Preliminary Establishment Of Two Multiplex Detection Methods For 16 Common Respiratory Virus Nucleic Acids

Respiratory tract infection is a kind of acute infectious disease caused by the invasion of a variety of pathogens in the upper and lower respiratory tract,mainly viruses,which has been reported worldwide.Economic development will inevitably bring about the migration of people and commodities,which also facilitates the spread of diseases,especially by simple means such as airborne droplets.For example,Ebola is prevalent in southeast Africa,and MERS prevails in the Middle East.In addition,there are many other severe respiratory diseases,which are all at considerable risk of importation.It has put great pressure on China’s epidemic prevention and control.Given that the initial symptoms after respiratory tract infection are relatively similar,and respiratory tract infection is often accompanied by co-infection of multiple pathogens,the establishment of multiple nucleic acid detection technology of common respiratory tract viruses is of great significance for the identification of unknown samples and the separation of co-infection.Hence,patients can get targeted treatment and avoid the abuse of antibiotics.For this purpose,this study is divided into two parts,and a total of 16typical respiratory viruses are selected:Respiratory syncytial virus A and B,two Influenza A subtypes H1 and H3,Two Influenza Virus B subtypes Victoria and Yamagata,Severe Acute Respiratory Syndrome Coronavirus 2,Human parainfluenza virus,HPIV1,2,3,Human Coronavirus 229E、NL63、OC43、HKU1,Human metapneumovirus,Human adenovirus.In the first chapter,based on the real-time quantitative fluorescence PCR technology,namely q PCR technology,Taqman probe method,Feipeng one-step rapid reverse transcription kit and Q5 q PCR instrument were used to establish the16-fold Taqman real-time quantitative fluorescence detection technology for 16common respiratory viruses in five tubes,and the established method was evaluated.The sensitivity of the method was evaluated by diluting the RNA standard of 16 respiratory viruses stored in the department.The minimum detection limit of each virus was calculated by the established standard curve.Except for H1 and SARS-Co V-2,which were 308 copies/μL and 184 copies/μL respectively,the remaining 14 viruses were all less than 64 copies/μL.This method produced a high sensitivity.The stability was evaluated using three concentration gradient copies of 10~4to 10~6 copies/μL.Three multiple Wells were set up in each experiment,and the intra-group stability was evaluated three times in batches.The coefficient of variation in the experimental group was less than3%,and the coefficient of variation between the groups was less than 6%,which showed good repeatability.11 other respiratory viruses were used to evaluate the specificity of the method.There was no cross reaction,indicating the specificity was good.25 respiratory clinical samples were used for preliminary validation,and the detection rate was 100%without cross reaction. The second part of the research is based on the emerging liquid chip technology as the core,combined with multiple PCR technology,using Feipeng one-step rapid reverse transcription kit,where 16 double liquid chip nucleic acid multiple detection technology was applied for 16 kinds of respiratory viruses.The sensitivity,repeatability and specificity of the established method were evaluated using the RNA standard in Part I,and 25 respiratory clinical samples were used for preliminary validation.The results showed that the sensitivity of the multichip detection technique for 16 viruses ranged from 10~2 to 10~4copies/μL,the coefficient of variation was below%,and the detection rate was 100%without cross reaction.In this study,based on Taqman fluorescent probe technology and liquid chip technology,q PCR rapid multiplex nucleic acid detection technology and 16 double liquid chip multiplex nucleic acid detection technology for 16 common respiratory viruses were preliminatively established,and the established methods were preliminatively evaluated.It has certain application value for the mixed detection and multiple detection in the prevention and control of respiratory diseases,which can also provide a new idea for the establishment of multiple detection methods for other viruses.