Study On The New Nucleic Acid Detection System Based On Bioluminescence Of Pyrophosphate

Nucleic acid(NA)detection plays an important role in cancer diagnosis,pathogen detection,plant pathogen detection,environmental microbial evaluation and and other important applications.Although nucleic-acid based detection methods have greatly improved over the years,there are still some disadvantages under certain circumstances:1.For samples containing very low number of targets,the sensitivity is not enough.2.The thermal cycling and optical systems are costly and usually large in physical size,therefore creates challenges for broader use in remote areas.1.For samples containing very low copy number of pathogenic microorganisms,the sensitivity is not high enough.2.Depending on variable temperature system and excitation light source system,the cost and equipment size are difficult to control,and it is difficult to popularize in remote areas.The method based on pyrophosphoric acid bioluminescence is a low cost and high sensitivity detection method with low requirements for equipment,and its sensitivity can reach single copy theoretically.The principle of it is to use pyrophosphoric acid(PPi),a byproduct produced in the process of nucleic acid amplification,to produce light signals under the action of ATP sulfase,5’-phosphoyl sulfate sodium salt,luciferase and triple double phosphatase.This method has the advantages of simple instrument,low detection limit,wide linear range and so on.It is widely used in chemical and biological analysis,especially suitable for the use of grass-roots units.Compared with fluorescence method,chemiluminescence method does not require external light source,thus reducing light scattering,reducing the interference of noise signal,improving the sensitivity of detection,and expanding the linear dynamic range.However,its sensitivity and stability are not high in practical application.Therefore,this paper carries out research based on the above problems and develops a new nucleic acid(NA)detection system based on pyrophosphate bioluminescence.Specific research contents are as follows:1.Establishment of a new NA detection method based on pyrophosphate bioluminescenceThrough the analysis of each component of pyrophosphoric acid bioluminescence method,the reasons for the low sensitivity of traditional pyrophosphoric acid chemiluminescence detection were found out,and through screening,dATPaS was confirmed to replace dATP for PCR amplification.It can not only eliminate the high background signal interference caused by dATP,but also participate in the amplification reaction well without affecting the amplification efficiency.Combined with terminal detection PCR,the detection limit of it was less than 10 Copies.The detection limit of new NA detection method based on pyrophosphate bioluminescence was nearly 1000 times than that of traditional one.2.Development of a new bioluminescent assay in real-time couple of loop-mediated isothermal amplification(LAMP-BART)The new NA detection system(LAMP-BART)was developed by modifying the weak light analyzer to detect nucleic acids quantitatively in real time,the detection limit of it was less than 10 Copies.The detection limit of this method is 1000 times higher than that of the existing BART nucleic acid detection system.Compared with the traditional bacterial culture method,the accuracy of the method for detecting Salmonella and Escherichia coli in poultry samples in animal hospital was up to 97%.When combined with the modified propylazide bromide reagent(PMAxxTM)for pretreatment,the accuracy was 100%.3.Simulation digital PCR based on new pyrophosphate bioluminescenceCombined with digital PCR technology,a new digital NA detection system based on pyrophosphoric acid bioluminescence can be developed and named simulation digital LAMP-BART system.The detection limit of new real-time of NA detection system was about 10 Copies/mL and achieved absolute quantification.Using the fake novel coronavirus reference plasmid(SARS-CoV-2 ORF1ab,containing complete SARSCoV-2 ORF1ab gene sequence)as the template,the system performed high sensitivity and good specificity.4.The development of solvent response intelligent magnetic beads and its application in the detection of new pyrophosphate bioluminescent nucleic acidThe solvent response intelligent magnetic beads were designed and developed with the double NA desorption effect of pH and organic solvent.Compared with traditional commercial magnetic beads for NA extraction,the NA efficiency of the new magnetic beads was improved by nearly 20%.Combined it with the digital LAMP-BART,the detection limit of this method was improved to reach 5 Copies/mL when using the fake novel coronavirus reference plasmid as template.This subject develops a rapid,accurate and sensitive nucleic acid analysis and detection system to realize the accurate detection of viruses and pathogenic microorganisms,which is of great significance for the prevention and control of bacterial and viruses with large area.