Release steps
1 Liquid samples such as serum, saliva, sputum, urine, swab collection fluid, cell culture fluid, etc.
(1) Take 150μL of 博坊在线 and place it in a 1.5mL centrifuge tube, and then take 50μL of the liquid sample to be tested, and mix thoroughly by pipetting and mixing 2-5 times. Place it vertically at room temperature (22~25℃) and stand still for ≥2min.
(2) If it is fresh whole blood, take 10μL of whole blood and add it to 100μL of 博坊在线 solution, and mix thoroughly by pipetting and mixing 2-5 times, and place it vertically at room temperature (22~25℃) for ≥2min.
Note: This method is not applicable to completely hemolyzed whole blood, and nucleic acid extraction must be performed before detection.
2 Tissue solid samples
Take 300μL of 博坊在线 and place it in a 1.5mL centrifuge tube, then add the soy to peanut-sized tissue sample into the buffer, cut it into pieces with scissors, and mix thoroughly by pipetting and mixing 3-5 times at room temperature (22~25℃) The static effect of vertical placement is ≥5min. After 5 min, take 2.5 μL of the supernatant of the mixture as a nucleic acid amplification template for real-time fluorescence or conventional PCR amplification, or constant temperature amplification (LAMP, RPA, RCA, etc.).
Note: You can also add Water or Normal saline tissue homogenate and then take the supernatant, and operate according to the liquid sample.
Amplification step
Directly take 2.5 μL of the supernatant of the mixed solution as a nucleic acid amplification template for real-time fluorescence or conventional PCR amplification, or constant temperature amplification (LAMP, RPA, RCA, etc.). Note: The volume of the template should be less than 10% of the total volume of the nucleic acid reaction. That is, the mixed supernatant added to the 25 μL reaction system does not exceed 2.5 μL, and the mixed supernatant added to the 50 μL reaction system does not exceed 5 μL.
Matters needing attention
1. Pathogen-free extraction and direct amplification is not only related to the sample pretreatment solution, but also closely related to the formula of the PCR reaction solution. The PCR reaction solution of the direct amplification method has a higher KCl concentration than the conventional PCR reaction solution to increase the possibility of direct amplification.
2. It is strictly forbidden to drink or touch 博坊在线, which will cause damage to the skin.
3. To ensure biological safety, the sample to be tested must be added to the sampling tube for 5 minutes, and the cover can be opened for testing after the pathogen is completely inactivated.
4. The collected specimens must be fresh and tested in time to improve the detection rate and sensitivity.