Method for diagnosing head and neck cancer via bacterial metagenomic analysis
A technology of head and neck cancer and bacteria, applied in the direction of biochemical equipment and methods, microbiological determination/inspection, etc.
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Embodiment 1
[0073] Example 1. Analysis of in vivo absorption, distribution and excretion patterns of bacteria and bacteria-derived extracellular vesicles
[0074] To assess whether bacteria and vesicles derived from bacteria are taken up systemically through the mucosa, experiments were performed using the following method. More specifically, 50 μg of fluorescently labeled bacteria and 50 μg of extracellular vesicles (EVs) derived from the bacteria were orally administered to the gastrointestinal tract of mice, and at 0 hours, and at 5 minutes, 3 hours, and 6 hours and measure fluorescence after 12 hours. As a result of observing the overall image of the mouse, such as Figure 1A As shown, bacteria were not absorbed systemically at the time of administration, but EVs derived from bacteria were absorbed systemically 5 minutes after administration, and strong fluorescence was observed in the bladder 3 hours after administration, thus confirming EVs are excreted via the urinary system and...
Embodiment 2
[0076] Example 2. Vesicle isolation and DNA extraction from saliva
[0077] To isolate extracellular vesicles from saliva and extract DNA, saliva was first added to a 10 ml tube, centrifuged at 3500 x g and 4°C for 10 minutes, the suspension was pelleted, and only the supernatant was collected. Place in a new 10ml tube. The collected supernatant was filtered with a 0.22 μm filter to remove bacteria and impurities, then placed in a centrifugal filter (50 kD) and centrifuged at 1500 × g and 4 °C for 15 min to discard materials with a size smaller than 50 kD, then Concentrate to 10ml. Bacteria and impurities were removed again using a 0.22 μm filter, and then the resulting concentrate was subjected to ultracentrifugation at 150,000 × g and 4 °C for 3 hours by using a 90ti type rotor to remove the supernatant, and the aggregated precipitate was washed with phosphoric acid Saline buffer solution (PBS) was dissolved, thereby obtaining vesicles.
[0078] 100 μl of extracellular ...
Embodiment 3
[0081] Example 3. Metagenomic analysis using DNA extracted from saliva
[0082] DNA was extracted using the same method as that used in Example 2, which was then subjected to PCR using the 16S rDNA primers shown in Table 1 to amplify the DNA, followed by sequencing (Illumina MiSeq sequencer). Export results as Standard Flowchart Format (SFF) files and use GS FLX software (v2.9) to convert SFF files to sequence files (.fasta) and nucleotide quality score files before determining credit ratings for reads , and remove fractions with window (20 bps) average base call accuracy of less than 99% (Phred score < 20). After removing low-quality parts, only reads with a length of 300 bps or greater (Sickle version 1.33) were used, and for operational taxonomic unit (OTU) analysis clustering was performed using UCLUST and USEARCH according to sequence similarity. Specifically, based on a sequence similarity of 94% for genera, 90% for families, 85% for orders, 80% for classes, and 75% f...
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