This product adopts a buffer system with a patented formula, which contains Lysis Buffer B1 and Lysis Buffer B2 that can directly lyse DNA or RNA viruses. It is suitable for direct molecular detection of DNA or RNA viruses from various blood and tissue sample materials. The entire extraction process does not involve protease digestion, high-temperature incubation, deproteinization or removal of other impurities. It requires no organic solvent extraction or anhydrous ethanol precipitation, making it simple, fast, and stable in quality with reliable performance.
Product Components and Storage Conditions:
N1508469
| Components
| 50T | Storage
|
N1508469A
| Lysis Buffer B1
| 5.5ml
| RT
|
N1508469B
| Lysis Buffer B2
| 5.5ml
| RT |
Product Features:
Simple and Fast: No liquid nitrogen is required, and viral DNA or RNA extraction can be completed in 5 minutes.
Strong Versatility: Suitable for various samples such as tissue materials, blood, etc.
Precautions:
1. This kit does not have an independent in vitro diagnostic function and shall not be used as a diagnostic reagent.
2. The sampling amount of tissue materials should be strictly controlled. The recommended dosage is 5-10 mg (equivalent to the size of one to half a grain of rice). Excessive sampling is likely to cause false-negative results. For specific details, refer to the figure below:
Experimental Procedure:
1. Tissue Samples:
Take a small amount of sample (5-10 mg, equivalent to the size of one to half a grain of rice) and place it in a 1.5 ml centrifuge tube. Add 100 μl of Lysis Buffer B1, ensure the buffer completely covers the sample, and grind thoroughly with a tissue grinder. After grinding, leave the tube at room temperature for 5 minutes. Then add 100 μl of Lysis Buffer B2, vortex to mix well, and centrifuge at 10,000 rpm for 2 minutes. After centrifugation, carefully pipette 100 μl of the supernatant into another clean 1.5 ml centrifuge tube to use as a template. The recommended volume of the template is 1/10 of the PCR reaction system (e.g., for a 50 μl PCR reaction system, use 5 μl of the template).
Note: The template must be prepared and used immediately.
2. Blood Samples:
Take 20 μl (or 10 μl) of tissue homogenate, plasma, or blood (fresh anticoagulated whole blood), add it to 100 μl of Lysis Buffer B1, vortex to mix well, and leave it at room temperature for 5 minutes. Then add 100 μl of Lysis Buffer B2, vortex to mix well, and centrifuge at 10,000 rpm for 2 minutes. After centrifugation, carefully pipette 100 μl of the supernatant into another clean 1.5 ml centrifuge tube to use as a template. The recommended volume of the template is 1/10 of the PCR reaction system (e.g., for a 50 μl PCR reaction system, use 5 μl of the template).
Note: The template must be prepared and used immediately.
3. Result Detection:
3.1 qPCR Amplification Reaction:
3.1.1 Recommended qPCR Amplification System:
Components
| Volume
|
ddH₂O
| Up to 50μl
|
Direct Amplification qPCR Reaction Mix
| 38μl
|
Direct Amplification HotStart Taq Enzyme
| 2μl
|
Direct Amplification qPCR Primers and Probes
| 5μl
|
Template
| 5μl |
After all reagents are added, mix well, perform a brief centrifugation, and collect all reagents at the bottom of the tube.
Preparation of Direct Amplification qPCR Primers and Probes (Recommendation):
Primer-Probe Stock Solution
| Final Concentration in Direct Amplification qPCR Primer-Probe Mix
| Single-Serve Volume
|
Primer F (40 pmol/μl)
| 4 pmol/μl
| 5μl
|
Primer R (40 pmol/μl)
| 4 pmol/μl
| 5μl
|
Taqman Probe (20 pmol/μl)
| 2 pmol/μl
| 5μl |
3.1.2 qPCR Reaction Program:
Steps
| Temperature
| Time
| Number of Cycles
|
| 1 | 95°C
| 3 min
| 1 cycle
|
| 2 | 95°C
60°C (Fluorescence is collected in this step)
| 15 sec
30 sec
| 40 cycles |
The annealing temperature is determined based on the design of the primers (and probes).
3.2 RT-qPCR Amplification Reaction:
3.2.1 Recommended RT-qPCR Amplification System:
Components
| Volume
|
ddH₂O
| Up to 50μl
|
Direct Amplification RT-qPCR Reaction Mix
| 38μl
|
Direct Amplification RT-qPCR Enzyme Mix
| 2μl
|
Direct Amplification RT-qPCR Primers and Probes
| 5μl
|
Template
| 5μl |
After all reagents are added, mix well, perform a brief centrifugation, and collect all reagents at the bottom of the tube.
Preparation of Direct Amplification RT-qPCR Primers and Probes (Recommendation):
Primer-Probe Stock Solution
| Final Concentrations in Direct Amplification RT-qPCR Primers and Probes Mix
| Single-Serve Volume
|
Primer F (40 pmol/μl)
| 4 pmol/μl
| 5μl
|
Primer R (40 pmol/μl)
| 4 pmol/μl
| 5μl
|
Taqman Probe (20 pmol/μl)
| 2 pmol/μl
| 5μl |
3.2.2 RT-qPCR Reaction Program:
Steps
| Temperature
| Time
| Number of Cycles
|
| 1 | 50°C
| 10 min
| 1 cycle
|
| 2 | 95°C
| 3 min
| 1 cycle
|
| 3 | 95°C
60°C (Fluorescence is collected in this step)
| 10 sec
30 sec
| 40 cycle |
The annealing temperature is determined based on the design of the primers (and probes).
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