De-host extraction kit for phlegm macrogenome
A technology of metagenomics and kits, applied in the field of dehostization extraction kits, which can solve the problems of inability to restore disulfide bonds, long time-consuming, short time-consuming, etc., to preserve cell integrity, reduce production costs, and improve detection rate effect
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[0042] Example 1 Dehost extraction method of sputum metagenomics.
[0043] Specific steps are as follows:
[0044] 1. Pretreatment of sputum
[0045] (1) Take 4 sputum samples, take 200μL of sputum, add 400μL of sputum pretreatment reagent and mix for 30s; the sputum pretreatment reagent used contains 10mM Tris-HCl, 50mM DTT, 15mM EDTA-Na2 , 0.1% SDC, 1% Triton X-100, 0.25% SDS, 20mM HEPS-KOH, used after filtration sterilization, pH 8.0. SDC is an ionic detergent used to increase protein solubilization.
[0046] (2) Add proteinase K to a final concentration of 0.5-5 mg / mL, mix well and incubate at 56°C for 20 minutes, shaking once every 5 minutes;
[0047] (3) Stop the reaction when the sputum becomes clear;
[0048] (4) Centrifuge at 10000g for 5 minutes and take the supernatant. By combining the reduction method and the enzymatic method, the sputum can be digested gently, with good effect and short time, while retaining the integrity of microbial cells, so that the homogenized sampl...
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