CN112961900A

CN112961900A - Virus aerosol grading sampling method

Info

Publication number: CN112961900A
Application number: CN202110150415.7A
Authority: CN
Inventors: 高玉伟; 郭振东; 张春茂; 赵宗正; 李佳明; 张�诚; 崔欢; 刘军; 万宗海; 李吉平; 李忠义; 孟轲音; 李楠; 郝镯; 周博; 向海洋
Original assignee: Military Veterinary Research Institute Academy Of Military Medical Sciences
Current assignee: Military Veterinary Research Institute Academy Of Military Medical Sciences
Priority date: 2021-02-03
Filing date: 2021-02-03
Publication date: 2021-06-15

Abstract

The invention belongs to the technical field of microbial aerosol sampling, and discloses a virus aerosol graded sampling method. The sampling method of the six-stage impact sampler is improved, so that the virus aerosol can be sampled in a grading manner; the sampled sample is a soluble gel film and can be dissolved by a small amount of liquid, so that the effect of concentrating and enriching the virus aerosol is achieved, and the detection sensitivity can be improved; the lysed sample can be subjected to nucleic acid extraction for PCR detection, or cells incubated for virus culture.

Description

Virus aerosol grading sampling method

Technical Field

The invention belongs to the technical field of microbial aerosol sampling, and relates to a virus aerosol graded sampling method.

Background

Some viruses that can be transmitted through the respiratory tract are very prone to generate aerosols, such as influenza virus, foot and mouth disease virus, new coronavirus of 2019, and the like. Aerosol exposure infection also becomes one of the important infection pathways for these viruses. Therefore, in the situation of epidemic outbreak of infectious disease, there is a great demand for sampling virus aerosol, however, the virus aerosol sampler on the market at present can only be used for measuring the concentration of virus aerosol, but cannot measure the particle size distribution characteristics thereof.

The six-stage impingement air microorganism sampler is a porous, laminar collision (air) sampler, commonly used for the measurement of aerobic bacterial and fungal concentrations in the environment and their particle size distribution, and is widely used as a standard instrument for counting viable microorganisms in microbial aerosols. The six-stage impact microorganism sampler is designed according to the aerodynamic principle and consists of six aluminum discs, and each impact disc is provided with accelerating jet pores with strictly designed diameters and number. The sampler can collect all particles according to the deposition condition of the human lung, no matter the physical size, the shape or the density. Each stage of the sampler may be placed in a petri dish containing an agar culture medium for collecting microbial particles in the sampled air, which may be left on the culture medium by the impact of the air stream. After sampling is completed, the culture dish can be taken out for culture and counted by a colony counter. At present, the six-stage impact type air microorganism sampler has the defect that virus sampling cannot be carried out.

Disclosure of Invention

The invention improves the sampling method of a six-stage impact sampler, so that the sampling method realizes the graded sampling of the virus aerosol; the sampled sample is a soluble gel film and can be dissolved by a small amount of liquid, so that the effect of concentrating and enriching the virus aerosol is achieved, and the detection sensitivity can be improved; the lysed sample can be subjected to nucleic acid extraction for PCR detection, or cells incubated for virus culture.

The technical scheme adopted by the invention is as follows:

a virus aerosol fractional sampling method comprises the following steps:

step 1, in a six-stage impact sampler, a collecting plate of each stage of sampler is sequentially provided with an agar layer, a nuclear pore filter membrane with the pore diameter of 0.05 mu m and a soluble gel membrane from bottom to top;

step 2, when the six-stage impact sampler runs at a flow rate of 28.3L/min, the aerosol particles are classified into six stages according to particle size screening, the first stage collects particles with the particle size larger than 7 microns, the second stage collects particles with the particle size of 4.7-7 microns, the third stage collects particles with the particle size of 3.3-4.7 microns, the fourth stage collects particles with the particle size of 2.1-3.3 microns, the fifth stage collects particles with the particle size of 1.1-2.1 microns, and the sixth stage collects particles with the particle size of 0.65-1.1 microns;

and 3, after sampling is finished, collecting the top layer water-soluble gel film, adding sterile normal saline, and adding water at 37 ℃ for 10 minutes to ensure full dissolution.

Further, the flow rate of the sampler is calibrated by using the airflow calibrator before each sampling.

Preferably, the temperature is maintained in 37 deg.C water for 10min to ensure sufficient dissolution.

Preferably, the agar layer has a volume of 27mL, the nucleopore membrane has a diameter of 90mm, and the sol-gel membrane has a diameter of 80 mm.

The lysed sample obtained as described above is used for nucleic acid detection or virus culture.

