Nucleic Acid Detection Method And Application Based On MALDI-TOF MS

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)is a new type of biological soft ionization mass spectrometry technique developed in recent years,which can detect and analyze biological macromolecules such as nucleic acids,peptides and proteins.The nucleic acid mass spectrometry system established by MALDI-TOF MS using the single base extension technology of dideoxyribonucleotide 3′end replication termination property combines the high precision mass spectrometry technology with biotechnology and computer intelligent analysis function,and becomes an important tool for the detection of single nucleotide polymorphism(SNP),gene mutation,DNA methylation and other molecules.Compared with foreign countries,the research and development of nucleic acid mass spectrometry in China is still at the basic stage,and the domestic market is still dependent on imports.In order to broaden the application direction of the team’s self-developed MALDI-TOF MS,meet the clinical testing needs,and break the situation that the domestic market of nucleic acid mass spectrometry detection system is occupied by foreign brands,the research participated in building a nucleic acid mass spectrometry detection system based on MALDI-TOF MS.The main contents of the study are as follows:1、The performance characterization experiments of nucleic acid mass spectrometry detection systems.The performance characterization experiments of the homemade mass spectrometry system were conducted using stainless steel target plates and silicon-based chip target plates,and the results showed that the detection performance parameters of the stainless steel target plates could meet the line standard requirements except for the sensitivity,while the performance of the homemade nucleic acid mass spectrometry system with silicon-based chip target plates far exceeded the requirements of the stainless steel target plates and the line standard.Specifically,the average resolution of the homemade nucleic acid mass spectrometry system with silicon-based chip target can reach 1000(FWHM)in the mass range of 3000-10000 Da,which is 30% higher than 750(FWHM)of the stainless steel target;the mass accuracy is within 120 ppm,which is also better than 180 ppm of the stainless steel target;the improvement in sensitivity is even more obvious,as the sensitivity of the chip The sensitivity of the chip target plate is as high as 1.5 fmol,which is more than 300 times that of the stainless steel target plate.Overall,the performance parameters of the silicon-based chip target plate can reach the international level of similar instruments.2、The development of mass accuracy algorithm.In order to improve the mass accuracy and ensure the accuracy of nucleic acid assay judgments,a calibration algorithm to improve the mass accuracy was developed.The conversion correction parameters of sample ion flight time and mass-to-charge ratio were obtained by external standard method to eliminate the quality deviation caused by internal differences of the instrument;then the theoretical flight time of sample extension primers and product sequences introduced in advance were used to calibrate the flight time obtained from the assay,which corrected the flight time offset caused by the difference of sample crystallization or the flatness of sample target and X-Y moving platform,and thus improved the mass accuracy of the instrument.After the second calibration step,the mass accuracy of the sample spectrogram can be further improved by 5-10 times.3、Application Research.(1)The homemade system was used to type 140 HPV samples,and the results showed that the homemade nucleic acid detection system was 97.9% consistent with the MassARRAY system and98.6% consistent with real-time fluorescence PCR.(2)A homemade nucleic acid mass spectrometry detection system based on the H.pylori(Hp)drug resistance gene detection method was established,and the method was used to detect drug resistance loci in 218 Hp-infected patients,and the results were highly consistent with those of the MassARRAY system.All of the above indicate that the performance of the in-house nucleic acid detection system is comparable to that of MassARRAY,a commercially available,clinically approved detection system.