A quantitative aerosolization release method for air pathogen transmission research and applications

Abstract

The present application relates to the technical field of biological detection and environmental monitoring, and particularly relates to a quantitative atomization release method for air pathogen transmission research and application. In view of the problems of low detection load, difficult sampling quantification and insufficient detection sensitivity of the existing air pathogen detection, the method uses an atomizer, first establishes an atomizer output characteristic standard curve by using an inverted cylinder method, then atomizes a pathogen-containing solution to prepare a controllable concentration aerosol, captures pathogen nucleic acid through a collection buffer, and completes pretreatment through a standardized process. The method realizes accurate control of pathogen aerosol and efficient capture of pathogen nucleic acid, adapts to subsequent nucleic acid detection, greatly reduces the detection limit, effectively solves the problem of air pathogen sampling quantification, provides a standardized and repeatable sample preparation scheme for air-borne pathogen monitoring and prevention and control, and has important public health significance.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a quantitative atomization release method and its application for research on the transmission of airborne pathogens. It is particularly suitable for constructing standardized pathogen aerosol simulation environments and can be widely applied to scenarios such as research on the transmission patterns of airborne pathogens, verification of air sampling and detection technologies, and evaluation of the effectiveness of epidemic prevention measures. Background Technology

[0002] Airborne pathogens are characterized by high infectivity and wide spread, posing a serious threat to public health and safety. To study the transmission patterns of airborne pathogens and evaluate the effectiveness of control measures, it is necessary to simulate real airborne pathogen environments under controlled conditions and develop a standardized quantitative atomization release method for airborne pathogen transmission research, along with its applications.

[0003] Existing aerosol release methods still have shortcomings in achieving standardization and quantification, mainly manifested in the following aspects: the lack of a universal method that can stably and accurately generate pathogen aerosols of known concentration and volume, resulting in poor comparability between different research results; limited adaptability of methods, mostly designed for specific pathogens, and have not yet formed a universal operating system covering different types of airborne pathogens; and less consideration of the impact of complex environments on aerosol release and subsequent capture, and the adaptability to efficient capture processes needs to be strengthened.

[0004] Therefore, there is an urgent need to develop an integrated atomization release method that is centered on precise quantification, has strong universality, and is adaptable to practical application scenarios. This would solve the key problems of insufficient quantitative accuracy and limited applicability in existing technologies, and provide a standardized technical solution for research on the spread of airborne pathogens. Summary of the Invention

[0005] This invention aims to solve the problems of complex operation, insufficient quantitative accuracy, and limited applicability in existing airborne pathogen atomization release technologies. It provides a quantitative atomization release method that is based on accurate calibration, simple to operate, and highly universal, so as to achieve standardized release of pathogen aerosols.

[0006] Another objective of this invention is to provide the application of the method in the detection of airborne pathogens, forming a standardized research method for the reliable detection and quantitative study of pathogens at extremely low concentrations in the air.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A quantitative atomized release method for research on airborne pathogen transmission specifically includes the following steps: (1) Establishing a standard curve of atomization time-gas volume: The gas volume generated by the atomizer at different time points was collected by the inverted graduated cylinder method. Each time point was independently measured ≥3 times and the average value was taken. The standard curve was established by linear regression analysis with atomization time as the abscissa and cumulative gas volume as the ordinate. The determination coefficient R² of the standard curve was ≥0.99. (2) Preparation of pathogen nebulization solution: The target pathogen solution is diluted with sterile diluent to a series of target concentrations to obtain pathogen nebulization solution; (3) Quantitative atomization release: Based on the standard curve established in step (1), calculate the atomization time required to generate a preset volume of air containing pathogens, add the pathogen atomization liquid to the calibrated atomizer, start the atomizer and run the atomization time to achieve quantitative atomization release of pathogens.

[0008] The different time points include at least five consecutive or intermittent time points of 10 s, 20 s, 30 s, 40 s, 50 s, 60 s and above, and the coefficient of variation of the gas output rate of the atomizer is ≤ 5%.

[0009] The target pathogen is a microorganism containing nucleic acid, including respiratory pathogens.

[0010] Furthermore, the respiratory pathogen is one or more of RNA viruses, DNA viruses, and drug-resistant bacteria; the RNA virus includes single-stranded RNA viruses and double-stranded RNA viruses; the DNA virus includes airborne DNA viruses such as herpesviridae and adenoviridae; and the drug-resistant bacteria include bacteria carrying carbapenemase resistance genes and quinolone resistance genes.

[0011] Furthermore, the sterile diluent is sterile physiological saline, phosphate buffer, or serum-free cell culture medium.

[0012] Furthermore, the preset volume is the target gas volume set according to actual needs.

[0013] In addition, the present invention also provides the application of the method in the detection of trace pathogen nucleic acids in ambient air, bioaerosols and object surface samples.

[0014] Furthermore, the application includes a pathogen capture step after aerosol release: passing a quantitatively aerosolized pathogen aerosol into a collection device containing a complex lysis buffer.

[0015] Furthermore, the composite lysis buffer solution is physically compatible with the solid-phase enrichment material, enabling the nanomaterials to be uniformly dispersed to form a stable suspension.

[0016] Furthermore, the solid enrichment material is a nanomaterial with a spiked structure on its surface, and the spike length of the nanomaterial is 30 nm to 60 nm, preferably a nanomaterial with a spike length of about 50 nm.

[0017] Furthermore, the application also includes compatibility with subsequent molecular detection, which includes at least one of reverse transcription combined with real-time quantitative PCR, isothermal amplification combined with CRISPR detection, and multiplex PCR.

