Daan Science: Application of isothermal amplification and fluorescence quantitative PCR in rapid detection of new crown nucleic acids
In the past two years, the rapid development of molecular biology technology under the global epidemic grip, the established diagnostic methods for rapid detection of neo-nucleic acids - fluorescent quantitative PCR and isothermal amplification are gaining attention day by day.
Loop-mediated iso-thermal amplification ( loop - mediated iso-thermal amplification , LAMP) is a nucleic acid isothermal amplification technique proposed by Japanese scholar Notomi, whose principle is based on DNA in dynamic equilibrium at about 65℃, using four different specific primers to identify six specific regions of the target gene by The principle is based on the dynamic equilibrium state of DNA around 65℃, and the amplification of the target fragment is achieved by the strand replacement function of Bst DNA polymerase and the interaction of Inter Primer and OuterPrimer.
In contrast, PCR uses DNA denaturation to single-stranded at 95°C in vitro, and primers bind to single-stranded at low temperature (often around 60°C) according to the principle of base complementary pairing, then adjust the temperature to the optimal reaction temperature of DNA polymerase (around 72°C), and DNA polymerase synthesizes the complementary strand along the direction of phosphate to pentose (5'-3'); its denaturation, denaturation and extension is actually a temperature-controlled process.
Main differences between the two.
But combined with the cunning and changeable nature of the new coronavirus, each time different "new" mutant strains, in the face of China's large population, its dense human traffic, the actual situation of many individual disease genotypes, in the control area of the emergence of confirmed patients, the need to achieve the first region-wide new coronavirus nucleic acid rapid test screening, and traceability investigation close contacts, and strictly control the occurrence of cross-regional transmission of the epidemic.
Therefore, for the actual rapid detection of new crown nucleic acid, qPCR due to the maturity of primer design and fluorescence channel diversity, its molecular diagnostic instruments also play an important role in high throughput; coupled with the current domestic sample volume of the General Assembly exists most laboratories can not strictly partition, LAMP method once the lid is open easy to form aerosol contamination, prone to false-positive problems.
Although LAMP solves the temperature control problem of PCR very well, it also brings other problems accordingly. Compared with the very mature technical system of PCR, we also look forward to the continuous optimization and development of isothermal amplification technology in the future, and the two technologies complement each other to develop better rapid detection kits for new crown nucleic acid diagnosis.
