Background
At the beginning of 2020, in the face of the sudden outbreak of COVID-19, nucleic acid testing became widely known. As one of the main detection methods in the field of IVD, nucleic acid detection kits have developed rapidly in recent years, especially in the field of molecular diagnosis POCT. At present, the main POCT nucleic acid detection technology on the market is qPCR technology, which has strong specificity and high sensitivity, can quantitatively detect pathogens, and can achieve high-throughput detection, but requires qPCR instruments. The instrument is large in size, high in price and relatively long in time, which greatly limits its promotion and on-site inspection at the grassroots level. Under this premise, loop-mediated isothermal amplification (LAMP) stands out in the POCT market with its advantages of rapid detection and low price.
The characteristic of LAMP is to design four primers for six regions on the target gene, and utilize strand displacement DNA polymerase (Bst DNA Polymerase) to carry out the amplification reaction under constant temperature (60 - 65℃), which can be realized within 15 - 60 min 109-1010-fold amplification. In addition to high specificity and high sensitivity, the advantages of the LAMP are simple to operate; in the application stage, the requirements for the instrument are low, and a simple constant temperature device can realize the reaction; the result detection is also very simple, no need to perform gel electrophoresis to observe the results like PCR. LAMP is a method suitable for on-site, grassroots rapid detection.
Vazyme Solutions
As a high-quality supplier of biological reagents, Vazyme has been committed to providing complete solutions for the development of LAMP/RT-LAMP assays to promote the development of molecular POCT. We have independently developed Bst DNA polymerase and reverse transcriptase suitable for RT-LAMP, which are applied to fast and efficient isothermal amplification of LAMP/RT-LAMP, accurately quantify DNA or RNA templates, and shorten detection time while improving sensitivity.
Figure 1. Experimental steps for fast extraction and detection
Performance Comparison of Conventional LAMP
1) Comparison of purified DNA
Method: Compare P703 performance with Supplier A using plasmid DNA template.
System: LAMP SYBR Green 25 μl/T
Table 1. Reaction system of P703 and Supplier A using plasmid DNA template
Test Instrument: ABI Q3 65℃, 60 min
Result:
Figure 2. Comparison of amplification performance between different gradients of DNA templates
Conclusion:
The amplification performance and specificity of P703 are good, mainly because the CT value of the same positive sample is smaller. In addition, the amplification of negative samples and No Template Control (NTC) do not peak or peak late.
2) Comparison of purified RNA template
Method: Compare RV101 + P703 performance with Supplier A using Human and bacterial RNA.
System: LAMP SYBR Green 25 μl/T
Table 2. Reaction system of RV101 + P703 and Supplier A using Human and bacterial RNA
Test Instrument: ABI Q3 65℃, 60 min
Result:
Figure 3. Comparison of RT-LAMP results between different species; left (bacterial samples); right (human samples)
Conclusion:
The plots show that RV101 + P703 have better amplification performance, specificity and repeatability. The amplification speed is mainly manifested in the relatively small CT value of positive samples, and the specific effect is mainly manifested in negative samples and NTC amplification does not peak.
Vazyme Products
Vazyme’s solutions for LAMP assays drive the development of faster, simpler and more efficient LAMP assays. At the same time, in order to meet the customers’ requirements for more convenient, fast and stable reagents, Vazyme is working on the reagent development of "RT-LAMP dye/color premix". We look forward to your continued attention.
Table 3. Vazyme’s LAMP/RT-LAMP products
