Thermostatic detection kit for detecting rabies virus subtypes
A technology of rabies virus and detection kit, which is applied in the field of constant temperature detection kit for rabies virus typing, can solve the problems of time-consuming detection and difficult identification of detection results, and achieve reliable results, low requirements for instruments, sensitivity and accuracy high effect
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Embodiment 1
[0047] Example 1: Rabies detection (using tissue of rabies-infected patients as samples)
[0048] 1. Sample pretreatment: Add 1ml of 0.9% normal saline to the tissue sample and grind it into a tissue suspension for testing;
[0049] 2. Take 250μl of the tissue suspension, centrifuge at 5000rpm / min for 10min, discard the supernatant, and keep the precipitate (if there is no precipitate, keep about 30ul of liquid).
[0050] 3. Add 100μl RNA extraction reagent, shake for 30S, centrifuge at 5000rpm / min for 10min, take the supernatant and put it in a 1.5mL centrifuge tube.
[0051] 4. Take three 200μl reaction tubes and mark them as No. 1, No. 2 and No. 3. Add 25 μl of PCR reaction solution, 5 μl of PCR reaction enzyme system, 5 μl of primer mixture (upstream primers for detection of type 1 rabies virus, downstream primers for detection of type 1 rabies virus, The upstream primers for detecting 2-7 types of rabies virus, the downstream primers for detecting 2-7 types of rabies vi...
Embodiment 2
[0053] Embodiment 2: repeatability test
[0054] Take three 200μl reaction tubes and mark them as No. 1, No. 2, No. 3, No. 4, No. 5, and No. 6. Add 25 μl of PCR reaction solution, 5 μl of PCR reaction enzyme system; 5 μl of primer mixture (upstream primers for detection of type 1 rabies virus, Downstream primers for detection of type 1 rabies virus, upstream primers for detection of type 2-7 rabies virus, downstream primers for detection of type 2-7 rabies virus, internal standard upstream primer, internal standard downstream primer concentration are 0.2 μmol / L), 5 μl Probe mixture (fluorescent probe 1, fluorescent probe 2, fluorescent probe 3, fluorescent probe 4, fluorescent probe 5, fluorescent probe 6, fluorescent probe 7, fluorescent probe 8, the probe concentration is 0.08 μmol / L). Add 10 μl of nucleic acid extracted from rabies tissue at a concentration of 1 ng / ul to reaction tubes No. 1, No. 2, No. 3, No. 4, No. 5, and No. 6; the final volume of each tube is 50 μl. ...
Embodiment 3
[0055] Embodiment 3: gradient test
[0056] Take six 200μl reaction tubes and mark them as No. 1, No. 2, No. 3, and No. 4. Add 25 μl of PCR reaction solution, 5 μl of PCR reaction enzyme system, 5 μl of primer mixture (upstream primer for detection of type 1 rabies virus, detection of type 1 rabies virus) into the tubes (No. Downstream primers, upstream primers for detection of 2-7 types of rabies virus, downstream primers for detection of 2-7 types of rabies virus, internal standard upstream primers, internal standard downstream primers at a concentration of 0.2 μmol / L), 5 μl probe mixture (fluorescence Probe 1, fluorescent probe 2, fluorescent probe 3, fluorescent probe 4, fluorescent probe 5, fluorescent probe 6, fluorescent probe 7, fluorescent probe 8 (probe concentrations are 0.08 μmol / L, respectively). Add 10 μl of nucleic acid extracted from rabies tissue at a concentration of 1000 ng / ul in reaction tube 1; add 10 μl of nucleic acid extracted from rabies tissue at a 1...
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