Background

Loop-mediated isothermal amplification (LAMP) is a versatile technique for detecting target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. LAMP technique does not require changing the reaction temperature and it's extremely fast, which is making them particularly well suited for field applications and point-of-care molecular diagnostics assays.

With well-designed 4-6 primers and the specific Bst DNA polymerase, the detection of the target of interest can be done within 30 min. There are many LAMP detection methods, including the fluorescent method, colorimetric method, fluorescent-probe-based method, turbidity method. HNB, etc. The LAMP solution provided by Hzymes is able to help achieve high sensitivity, good primer compatibility, and excellent performance in specificity, tolerance, and stability.

Advantages

Bst DNA polymerase V2

Bst DNA polymerase V2 is derived from Bacillus stearothermophilus DNA Polymerase l, which contains 5 '→3’DNA polymerase activity and strong strand displacement activity but lacks 5'→3exonuclease activity. lt can be used for DNA strand displacement reaction and isothermal amplification. Hzymes Biotech's Bst DNA polymerase V2 has fast amplification speed, strong resistance to PCR inhibitors, and good sample compatibility. In addition, the WS Bst DNA polymerase V2 is modified by aptamers, which can effectively prevent false positive results. The glycerol-free Bst DNA PolymeraseV2 version is more suitable for developing lyophilized LAMP kits.

Fast polymerization

Blue: Company N; Green: HMD7006-WS Bst DNA polymerase V2

Polymerization speed is a crucial indicator for evaluating Bst DNA polymerase. The SARS-CoV-2 N gene target (0.5 cp/uL, 1 cp/uL, and 5cp/uL) can be successfully detected using Ws Bst DNA polymerase V2 (HMD7006). And the whole detection performs faster than company N at the same template concentration (5 cp/uL).

Specificity

Hzymes Biotech's WS Bst DNA polymerase V2(HMD7006 and HMD 7007) user aptamer-based blocking technology. lt blocks the enzyme's activity through the tight bond of the aptamer and the polymerase at a lower temperature. But at the working temperature, the binding of the aptamer to the enzyme is released, and the enzyme becomes active.

Example: NTC diagram when detecting SARS-CoV-2 N gene target

Comparison of enzyme activity with Company N at 30°C and 65°C

After aptamer modification, WS Bst DNA polymerase V2(HMD7006 ancHMD7007) has lower enzymatic activity than Company N at 30 °C and higher blocking efficiency(More beneficial to prevent non-specific amplification). At the working temperature(65°C), the activity is released and is higher than that of Company N. In an RT-LAMP reaction. NTC does not appear within 60 minutes.

Inhibitor tolerance

Hzymes Biotech's Bst DNA polymerase V2(HMD7005) is compared with competing products in different inhibitory environments (guanidine hydrochloride, guanidine isothiocyanate, KCl, etc.), Hzymes Biotech's Bst DNA polymerase V2(HMD7005) is more robust than the competitive products and more suitable for different application scenarios, such as direct amplification. it is easier for the subsequent development of LAMP/RT-LAMP reagent.

Glycerol-free and Stability

Bst DNA polymerase V2 and WS Bst DNA polymerase V2 products maintain the same activity after incubation at 37'c for 0. 3. and 7days. To ensure the development of LAMP/RT-LAMP lyophilized reagents, the activity of glycerol-free Bst DNA polymerase V2 series (HMD7005, HMD7006, HMD7007) remains unchanged after repeated freeze-thaw cycles 40 times.

RTL Reverse Transcriptase 2.0

RTL Reverse Transcriptase 2.0 is a brand-new reverse transcriptase from a different source. lt can maintain enzyme activity at higher temperatures (RT-LAMP working temperature) and has RNase H activity. lt has the advantages of high reverse transcription efficiency, good freeze-thaw, thermal stability, and strong inhibitor tolerance. Compared with other reverse transcriptase RTL reverse transcriptase 2.0 is more suitable for developing the RT-LAMP reagent. In addition, the RTL Reverse Transcriptase 2.0 series products include glycerol-free and aptamer-modified versions, which are more helpful for developing lyophilized RT-LAMP reagents and preventing false positive results.

Reverse transcription efficiency

The activity of RTL reverse transcriptase 2.0 at different temperatures

Hzymes Biotech's RTL reverse transcriptase 2.0 (HMD5302) has excellent heat resistance, and its optimum activity range perfectly adapts to the reaction conditions of RT-LAMP (60-65 °C). lt’s reverse transcription efficiency is high, and can be completed within 1-3 minutes.

Thermal stability at higher temperature

Compared with Company N, Hzymes Biotech's RTL reverse transcriptase 2.0 (HMD5302) has higher activity and better stability when incubated at 37 'c and 50 °C for 30 min and 10min).

Glycerol-free and stability

37 °C Thermal stability for 7 days

Freeze-thaw stability at -20 °C

30-days stability at 25°C

Hzymes Biotech's RTL reverse transcriptase 2.0 (HMD5302), incubated at 25'c for 30 days and incubated at 37'c for 7 days, the reverse transcription performance of RT-LAMP did not change, and the activity maintained the same. To ensure the development of RT-LAMP lyophilized reagents, the activity of glycerol free RTL reverse transcriptase 2.0 (HMD5302) remained unchanged after repeated freeze-thaw cycles 50 times.

Inhibitor tolerance

Guanidine hydrochloride

Guanidinium isothiocyanate

Whole blood

NaCl

Guanidine hydrochloride and guanidine isothiocyanate are common nucleic acid extraction residues. RTL reverse transcriptase 2.0 (HMD5202) activity is higher than that of Company N under the same inhibition concentration. lt is better than competing products with NaCl and whole blood including single components. it is more suitable for various application scenarios, such as direct amplification, and easier for the subsequent development of RT-LAMP reagent.

RT-LAMP Master Mix (Fluorescent) Liquid & Lyophilized

High sensitivity and high inhibitor tolerance

RT-LAMP reaction is performed using a nasal swab sample spiked with SARS-CoV-2 pseudovirus (50copies/T,100 copies/T, and 250 copies/T) after a simple treatment with Hzymes Sample release buffer (HMD3504).

High specificity

There is no NTC amplification while using RT-LAMP fluorescent master mix (Liquid, lyophilization ready) (HMD5207).

Performance consistency with lyophilized reagent

The data illustrates that the lyophilization process does not affect the performance of the RT-LAMP reaction (The same template concentration: 250 copies/T,100 copies/T, and 50 copies/T).

dUTP tolerance

Hzymes RT-LAMP fluorescent master mix (Liquid, lyophilization ready) (HMD5207) performs better dUTP tolerance than company N, while using the same primer set and template.

RT-LAMP Master Mix (Colorimetric) Liquid & Lyophilized

Selection Guide – For Your LAMP Application

LAMP/RT-LAMP raw materials

LAMP/RT-LAMP related CDMO service

LAMP/RT-LAMP master mix