The invention realizes virus aerosol sampling based on a six-stage impact sampler. The traditional sampling mode uses the agar culture dish as the sampling medium, can be with on the agar culture medium that planktonic bacteria directly gathered, but appraise cultivateable colony concentration, flora and particle size distribution after the cultivation. The method improves the traditional method and uses the soluble gel membrane for sampling, realizes the graded sampling of the virus aerosol, and is convenient for recovering the sample after the collection is finished. The method can directly dissolve the soluble gel film in a small amount of liquid after the sample is collected, realize the conversion from a large amount of aerosol to a small amount of hydrosol, concentrate the sample, improve the detection sensitivity and perform nucleic acid extraction or virus culture subsequently.

Detailed Description

Example 1

The virus aerosol fractional sampling method comprises the following steps:

step 1: preparation of reagents

Viral RNA extraction kits were purchased from Qiagen; prime Script^TMThe RT reagen Kit with gDNA Eraser reverse transcription Kit is purchased from Dalian Bio-engineering (Dalian) Co., Ltd; power SYBR Green PCR Master Mix was purchased from ABI, USA; water-soluble gel films were purchased from sartorius, germany; 0.05 μm nuclear pore membranes were purchased from Whatman; TSA medium was purchased from BD corporation, USA.

Step 2: preparing instrument

A six-stage impactor was purchased from Tisch, usa; the quantitative sampling pump ZR2000 is purchased from Qingdao Zhongrui intelligent instrument Co., Ltd; the ordinary PCR instrument and the fluorescent quantitative PCR instrument are purchased from ABI company of America; clean bench was purchased from hel corporation;

and step 3: preparing a sampling plate, and preparing an agar layer

20g of TSA solid medium was placed in an Erlenmeyer flask, and 500mL of ddH was added₂And O, uniformly mixing until no attachment exists at the bottom of the conical flask, putting the conical flask into an autoclave for sterilization (121 ℃, 15min), taking out the conical flask after sterilization is finished, measuring 27mL of culture medium by using a measuring cylinder, pouring the culture medium into a 9cm plate, and waiting for natural cooling.

And 4, step 4: collecting samples

Placing a layer of 0.05-micron nuclear pore membrane on a solid culture medium, then placing a water-soluble gel membrane, sequentially loading into each level of a 6-level impact sampler, connecting a six-level sampler and a ZR-2000 sampling pump by using a colloid hose, opening the sampling pump, setting the sampling rate (28.3L/min) and the sampling time, and clicking a start button to start sample collection.

And 5: recovering the sample

And after sampling is finished, taking out the water-soluble gel film in each level, transferring the water-soluble gel film into 50mL centrifuge tubes, marking, adding 5mL sterile physiological saline into each centrifuge tube, performing vortex oscillation, and incubating in a water area at 37 ℃ for 10min until the water-soluble gel film is completely dissolved.

Step 6: viral nucleic acid detection

Viral RNA in the sample was extracted using a Qiagen viral RNA extraction kit and the RNA was reverse transcribed into cDNA using a TaKaRa reverse transcription kit. Followed by fluorescent quantitative PCR detection using specific primers. Absolute quantification of viral copy number can be achieved by setting standards.

And 7: titration of viral virulence

The susceptible cells were inoculated into 6-well cell culture plates, after the cell density was 100%, the medium was discarded, 1mL of the sample was incubated for 1 hour, then the cells were washed 3 times with sterile PBS, and low melting agarose medium was added. And putting the agarose into a cell culture box for inverted culture for 48-72h after the agarose is solidified at room temperature. Finally, cell staining is carried out, and the number of plaques is counted, so that virus virulence is obtained.

Claims

1. A virus aerosol fractional sampling method is characterized by comprising the following steps:

step 1, improving six-stage impact samplers, wherein a collecting plate of each stage of sampler is changed from an original single agar layer to an agar layer, a nuclear pore membrane with the pore diameter of 0.05 mu m and a soluble gel membrane from bottom to top in sequence; sequentially loading the collecting plates into each level of a six-level impact sampler;

step 2, when the six-stage impact sampler runs at the flow rate of 28.3L/min, the aerosol particles are classified into six stages according to the particle size: the first stage collects particles with the particle size of more than 7 mu m, the second stage collects particles with the particle size of 4.7 mu m-7 mu m, the third stage collects particles with the particle size of 3.3 mu m-4.7 mu m, the fourth stage collects particles with the particle size of 2.1 mu m-3.3 mu m, the fifth stage collects particles with the particle size of 1.1 mu m-2.1 mu m, and the sixth stage collects particles with the particle size of 0.65 mu m-1.1 mu m;

2. The method for fractional sampling of a viral aerosol according to claim 1, wherein each collection plate has a agar layer volume of 27mL, a nucleopore membrane diameter of 90mm, and a sol gel membrane diameter of 80 mm.

3. The virus aerosol fractional sampling method of claim 1 or 2, wherein the flow rate of the sampler is calibrated before each sampling using an airflow calibrator.

4. A lysed sample obtained by the method of viral aerosol fractionation according to any one of claims 1 to 4 for use in nucleic acid detection or virus culture.