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0Isothermal nucleic acid amplification technology can be divided into many categories according to different amplification principles. At present, the most common and widely used is the loop-mediated isothermal amplification technology(LAMP) developed by Japanese scholar Notomi et al. in 2000. Bst DNA polymerase is the core enzyme of isothermal amplification technology. What is the function and performance of Bst DNA polymerase in LAMP?
1. How does Bst DNA polymerase plays a role in LAMP？
2. What is Lyophilized Bst Plus DNA Polymerase?
3. What is the product feature of Lyophilized Bst Plus DNA Polymerase(14405ES)?
4. What is the product performance of Lyophilized Bst Plus DNA Polymerase(14405ES)?
5. Related products
6. Regarding reading
1. How does Bst DNA polymerase plays a role in LAMP？
The technical feature of LAMP is to design four primers for six regions of the target gene and carry out the amplification reaction using BST DNA polymerase under the constant temperature condition of 60~65 ℃, achieving 109-1010 times amplification within 15-60 minutes. Bst DNA polymerase has strong thermal stability, strand displacement activity, and polymerase activity. It can be a rapid, efficient, and specific amplification template under constant temperature conditions without PCR thermal denaturation amplification cycle, and complete the entire LAMP experiment within 1 hour.
2. What is Lyophilized Bst Plus DNA Polymerase?
Lyophilized Bst Plus DNA Polymerase is used to freeze glycerol-free Bst Plus DNA Polymerase, which solves the problem of glycerol-free Bst enzyme instability. At the same time, this product has strong 5 '- 3' DNA polymerase activity, strand displacement activity, and dUTP tolerance, which is more suitable for the anti-pollution isothermal amplification reaction, It is convenient for customers to use in the production of freeze-dried products.
Figure1: 14405ES-Lyophilized Bst Plus DNA Polymerase ( 60 U/μL，Glycerol-Free)
3. What is the product feature of Lyophilized Bst Plus DNA Polymerase(14405ES)?
- The glycerol-free enzyme, suitable for lyophilization
- Transport at normal temperature to reduce costs
- Strong strand displacement activity, sensitive and stable
- Fast re-dissolution and convenient operation
4. What is the product performance of Lyophilized Bst Plus DNA Polymerase(14405ES)?
4.1 Freeze dried powder shape: compared with T0 moment, the shape of powder remains unchanged after 45 ℃ - 15DAYS freeze drying.
Figure 2. Freeze-dried powder shape
4.2 stability
The performance of freeze-dried powder after thermal acceleration can still be consistent with that of liquid reagent, and freeze-dried powder(14405ES) is
universal and can be used for the fluorescent dye method and pH indicator method.
4.2.1 Fluorescent dye method——stability
In the RT-LAMP fluorescent dye amplification system, the performance of glycerol-free Bst freeze-dried powder after T0, 45℃-7days and 45℃ -15 days of thermal acceleration is consistent with that of the liquid version of glycerol-free Bst enzyme, and NTC does not peak.
Experimental conditions:
| System | RT-LAMP Fluorescent dye method | Instrument | Q5, SLAN |
| Template | COVID-19 RNA | Concentration | 50copies/T |
| Procedure | 37℃, 2 minutes. 65℃, 60 minutes |
Note: The control group is a liquid version without glycerol Bst enzyme of the same concentration.
Figure 3. Stability results
4.2.2 Fluorescent dye method——Performance of Bst enzyme after freeze-drying of RT-LAMP system
In the RT-LAMP fluorescent dye amplification system, the product performance of glycerol-free Bst freeze-dried powder freeze-dried in the RT-LAMP system after 45 ℃ - 15 days was consistent with that of the liquid version of glycerol-free Bst enzyme. The detection rate of the product in the control group and the RT-LAMP system after freeze-dried was 100% (16/16) and 94% (15/16) respectively.
Experimental conditions:
| System | RT-LAMP Fluorescent dye method | Instrument | Q5, SLAN |
| Template | COVID-19 RNA | Concentration | 50copies/T |
| Procedure | 37℃, 2 minutes. 65℃, 60 minutes |
Note: The control group is a liquid version without glycerol Bst enzyme of the same concentration.
Figure 4. Performance of Bst enzyme after freeze-drying of RT-LAMP system
4.2.3 PH indicator method ——Stability
In the RT-LAMP pH indicator amplification system, compared with T0 time, the detection rate of Bst freeze-dried powder was 88% (7/8) after 45 ℃ - 15 days of thermal acceleration.
Experimental conditions:
| System | RT-LAMP PH indicator method | Template | COVID-19 RNA |
| Procedure | 37℃, 2 minutes. 65℃, 60 minutes | Concentration | 50copies/T |
Note: The control group is a liquid version without glycerol Bst enzyme of the same concentration.
Figure 5. Stability results
4.2.4 PH indicator method——Refreezing stability
In the RT-LAMP pH indicator amplification system, after 45 ℃ - 7 days of refreezing, the performance is stable. The control group is liquid Bst enzyme. Compared with the control group, the detection rate of 14405ES after thermal acceleration is 94% (30/32).
Experimental conditions:
| System | RT-LAMP PH indicator method | Template | COVID-19 RNA |
| Procedure | 37℃, 2 minutes. 65℃, 60 minutes | Concentration | 50copies/T |
Note: The control group is a liquid version without glycerol Bst enzyme of the same concentration.
Figure 6. Refreezing stability results
4.3 Specificity
In the RT-LAMP fluorescent dye amplification system, the 48-well NTC did not peak, and the specificity of f14405ES was 100%.
Experimental conditions:
| System | RT-LAMP Fluorescent dye method | Instrument | Q5, SLAN |
| Template | COVID-19 RNA | Concentration | 50copies/T |
| Procedure | 37℃, 2 minutes. 65℃, 60 minutes |
Note: The control group is a liquid version without glycerol Bst enzyme of the same concentration.
Figure 7. Specific results
5. Related products
The related products that Yeasen can provide are shown in Table 1.
Table 1. Related products
| Product Positioning | Product name | Cat# |
| Highly sensitive Bst enzyme | Hieff™ Bst Plus DNA Polymerase (40 U/μL) | 14402ES |
| Hieff™ Bst Plus DNA Polymerase (2000 U/μL) (Inquire) | 14403ES | |
| Lyophilized Bst Plus DNA Polymerase ( 60 U/μL， Glycerol-Free) | 14405ES | |
| Fluorescent dye method | RT-LAMP Dye Assay Kit (UDG plus) | 13762ES |
| PH indicator method | RT-LAMP pH Sensitive Dye Chromogenic Version Freeze-Dryable Reagent | 13920ES |
| COVID-19 | RT-LAMP COVID 19 (pH Indicator Method) Lyophilized Beads | 13919ES |
| RT-LAMP COVID-19 Primer Master Mix (N) | 13995ES | |
| Reverse transcriptase suitable for RT-Lamp | Hifair™ Ⅲ Reverse Transcriptase (Inquire) | 11111ES |
| Hifair™ Ⅲ Reverse Transcriptase, Glycerol-free (Inquire) | 11297ES | |
| Murine RNase Inhibitor | Murine RNase inhibitor (40 U/μL) | 10603ES |
| Murine RNase inhibitor (200 U/μL, Glycerol-free) (Inquire) | 10703ES | |
| Heat-labile UDG | Uracil DNA Glycosylase (UDG/UNG), heat-labile, 1 U/μL (Inquire) | 10303ES |
| High-purity dUTP | dUTP (100 mM Solution) | 10128ES |
6. Regarding reading
Choosing the right key enzyme raw materials to make LAMP /RT-LAMP more sensitive and faster!
