Respiratory Virus Nucleic Acid Capture And Enrichment Technology Research

Respiratory viruses have frequent mutations and are the main cause of of respiratory infectious diseases.Mutation can increase the ability of virus transmission,pathogenicity,drug resistance and developing new types,and is an important cause of emerging infectious diseases.Therefore,in the prevention and control of respiratory infectious diseases,the detection and identification of the genome sequence information is the key to identify the characteristics of gene variation and assess the potential epidemic transmission characteristics and pathogenicity of the virus.The next generation sequencing technology breaks through the limitations of traditional isolation culture and the design of primers based on known sequences to identify pathogens,and has been widely used in the acquisition of pathogenic genome sequences in various samples,but the high abundance of host genomes interferes with deep sequencing of viral genomes from clinical samples.In recent years,developed a variety of enrichment technology,in order to improve the depth of sequencing of viral genome in clinical samples,which is based on the second generation sequencing of enrichment of target capture probe system is considered to be opened up a new era of molecular detection.At present,two reports have proposed the design strategy of virus capture system,which has good results in sequencing sensitivity and genome coverage.Due to its for all vertebrate viruses,restricted to the current commercial synthetic probe storage capacity up to 200 million limit,probe design with only one or several of the genome sequence of each species for reference,which is insufficient to identify gene polymorphism.Therefore,it is a trend to design a targeted enrichment system for virus pathogens of different infection syndromes.Due to the polymorphism of respiratory viruses,how to optimize the design of the probe,which probe synthesis strategy to adopt,how to optimize database construction conditions are the technical difficulties of establishing the enrichment system needs to solve.In order to provide technical support for the prevention and control of infectious diseases,a probe set for enrichment and capture of human respiratory virus based on high throughput sequencing(HTS)technology was established in this study by solving the above technical difficulties..The reference library for probe design contains all the human respiratory virus sequences which are collected from the public database,contains rhinovirus,enterovirus,coronavirus,influenza virus,parainfluenza virus,metapneumovirus,respiratory syncytial virus,adenovirus,and boca virus,fragment the sequence with the 120-mer analysis.According the principle that the coverage of the standard strain reference sequence is not less than 90%,we tested the degree of redundancy by 60%,70%,80%,90%,99%and determined the redundancy with similarity of 95%or 99%,The quality of the probe sequence library was evaluated,according to the standard of 15 consecutive bases that were complementary to the viral sequence and the similarity between the probe sequence and the reference sequence was>75%.The capacity of the RNA probe library is 8.35M.The enrichment efficiency of respiratory tract virus probe set was verified by experiment.DNA and RNA virus acquired from cultivation and clinical samples were selected and the concentration of viral nucleic acid was diluted to 107/ul,105/ul and 104/ul according to copy number.The results showed that when the number of nucleic acid copies was 107,105 and 103,the average coverage of the whole genome after targeted enrichment was 2.7 times,1.17 times and 3.12 times higher than that of the unenriched nucleic acid sequences.Genome coverage depth increased 5.10-256 times,2.69-257.55 times and 1.00-428.86 times respectively.Finally,in clinical samples test,only 3 reads in the 2009 a(H1N1)genome were found and only PB2 segment information is available.using the non-offset amplification enrichment system,and 4 gene segments were obtained through targeted capture.This indicates that the capture system is more sensitive to confirm virus infection.In the adenovirus samples with positive test results,the coverage of the virus sequences obtained by targeted enrichment technology increased by nearly five times compared with that of the non-offset amplification enrichment system and sequencing depth increased from 5.37 to 18.73.In conclusion,the human respiratory virus enrichment technology system based on high-throughput deep sequencing technology was established to improve the detection of low-abundance virus nucleic acid in clinical samples.Virus genome sequence coverage especially in polymorphism cover compared with the existing sequence independent amplification techniques have obviously improved,helps to obtain a more comprehensive virus genome sequence information.This technique system is suitable for the capture of gene sequences of common respiratory virus with extremely low abundance in samples,and more viral gene mutations are found in clinical case samples,which is is helpful to elucidate functional sites including drug resistance and the change of virulence and to identify quasispecies.The technical system established in this study provides a practical technical reserve for the prevention and control of respiratory tract infectious diseases and the study of etiology.