Research And Development Of Pathogen Detection Method Based On Metagenome High-throughput Sequencing

In biology,a pathogen,or a causative agent,refers to a germ that can attack human body,produce infectious disease or even epidemics.Typically,the term pathogen includes bacterium,virus,fungus,parasite,mycoplasma,chlamydia,rickettsia,spirochete or other micro-organism,among which bacterium and virus are the most detrimental ones.Currently infectious diseases have become a severe threat for human beings and epidemics remain the chief culprit of human demise.According to the survey of the World Health Organization(WHO),in 1955 at least 17 million people died from epidemics all over the world.By contrast,based mainly on empirical and directional means,traditional pathogen detection and identification methods fail to meet the comprehensive need that is not only instant and precise but has a wide coverage.Thus,this study explores for the hospital and patients,original detection is in great demand.This study explores methods that can verify the causative agent in a more comprehensive,accurate and efficient way,as well as help doctors with diagnosis and prescription in the treatment.In this way,the course of treatment can be shortened,death rate descended and medical cost lowered.In light of the fact that pre-existing knowledge of pathogen in the samples is not requisite in pathogenic detecting methods based on high-through sequencing of metagenome,we can commence sequencing and identifying once obtain the patient’s sample,saving the process of separation,culture or enrichment and purification.As an improvement of traditional methods,they are characterized by a wide range,high throughput and relatively short period of detection.However,due to the lack of specific design and according practical exploration,it does not yet represent an effective clinical way to analyze the efficiency,examine the accuracy and readability of the report.We have,therefore,made efforts as follows: First,we developed genome database and bioinformatic pipeline for clinical use,followed by pre-processing samples with self-developed pathogenic detecting as well as sequencing;Besides,we strove to evaluate the sequencing quality,appropriate sequencing length and core algorithms;Finally,we set up pathogenic detecting methods based on high-through sequencing of metagenome.In order to validate the methods,this analysis utilizes simulated and clinical samples in the experiment.The results indicate that:(1)the throughput of detection is about 8-10 samples per batch;(2)the detective period is less than 28 hours;(3)the detection sensitivity of 20 M sequences reaches up to 50 copies/m L,at the detection rate of about 87.80%(36/41).Although further validation is needed so as to heighten the property,with the pathogenic detecting methods based on high-through sequencing of metagenome constructed in this study,both direct high-through sequencing of different kinds of clinical samples and feasible detection for a variety of pathogens are made come true.Overall,this study brings referential value to the ultimate fulfillment of the regular use of pathogenic detecting methods based on high-through sequencing of metagenome in clinic.