[0018] The present invention has the following beneficial effects: This invention provides a quantitative nebulization release method and its application for research on airborne pathogen transmission. By precisely calibrating the nebulizer, a pathogen nebulization solution is prepared and then quantitatively released through nebulization. Simultaneously, a dedicated lysis buffer and a nanomaterial with a specific structure are used to adapt to subsequent molecular detection, achieving quantitative and controllable release of aerosols and detection of airborne pathogens. This method improves the detection sensitivity of airborne RNA viruses to 10 pfu / L and exhibits good tolerance to complex environmental interferences. The operation is simple and controllable, and it can be widely applied to the detection of trace pathogen nucleic acids in ambient air, bioaerosols (such as human exhaled air), and object surface samples. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the process of this method.

[0020] Figure 2 The images show TEM images (A) of silica nanoparticles with different spike lengths in Example 1 of the present invention, electrophoresis results (B) of material-2 capturing complex structure RNA (18S rRNA and VSV genomic RNA), and electrophoresis results (C) of silica nanoparticles with four different spike lengths capturing siRNA.

[0021] Figure 3 The diagram (A) shows the process of calibrating the atomized gas rate using the inverted graduated cylinder method in Embodiment 2 of the present invention, along with the quantitative equation relating atomization time and gas volume and the coefficient of determination R² (B). Figure 4 The images show a comparison of the dispersibility of material-2 with lysis buffer B and commercial Trizol reagent in Example 3 of this invention (A), the electrophoresis results of material-2 capturing vesicular stomatitis virus (VSV) nucleic acid after treatment with lysis buffer B (B), and a statistical graph of capture efficiency in a simulated complex environment (C).

[0022] Figure 5 The figure shows the standard curve (A) of VSV genomic RNA in the RT-qPCR method of Example 4 of the present invention, and the Ct value results of qPCR detection of VSV virus aerosols at different concentrations (B).

[0023] Figure 6 The graph shows the standard curve (A) of PEDV genomic RNA in the RT-qPCR method of Example 5 of the present invention, and the Ct value results of RT-qPCR detection of PEDV virus aerosols at different concentrations (B). Detailed Implementation

[0024] The following detailed description, in conjunction with the accompanying drawings and embodiments, illustrates a quantitative atomized release method and its application in research on the transmission of airborne pathogens, but the scope of protection of this invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field.

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

Claims

1. A quantitative atomized release method for studying the transmission of airborne pathogens, characterized in that, Specifically, the following steps are included: (1) Establishing a standard curve of atomization time-gas volume: The gas volume generated by the atomizer at different time points was collected by the inverted graduated cylinder method. Each time point was independently measured ≥ 3 times and the average value was taken. The standard curve was established by linear regression analysis with atomization time as the abscissa and cumulative gas volume as the ordinate. The determination coefficient R² of the standard curve was ≥ 0.

99. (2) Preparation of pathogen nebulization solution: The target pathogen solution is diluted with sterile diluent to a series of target concentrations to obtain pathogen nebulization solution; (3) Quantitative atomization release: Based on the standard curve established in step (1), calculate the atomization time required to generate a preset volume of air containing pathogens, add the pathogen atomization liquid to the calibrated atomizer, start the atomizer and run the atomization time to achieve quantitative atomization release of pathogens.

2. The method according to claim 1, characterized in that, The different time points mentioned in step (1) include at least 5 consecutive or interval time points of 10 s, 20 s, 30 s, 40 s, 50 s, 60 s and above. The calibration step also includes independently repeating the measurement at least 3 times for each time point and taking the average value. The coefficient of variation of the gas output rate of the atomizer is ≤ 5%.

3. The method according to claim 1, characterized in that, The target pathogen mentioned in step (2) is an airborne pathogen containing nucleic acid, including but not limited to respiratory pathogens.

4. The method according to claim 3, wherein the respiratory pathogen is one or more of RNA viruses, DNA viruses, and drug-resistant bacteria; the RNA virus includes single-stranded RNA viruses and double-stranded RNA viruses; the DNA virus includes airborne DNA viruses such as herpesviridae and adenoviridae; and the drug-resistant bacteria include bacteria carrying carbapenemase resistance genes and quinolone resistance genes.

5. The method according to claim 1, characterized in that, The sterile diluent mentioned in step (2) is sterile physiological saline, phosphate buffer, or serum-free cell culture medium.

6. The method according to claim 1, characterized in that, The preset volume mentioned in step (3) is the target gas volume set according to actual needs.

7. The application of the method according to any one of claims 1 to 6 in the detection of trace pathogen nucleic acids in ambient air, bioaerosols (such as human exhaled air) and object surface samples.

8. The application according to claim 7, characterized in that, Its application includes the pathogen capture step after aerosol release: the pathogen aerosol released by quantitative aerosol is passed into a collection device containing a composite lysis buffer solution, the composite lysis buffer solution being optimized in Chinese patent application CN117286219A. The composite lysis buffer solution has physical compatibility with solid phase enrichment materials, enabling the nanomaterials to be uniformly dispersed to form a stable suspension.

9. The application according to claim 8, characterized in that, The solid enrichment material is a nanomaterial with a spiked structure on its surface. The spike length of the nanomaterial is 30 nm to 60 nm, preferably a nanomaterial with a spike length of about 50 nm.

10. The application according to claim 7, characterized in that, Its applications also include compatibility with subsequent molecular detection, which includes at least one of reverse transcription combined with real-time quantitative PCR, isothermal amplification combined with CRISPR detection, and multiplex PCR.

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