The Establishment Of The Screening Process About Pathogen's Specific Nucleotide Signatures And Verify The Sigantures' QPCR Arrays

Pathogen detection is an important point in the infectious diseases' prevention and control,in the face of thousands of known or unknown pathogens,the traditional method of pathogens' separation and cultivation has the defect of blindness,low-throughput and long cycle.Pathogens' confirmation needs of the evidences about epidemiology,etiology,serology,morphological and immunohistochemical.But some pathogens cannot be separated succed,some pathogens have not susceptible animal model to be verified.If in the early stages of the infectious disease we can rapid high-throughput detect the pathogens' nucleic acid or protein,which could make the scope of the identification of suspicious infection pathogens be narrowed,also could guide the pathogen identification,serological detection,clinical diagnosis and symptomatic treatment,after that.With the widely application of the next-generation sequencing technology,the genome information exploding of species and pathogens.The research about genome data information to be the foremost field of the biological and medical field.In order to achieve the purpose of rapid and high-thoughput to test the pathogens,the base is to build a complete and efficient pathogens signatures sequence database.Signatures are nucleotide sequences that can be used to detect the presence of an organism and to distinguish that organism from all other species.And to build a complete and efficient pathogens signatures database,which must be based on intelligent computing process of big data,to establish a suitable algorithm and the signatures output alignment.Our study based on <The main pathogenic microorganism catalogs > which released by the ministry of health in China 2006,mege the < Pathogenic Biology > edited by en-jie luo and the < Medical microbiology > edited by fan Li and zhi-kai Xu.567 kinds of pathogens are list,combining with the 16 sr RNA sequence library Silva(http://www.arb-silva.de/contact/),bacterial virulence genes VFDB library(http://www.mgc.ac.cn/VFs/main.htm),drug resistance gene ARDB library(http://ardb.cbcb.umd.edu/).Our study aims to build a set of efficient and practical pathogen signature screening system.And on the basis of screening system,rating signatue is put forward,family signatures + species signature + virulence genes signature + resistance gene signature together detect pathogens,eventually through multiple targets realizing a complete coverage to achieve one-time test all pathogens and their drug resistance and pathogenic characteristics.Through our research,has obtained the following achievements:1)Based on MUMmer + Insignia we groped and established our signature screening process “TMMU signature workflow”,and implement workflow visualization.all target pathogens nucleotid sequence were beening background in the workflow,at the same time in order to speed up Insignia process after match by MUMmer,the files have been slit according the taxon.To reduce the loss of specific signatures,merge the Nt and WGS to be BLAST library of our workflow,and eliminate all synthetic sequence.Two important parameters of BLAST have been setting: one was by name(dual)to determine whether the same pathogen specie;the other was 90% coverage,if other species sequence with the signature's coverage reached 90%,this signature was deemed to not specific.2)Through a variety of computing verification,TMMU signature workflow precise calculation process is not suitable for bacteria genus signature comparison(both based on 16 s sequences and whole genome sequence).But clustal Omega could find the conservative segment of 16 S sequences,then through multiple signatures to cover the individual base differences,the genus detect purpose could been achievement.3)TMMU signature workflow precise calculation process applies not only DNA genome sequence and RNA genome sequences,also applies to short sequences(except 16 s sequence,which can't distinguish the pathogens between species).Pathogens have no genome sequence and the virulence genes,resistance genes sequences,also could obtain signature sequences by differents strategy that don't make intersection between these sequences.4)15 kinds of highly pathogenic haemorrhagic fever virus qPCR arrays and 29 kinds of mycobacterias qPCR arrays have been established.Highly pathogenic haemorrhagic fever virus qPCR arrays standard curve's correlation coefficient greater than 0.99 and the sensitivity can be up to 5 x 101 copies.Repeatability and accuracy test's result,the CV of the intra assay reproducibility was 0.88%-2.50%,the CV of the inter assay reproducibility was 1.75%-3.31%.29 mycobacterias qPCR arrays standard curve's correlation coefficient greater than 0.99 and the sensitivity can be up to 5 x 103 copies.Two tuberculosis probes detected 23 cases of TB smear-positive samples all positive,20 patients with TB smear-negative all negative,specificity 100% and sensitivity 100%.9 positive samples detected by Gene X-pert MTB for comparison,probe1 tested 7 positie samples,and probe2 tested 7 positive samples.However,the positive results of two probes are not completely overlap,actually their complement by each other,so 8 positive samples were detected totally.Which indicated more pathogens targets detecting is superior to the single target.