Research And Application Of DNA Nanoball Sequencing Technology In Detection Of Pathogenic Microorganisms

With the change of population structure,abuse of antibiotics,emergence of new pathogens and the increase of drug-resistant bacteria,the impact of infectious diseases on human health is increasing.According to WHO's announcement of the top ten global health threats in 2019,influenza,drug resistance,Ebola,dengue fever,AIDS,vaccination hesitation and other diseases closely related to infection occupy 6 of the top 10 by absolute advantage.However,in the field of pathogenic microorganism detection,there are still many critical infections that cannot be accurately diagnosed,such as: 15%-25% of pneumonia pathogenic cause is unknown,20% of clinical fever pathogenic cause is unknown.In the field of complex pathogenic infections,the positive rate of traditional methods for blood culture samples is 8-12%,and the positive rate of cerebrospinal fluid samples is 8-10%.The Conventional pathogenic microorganism examination includes morphology and serology.These techniques play an important role in the field of pathogenic microorganism detection,but there are also some shortcomings,such as the difficulty of isolation and cultivation of pathogens,long detection period,detection range limited to specific known pathogens,and cannot discover new pathogens.Metagenomics Next Generation Sequencing(mNGS)based on pathogen nucleotide sequence analysis,mNGS not only greatly simplify the detection process and shorten the detection period,but also has great significance for the detection of new pathogens or difficult to identify by conventional methods.In this study,DNA nanosphere sequencing technology was used to establish a set of mNGS pathogenic microorganism detection process based on domestic MGISEQ-2000 sequencer.We have improved the detection process of mNGS,such as improved the sample preparation,DNA extraction and library construction,including: 1)Increase 10 times the sensitivity of fungi detection by using 0.5mm glass beads and ultrasound interruption.2)Using 10 um filter membrane and DNase to decrease the host background DNA,increase microbial sequences by 2 times.3)Adjust the concentration of dNTP and adapter to improve the success rate of onetime detection of low nucleic acid sample.4)Increasing the amount of sequencing output data is helpful to detect low pathogens and reduce false negative.Finally,bacterial,fungal standard strains and virus-positive serum reference materials were used to verify the sensitivity,repeatability and specificity of the procedure,and the detection accuracy of the detection procedure was verified by comparison with clinical results.