Sample pretreatment method for extracting microorganism total DNA in biological aerosol

A sample pretreatment and bioaerosol technology, which is applied in the field of microbial research in air aerosol, can solve the problems of complex buffer configuration process, large DNA loss, large filter membrane area, etc. Effects of poisoning and simplification of operation steps

Active Publication Date: 2016-04-06
BEIJING AGRO BIOTECH RES CENT
0 Cites 5 Cited by

AI-Extracted Technical Summary

Problems solved by technology

However, the first method has a large filter membrane area, which leads to a large amount of DNA loss during the DNA extraction process; although the second method has a small loss during the extraction process, the buffer used in the ...
View more

Abstract

The invention relates to a sample pretreatment method for extracting microorganism total DNA in biological aerosol. The method includes the steps that a filter membrane is adopted to collect air microorganisms, 0.8% normal saline is used as a buffer solution, the microorganisms are fully separated under the oscillation action of a shaking table supplemented by glass beads, and membrane stripping liquid is filtered through a 25-mm filter membrane, so that the microorganisms are concentrated on the surface of a membrane with the area of 1.96 cm<2> and oscillated, walls of the microorganisms, with thick cell walls, on the surface of the membrane are broken preliminarily, and follow-up DNA extraction is facilitated. The sample pretreatment method is easy to operate, harmless to the human body and capable of effectively increasing the DNA extraction amount.

Application Domain

DNA preparation

Technology Topic

Saline waterChemistry +10

Examples

  • Experimental program(2)

Example Embodiment

[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.

Example Embodiment

[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.

PUM

PropertyMeasurementUnit
Diameter5.0mm
Diameter25.0mm
Aperture0.22µm

Description & Claims & Application Information

We can also present the details of the Description, Claims and Application information to help users get a comprehensive understanding of the technical details of the patent, such as background art, summary of invention, brief description of drawings, description of embodiments, and other original content. On the other hand, users can also determine the specific scope of protection of the technology through the list of claims; as well as understand the changes in the life cycle of the technology with the presentation of the patent timeline. Login to view more.

Similar technology patents

Stripping device and stripping method for welding quality detection

PendingCN113295136AImprove stripping efficiencyReduces the chance of breaking off or falling off the retainer
Owner:LINKDATA NEW ENERGY CO LTD

Stripping machine for gleditsia sinensis seed husks

ActiveCN110313621AImprove stripping efficiencyavoid stranded
Owner:河南省林业科学研究院

Gynecological tumor operation tumor stripper easy and convenient to operate

PendingCN114259281AImprove stripping efficiencyspeed up surgery
Owner:赵惠杰

Pair of quick optical cable stripping pliers

PendingCN112615312AImprove stripping efficiencyReduce Tooling Costs
Owner:国网浙江省电力有限公司开化县供电公司

Full-automatic adhesive tape stripping device

PendingCN114506729AImprove stripping efficiencyHigh stripping precision
Owner:东莞松山智能机器人有限公司

Classification and recommendation of technical efficacy words

  • Improve stripping efficiency
  • Simplify operation steps

Process for extracting rare earth element by non-saponifiable phosphorous mixing extractant

ActiveCN1804063AImprove stripping efficiencyReduce acid consumption
Owner:GRIREM ADVANCED MATERIALS CO LTD

Long-distance wire stripper

PendingCN107275989AImprove stripping efficiencyReduce security risks
Owner:HUBBELL ELECTRIC WUHU

Stripping machine for gleditsia sinensis seed husks

ActiveCN110313621AImprove stripping efficiencyavoid stranded
Owner:河南省林业科学研究院

Stripping device and stripping method for welding quality detection

PendingCN113295136AImprove stripping efficiencyReduces the chance of breaking off or falling off the retainer
Owner:LINKDATA NEW ENERGY CO LTD

Full-automatic adhesive tape stripping device

PendingCN114506729AImprove stripping efficiencyHigh stripping precision
Owner:东莞松山智能机器人有限公司

Icon batch processing method and device and mobile terminal

InactiveCN106201191ASimplify operation stepsImprove icon management efficiency
Owner:BEIJING ANYUNSHIJI TECH CO LTD

Method and device for measuring hyperpressure of flammable gas explosion shock waves

InactiveCN102879144ASimplify operation stepsMeasurement results are intuitive
Owner:CHANGZHOU UNIV

Method for separating and purifying chlorogenic acid from eucommia bark leaves

ActiveCN102040520ASimplify operation stepsSingle type of equipment
Owner:ZHEJIANG UNIV OF TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products