Rapid Fungus detection kit and detection method using same
A detection kit and detection method technology, applied in the field of clinical microorganism identification, can solve the problems of prolonged hospitalization, drug resistance test costs, treatment failure, etc., and achieve the effects of shortened identification time, low cost, and convenient operation.
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Embodiment 1
[0063] Embodiment 1: the preparation method of kit.
[0064] (1) DNA extraction reagents:
[0065] Buffer I is 9g / L NaCl aqueous solution;
[0066] Buffer II is 10g / L SDS, 2% (v / v) Triton X-100, 100mmol / L NaCl, 10mmol Tris-HCl (pH 8.0), 1mmol / L EDTA, and the solvent is water;
[0067] Buffer III is a saturated NaCl aqueous solution (about 6mol / L);
[0068] Buffer TE is 10mmol / L Tris-HCl (pH 8.0), 1mmol / L EDTA, and the solvent is water;
[0069] Among them: NaCl (purchased from Shanghai Shisihewei Chemical Co., Ltd.), SDS (purchased from Sigma Company in the United States), EDTA (purchased from Hangzhou Chemical Reagent Co., Ltd.), Tris-HCl (purchased from Sigma Company in the United States), Tris-HCl (Tris-base was purchased from Sigma Company in the United States), hydrochloric acid purchased from Hangzhou Chemical Reagent Co., Ltd.), Triton X-100 (purchased from Sigma, USA)
[0070] (2) Reaction solution:
[0071] PCRBuffer: 0.1% (v / v) NP-40 (purchased from Sigma, USA),...
Embodiment 2
[0088] Embodiment 2: detection method.
[0089] Instruments: Bio-Rad S1000 PCR instrument, Beckman Microfuge 22R desktop micro-centrifuge, Beijing Liuyi agarose gel electrophoresis instrument, Shanghai Peiqing gel imaging system, QIAGEN PyroMark Q96ID sequencer.
[0090] (1) Extracting fungal DNA, taking clinical urine samples as an example, specifically includes the following steps:
[0091] (1a) Take 2ml of clinical urine sample in a centrifuge tube and centrifuge at 12000rpm for 10min at high speed;
[0092] (1b) Discard the supernatant, add 800ul Buffer I, and vortex for 30 seconds;
[0093] (1c) 12000rpm high-speed centrifugation for 5min;
[0094] (1d) Discard the supernatant, invert and blot dry as much as possible, add 300ul Buffer II, and shake on a high-speed oscillator for 10min;
[0095] (1e) Add 300ul Buffer III to the centrifuge tube and vortex for 30s;
[0096] (1f) 4000rpm centrifugal 15min;
[0097] (1g) Put the supernatant obtained in (1f) into another c...
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Abstract
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