Rapid Detection Method Of Pathogenic Microorganism By Next-generation Sequencing (NGS)

Respiratory tract infections can be caused by a large range of pathogens,including viruses,bacteria and(less often)fungi,which lead to its prevalence in epidemic and difficulties in prevention.In 2012,the world health organization(WHO)ranked the respiratory tract infection as the top third cause of mortality worldly.The conventional pathogen diagnosis methods in clinic are time consuming and low efficient.Therapy treatment of respiratory tract infection usually requires empirically testing a large collection of antibiotic agents meanwhile it also increases the risk of generating broad-spectrum antibiotic resistant pathogens.To address this limits,here,we propose to develop a deep sequencing-based method to identify the disease causing pathogens of individual patients,which will provide guidance for effective therapeutic treatments,as a way of precision medicine.Using deep sequencing methods,we can comprehensively map all the identities of pathogens in individual patient in a high-throughput and robust fashion;meanwhile we can easily exclude the host DN A/RNA contamination through bioinformatics analysis.This research has optimized and perfected the key technical points of preprocessing,extraction and database construction.The sa mples of nasopharyngeal swabs and cerebrospinal fluid were concentrated by Millipore ultrafiltration tube,and the content of pathogenic nucleic acid was increased by filtration.The results showed that sputum could be pretreated by 5% dithiothreitol before nucleic acid extraction.The use of the TI ANamp Micro DNA Kit can be used at a lower cost to achieve better extraction results,DNA in the library when the best ratio of the amount of purified beads is the end of the repair with 1.6 times the volume of magnetic beads,After the connection with 1.2 times the volume of magnetic beads purification,the use of magnetic beads on the database after the DNA fragment selection cost savings and save time.Finally,a complete process of receiving reports from the samples is formed.To achieve the entire process in 48 hours to complete.And the feasibility and accuracy of the technology are verified from the aspects of sensitivity,stability,specificity and accuracy.