本产品采用了具有专利配方的缓冲体系，包含了直接裂解DNA或RNA病毒的裂解液B1和裂解液B2，适用于从各种血液及组织病料样本中直接进行DNA或RNA病毒的分子检测。整个提取过程不包含蛋白酶消化，高温孵育，去蛋白及其它杂质的过程，无需有机溶剂抽提，无需无水乙醇沉淀，简便、快捷，而且质量稳定可靠。
产品组分及储存条件：
| N1508469 | Components
| 50T | Storage
|
N1508469A
| 裂解液B1
| 5.5ml
| RT
|
N1508469B
| 裂解液B2
| 5.5ml
| RT |
产品特点：
简单快速：无需液氮，5min即可完成病毒DNA或RNA提取。
普适性强：适用于各种组织病料、血液等样本。
注意事项：
1. 试剂盒不具备独立的体外诊断作用，不可作为诊断试剂。
2.应严格控制组织病料的取样量，建议用量为5-10mg(一颗到半颗大米大小)，取样量太多容易造成假阴性结果。具体情况参考下图：
实验流程：
1. 组织类样本：
取少量样品 (5-10mg，一颗到半颗大米大小) 于1.5ml的离心管中，加入100μl裂解液B1，确保缓冲液可以完全覆盖样品，用组织研磨器充分研磨。研磨结束后室温放置5min，然后加入100μl裂解液B2，震荡混匀，10,000rpm离心2min。离心结束后，小心吸取100μl上清液于另一干净的1.5ml离心管中作为模板使用，使用量为PCR反应体系的1/10（例如50μlPCR反应体系，模板使用量为5μl）。
注意：必须现处理现用。
2. 血液类样本：
取组织匀浆液、血浆或血液 (新鲜抗凝全血) 20μl (或10μl) 加入到100μl裂解液B1中震荡混匀后室温放置5min，然后加入100μl裂解液B2，震荡混匀，10,000rpm离心2min。离心结束后，小心吸取100μl上清液于另一干净的1.5ml离心管中作为模板使用，使用量为PCR反应体系的1/10 (例如50μlPCR反应体系，模板使用量为5μl)。
注意：必须现处理现用。
3. 结果检测：
3.1 qPCR扩增反应：
3.1.1 推荐使用qPCR扩增体系：
组分
| 体积
|
ddH₂O
| Up to 50μl
|
直扩qPCR反应液
| 38μl
|
直扩HotStart Taq酶
| 2μl
|
直扩qPCR引物探针
| 5μl
|
模板
| 5μl |
试剂全部加好后，混匀后瞬时离心，将所有试剂收集到管底。
直扩qPCR 引物探针配制 (建议)：
引物探针母液
| 直扩qPCR引物探针mix中终浓度
| 单人份用量
|
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反应程序：
步骤
| 温度
| 时间
| 循环数
|
| 1 | 95°C
| 3 min
| 1 cycle
|
| 2 | 95°C
60°C (此步收集荧光)
| 15 sec
30 sec
| 40 cycles |
根据引物 (探针) 设计确定退火温度。
3.2 RT-qPCR扩增反应：
3.2.1 推荐使用RT-qPCR扩增体系：
组分
| 体积
|
ddH₂O
| Up to 50μl
|
直扩RT-qPCR反应液
| 38μl
|
直扩RT-qPCR酶混合液
| 2μl
|
直扩RT-qPCR引物探针
| 5μl
|
模板
| 5μl |
试剂全部加好后，混匀后瞬时离心，将所有试剂收集到管底。
直扩RT-qPCR引物探针配制 (建议)：
引物探针母液
| 直扩RT-qPCR引物探针mix中终浓度
| 单人份用量
|
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 |
3.2.2RT-qPCR反应程序：
步骤
| 温度
| 时间
| 循环数
|
| 1 | 50°C
| 10 min
| 1 cycle
|
| 2 | 95°C
| 3 min
| 1 cycle
|
| 3 | 95°C
60°C (此步收集荧光)
| 10 sec
30 sec
| 40 cycle |
根据引物(探针)设计确定退火温度。
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).
ZJ26F0433900
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