Indoor micro-ecological environment livability detection method based on human conditioned pathogens
A detection method and technology for pathogenic bacteria are applied in the field of indoor micro-ecological environment livability detection based on human conditional pathogenic bacteria, which can solve the problems of long time and achieve the effect of rapid judgment.
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Embodiment 1
[0029] Example 1: Collection of environmental flora samples
[0030] 1. Determine the designated sampling position in the process of collecting samples with swabs (for some clean environmental samples, ≥25cm 2 ), mark 16cm with a ruler / template 2 Area.
[0031] 2. Label all collection tubes.
[0032] 3. Put on rubber gloves, take the disposable swab out of the package, and be careful not to touch the surface of the swab with others.
[0033] 4. Immerse the swab head into the mixed solution of 0.01mol / L NaCl and 0.1% Tween20, and then press the swab into the tube to remove excess liquid.
[0034] 5. Smear the swab on the marked area horizontally and vertically (total 50 times, about 30-35s) for sampling.
[0035] 6. Cut off the swab head, put it into a marked, 2mL empty tube, and cap the tube tightly.
[0036] 7. Place the sample in an ice box, and then transfer it to a -80°C refrigerator for storage.
Embodiment 2
[0037] Example 2: Extract the DNA of the sample and perform high-throughput sequencing
[0038] 1. Pipette 350 μL of PBS solution and add it to the above 2 mL centrifuge tube containing the sample.
[0039] 2. Add 350 μL AL buffer, 40 μL lysozyme (10 mg / mL), 6 μL mutanolysin (25000 U), and 300 mg glass beads. Mix well, and incubate the sample at 37°C for 1h.
[0040] 3. Transfer the centrifuge tube to a tissue grinder (Qiagen) for 3 min at 26 Hz,
[0041] 4. Add 20 μL proteinase K (provided by QIAGEN kit) to the tube, and shake to mix.
[0042] 5. Incubate at 56°C for 3h.
[0043] 6. Transfer the supernatant to a new tube and discard the swab. with ddH 2 O wash the beads 2 times with 200 μL each.
[0044] 7. Add 1 / 2 volume of alcohol and mix well.
[0045] 8. Put the 2mL collection tube (provided in the kit) under the Dneasy centrifugal column, draw the mixture in step 7 to the spin column, centrifuge at 8000rpm / min for 1min, and discard the waste liquid and the collect...
Embodiment 3
[0050] Example 3: Using bioinformatics to analyze community structure
[0051] The off-machine data is based on the GreenGenes 16S rRNA full-length sequence database. Using software, the bacteria contained in the sample are classified, and the structure of the genus layer in the evolutionary classification is restored, and finally the relative abundance of the bacteria contained in the sample is obtained. further said to use QIIME software (Quantitative Insights Into MicrobialEcology, the coding sequence (barcode) of the original data is split, use Trimmomatic software to carry out the first quality control (parameter is set to Trimmomatic: SLIDINGWINDOW:30:25MINLEN:25), Use FLASH software to fuse the sequence data at both ends (parameter setting is FLASH: -M 200 -m 5 -x 0.1), and then use Fastx Toolkit software for secondary quality control (parameter setting is Fastx Toolkit: -Q 33 -q 25 -p 80). Finally, the QIIME software was used to remove the chimera (chimera), and finall...
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