experiment was repeated three times, and the average value of the three experiments was calculated.

[0035] In the prototype electrostatic air sterilizer, with a wind speed of 1 (0.45 m / s) and without air purification and sterilization mode activated, the initial viral concentration of SARS-CoV-2 was 1.22 × 10⁻⁶. 5 TCID 50 / m 3 It dropped to 8.17 × 10 at 60 minutes. 4 TCID 50 / m 3 After the air purification and disinfection mode was activated, samples collected after 60 minutes did not induce cytopathic effects (CPE), consistent with the cell growth observed in the negative control group. Figure 2 As shown.

[0036] Therefore, when the air purifier is set to fan speed 1 and the air disinfection mode is turned on, the kill rate of SARS-CoV-2 Omicron virus is >99.99% after 60 minutes (Table 3).

[0037] Table 3. Results of SARS-CoV-2 Omicron aerosol disinfection when the air purifier is set to fan speed 1.

[0038] When the prototype electrostatic air sterilizer was set to a wind speed of 2 (0.84 m / s) and the air purification and sterilization mode was not activated, the initial viral concentration of SARS-CoV-2 was 1.22 × 10⁻⁶. 5 TCID 50 / m3 The viral concentration dropped to 7.0 × 10⁶ after 60 minutes. 4 TCID 50 / m 3 When the air purification and disinfection mode is activated, samples collected after 60 minutes do not induce cytopathic effects (CPE), consistent with the cell growth observed in the negative control group. Figure 3 As shown.

[0039] Therefore, when the wind speed of the prototype electrostatic air sterilizer was set to 2, the kill rate of SARS-CoV-2 Omicron virus was >99.99% after 60 minutes of operation (Table 4).

[0040] Table 4. Results of SARS-CoV-2 Omicron aerosol disinfection when air purifier fan speed is set to 2.

[0041] 4. Conclusion 4.1 When the wind speed was set to 1 (0.45 m / s) on the prototype electrostatic air sterilizer, the sterilization rate of the SARS-CoV-2 virus exceeded 99.99% after the sterilization function was turned on for 60 minutes.

[0042] 4.2 When the wind speed was set to 2 (0.84 m / s) on the prototype electrostatic air sterilizer, the sterilization rate of the SARS-CoV-2 virus exceeded 99.99% after the sterilization function was turned on for 60 minutes.

[0043] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for evaluating the disinfection effectiveness of an air cleaner against virus-containing aerosols, characterized by, The method comprises the following steps: (1) The layout of the aerosol infection chamber in the four-level biosafety laboratory: Place the desktop air plankton sampler, air purifier and Collison aerosol generator in sequence at the center position of the aerosol infection chamber in the four-level biosafety laboratory, and the direction from the Collison aerosol generator to the desktop air plankton sampler is the airflow direction; close the air supply and exhaust airtight valves and the airtight door of the aerosol infection chamber at the beginning of each experiment to ensure the airtightness of the aerosol infection chamber; (2) Start the Collison aerosol generator to generate virus aerosols, and inject the virus aerosols into the aerosol infection chamber for a set time; (3) Start the air purifier fan to circulate, and under the condition of starting the disinfection mode, let the air purifier act for a set time as the experimental group; at the same time, under the condition of not starting the disinfection mode, let the air purifier act for the same time as the control group; Start the desktop air plankton sampler to sample the virus aerosols for the control group and the experimental group respectively, take down the gelatin filter membrane after sampling, immediately add 10 ml of DMEM solution 37℃ to act for 10 min to completely dissolve the gelatin filter membrane, and then perform infectious titer detection; (4) Disinfect and air filter purify the laboratory aerosol infection chamber; (5) According to the initial virus concentration detected under the condition of starting the disinfection mode and the virus concentration detected after a period of time under the condition of starting the disinfection mode, the virus killing rate can be calculated.

2. The method as recited in claim 1, characterized in that The virus aerosol in step (2) is a novel coronavirus aerosol, and the volume of the novel coronavirus solution is 5 ml, and the air compressor is stopped to stop the aerosol generation after 3 min of injection.

3. The method as recited in claim 1, wherein In step (3), the MD8 airscan type desktop air plankton sampler and the special gelatin filter membrane 17528-080ACD are used for sampling, the sampling flow rate is 30 L / min, and the sampling volume is 100 L.

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