Sample pretreatment method for extracting microorganism total DNA in biological aerosol

[0020] Example 1

[0021] Sample collection: Air bioaerosol samples were collected in the Beijing Academy of Agriculture and Forestry Sciences. The sampling instrument is the air total particulate matter collector (Qingdao Laoying Environmental Protection Technology Co., Ltd.), and the sampling time is 48 hours. Filter the sampled microorganisms onto a quartz fiber filter membrane with a diameter of 90 mm, and then divide the quartz fiber filter membrane into 3 parts.

[0022] In the first part, according to the method of this application, the fibrous filter membrane that adsorbs bio-aerosol is laid flat along the tube wall in a 50ml sterile centrifuge tube. The side of the filter membrane that does not adsorb microorganisms is attached to the centrifuge tube wall, and the particles are absorbed Face up, making sure that the glass beads directly touch the absorption surface. Add 40 sterile glass beads with a diameter of 5 mm and 0.8% sterile normal saline. The amount of sterile normal saline added is based on the complete infiltration of the fibrous filter membrane and the remaining amount. The centrifuge tube was fixed on a constant temperature shaker and treated at 100 rpm for 12 hours. During the operation, it is necessary to ensure that the glass beads are in full contact with the air microorganism adsorption surface during the rotation of the shaker. Vibrate treatment on a constant temperature shaker; filter the eluate after the above treatment with a smaller filter membrane (a microporous cellulose acetate filter membrane with a diameter of 25 mm and a pore size of 0.22 μm). Then put the above-mentioned microporous filter membrane into a 1.5ml centrifuge tube, and process it on a shaker at a speed of 6.0m/s for 50s. Use the DNA extraction kit (MO-BIO DNAIsolationKit) for DNA extraction.

[0023] The second part, without processing, directly perform DNA extraction operation on the filter membrane that adsorbs bio-aerosol;

[0024] In the third case, the reported method was used for pretreatment. Lay the fibrous filter membrane that adsorbs the bio-aerosol in a 90 mm sterile plate, add a stripping buffer, and use 0.1% TritonX-100. The plate was fixed on a constant temperature shaker, and after the treatment, the eluate after the above treatment was filtered with a microporous acetate filter membrane with a diameter of 25 mm and a pore size of 0.22 μm. Directly perform DNA extraction operation on the filter membrane that adsorbs biological aerosol.

[0025] After testing (ThermoNANODROP2000, USA), the pre-treated filter membrane of the present invention, that is, the concentration of DNA extracted in the first part is 40.5ng/μl, the filter membrane without pre-treatment, the concentration of DNA extracted in the second and third parts They are 18.5ng/μl and 30.7ng/μl respectively.

[0026] Example 2

[0027] Sample collection: Air bioaerosol samples were collected in a chicken house in a chicken farm in Beijing. The sampling instrument is the air total particulate matter collector (Qingdao Laoying Environmental Protection Technology Co., Ltd.), and the sampling time is 24 hours.

[0028] The first part, according to the method of this application, spread the bio-aerosol-absorbing quartz fiber filter membrane in a 50ml sterile centrifuge tube along the tube wall. The side of the filter membrane that does not adsorb microorganisms is attached to the centrifuge tube wall, and the particles are absorbed Face up, making sure that the glass beads directly touch the absorption surface. , Add 50 sterile glass beads with a diameter of 5mm and 0.8% sterile normal saline. The amount of sterile normal saline added is based on the complete infiltration of the quartz fiber filter and the remaining. The centrifuge tube was fixed on a constant temperature shaker and treated at 100 rpm for 12 hours. During the operation, it is necessary to ensure that the glass beads are in full contact with the air microorganism adsorption surface during the rotation of the shaker. Shake treatment on a constant temperature shaker; filter the eluate after the above treatment with a smaller filter membrane (a microporous cellulose acetate filter with a diameter of 25 mm and a pore size of 0.22 μm). Then put the above-mentioned microporous filter membrane into a 1.5ml centrifuge tube, and process it on a shaker at a speed of 6.0m/s for 45s. Use the DNA extraction kit (MO-BIO DNAIsolationKit) for DNA extraction.

[0029] The second part, without processing, directly perform DNA extraction operation on the quartz fiber filter membrane that adsorbs bio-aerosol;

[0030] In the third case, the reported method was used for pretreatment. The fibrous filter membrane that adsorbs the bio-aerosol is laid flat in a 90 mm sterile petri dish, and a stripping buffer is added, using 0.1% TritonX-100. The plate was fixed on a constant temperature shaker, and after the treatment, the eluate after the above treatment was filtered with a microporous acetate filter membrane with a diameter of 25 mm and a pore size of 0.22 μm. Finally, directly perform DNA extraction on the filter membrane that adsorbs bioaerosol.

[0031] After testing (ThermoNANODROP2000, USA). The concentration of DNA extracted from the first part, the second part and the third part were 65.7ng/μl, 40.6ng/μl and 49.2ng/μl, respectively.

[0032] Result analysis

[0033] A comparative analysis of the above-mentioned concentration of bio-aerosol microbial DNA extracted by different pretreatment methods in the two environments shows that the concentration of DNA extracted by the fiber filter membrane treated by the pretreatment method of the present invention is higher than that of the unpretreatment Fiber filter membrane; compared to the treatment method using organic solvent as the stripping solution, the concentration of DNA extracted by the method of the present invention is higher than that of the prior art using organic solvent as the stripping solution, and the stripping solution configuration of the present invention It is simple and safe to use; at the same time, the method of the present invention can be applied to the pretreatment of bio-aerosol-absorbing filter membranes collected in different environments.