Establishment And Application Of A Novel Technology For Pathogen Nucleic Acid Detection

Infectious diseases are usually caused by the infection of pathogenic microorganisms and pose a serious threat to human health.With the rapid development of the economy of China,the lifestyles of people have also undergone major changes.This has brought new problems and challenges to the prevention and control of infectious diseases in China.Reliable and effective diagnostic methods for pathogens are important tools for the prevention and control of infectious diseases.Traditional methods for detecting pathogenic microorganisms often have problems such as low sensitivity or low specificity,which has led to limitations in the diagnosis of low conentration samples,mixed infections,or unexplained infections.With the continuous development of molecular biology and genomics,various molecular diagnostic techniques and nucleic acid detection methods have gradually become an important tool in the field of public health and clinical testing,especially in the diagnosis of infectious diseases,infectious disease prevention and control,syndrome monitoring,unknown infectious pathogen diagnosis and microbial genomics have become mainstream methods.In recent years,molecular biology techniques such as polymerase chain reaction and nucleic acid molecular hybridization have been widely used in the diagnosis of pathogenic biology.More advanced gene chip technologies and high-throughput sequencing technologies are also in a rapid development stage.One single nucleic acid detection method is difficult to meet different experimental and clinical needs.How to make full use of the advantages of different molecular diagnostic techniques to carry out detection and monitoring of pathogens is of increasing concern.Under this demand,this research establishes a series of innovative technologies for pathogen nucleic acid detection,in order to apply different detection methods and strategies in different situations,and to solve the corresponding science in the most rapid,economical,accurate,and scientific manner problem.These innovative nucleic acid detection methods,each of which differs in terms of equipment requirements,detection throughput,detection cost,difficulty of operation,and experimental turnaround time,can achieve different experimental objectives.The pathogen detection work of medical institutions at all levels in China provides a powerful tool.New methods include:1.One step nested PCR(LNA-OSN-PCR)based on locked nucleic acid technology.This method does not need to open the cap during the reaction,which greatly reduces the risk of contamination compared to conventional nested PCR(N-PCR),while reducing the reagent cost and reducing the workload.LNA-OSN-PCR is a general-purpose strategy.The principle of primer design is the same as that of the classical N-PCR method.Any laboratory can establish corresponding methods according to their own needs.Researchers can also upgrade existing mature N-PCR methods to one-step strategies.This study demonstrated that LNA-OSN-PCR is suitable for the detection of single-stranded RNA viruses,double-stranded RNA viruses,and DNA viruses in cerebrospinal fluid,buccal swabs,or fecal specimens.Both N-PCR and semi-N-PCR methods can be upgraded.optimization.LNA-OSN-PCR is versatile and does not require special consumables or equipment.It does not require pretreatment of the reaction tubes.It does not require modification or redesign of internal and external primers,and no additional components need to be added to the system.From primer design to experimental manipulation,classic PCR principles can be followed.Any microbiological investigator with N-PCR requirements can try this new method.2.One step nested real-time PCR(LNA-OSN-q-PCR)based on locked nucleic acid technology.In this method,LNA acid external primers,DNA primers and Taqman probes are added simultaneously in the system,and fluorescence signals are detected in real time while being amplified.LNA-OSN-q-PCR requires that all 5 oligonucleotides match the template sequence to be successfully performed.It also combines the advantages of N-PCR nested template enrichment strategy and q-PCR fluorescence detection method.Extremely high specificity and sensitivity.The establishment of LNA-OSN-q-PCR method can redesign probes based on established mature N-PCR,or redesign primers based on mature q-PCR.LNA-OSN-q-PCR does not need to open the lid during the whole experiment,and has almost no risk of contamination.It can be used on a large scale as a routine molecular diagnostic method in microbiology laboratories and has the potential to become a commercial kit.3.Resequencing Pathogen Microarray for the diagnose of central nervous system infection(RPM-IVDC4).The method combines the ability of multiplex PCR to enrich high-throughput nucleic acid samples and the high-throughput nucleic acid hybridization detection capability of the gene chip technology.It can simultaneously perform molecular diagnostics for hundreds of types of encephalitis/meningotrophic pathogens in one experiment.The sequence information of the hybrid DNA can be obtained.The detection spectrum of RPM-IVDC4 covers the vast majority of pathogens that infect the central nervous system,including viruses,bacteria,fungi,and parasites.The species-specific primers were designed not only for more than 100 pathogens,but also general-purpose generic families/genus-specific primers were designed for 45 taxa and 30 taxonomic families,thus greatly expanding the potential detection of RPM-IVDC4.range.RPM is well suited for use as a molecular diagnostic method for the monitoring of syndromes,particularly against encephalitis/meningitis syndrome.Because of the wide variety of pathogens involved in CNS infection and the rapid development of diseases after infection,severe sequelae and deaths are often caused.RPM does not require users to have bioinformatics analysis capabilities.Through standard experimental procedures,a large amount of diagnostic information can be obtained,which makes it easier for microbiologists or health care workers to accept.Therefore,RPM-IVDC4 can be used not only as an effective means for detecting encephalitis/meningitis pathogens,but also as a clinical diagnostic tool for CNS infections.4.Amplicon sequencing method based on MinION.We took the case of enterovirus 71 and Coxsackievirus A16 as examples to introduce how to perform deep sequencing of the whole genome of known pathogens based on nanopore sequencing technology,and to describe in detail the sample processing in the nanopore sequencing experiment and amplicon preparation.,Library construction and on-machine sequencing and other steps.During the sequencing process,we performed a comprehensive assessment of the comprehensive performance of the MinION sequencing platform,and analyzed the sequencing characteristics of MinION from the perspective of data quality,sequencing efficiency,sequencing accuracy,and multi-sample detection capabilities.5.Metagenomics Sequencing method based on MinION.We established a metagenomic DNA sequencing method based on MinION and detected pathogens directly from clinical samples that have never been cultured.We used MinION to directly perform pathogen detection in 12 clinical samples of cerebrospinal fluid.Among them,reads of Mycobacteria were found in 10 cases,and related pathogens such as herpes simplex virus 2,Neisseria and Streptococcus were found in other samples.clue.The positive results of mycobacteria were confirmed by q-PCR review,demonstrating that the metagenomic sequencing method based on MinION has the ability to directly detect pathogens from clinical samples.