Microfluidic Isothermal Amplification Based Fluorescent Technology Assay For Severalrespiratory Infection Viruses

Background:In recent years,frequent outbreaks of respiratory infectious diseases have seriously endangered public health.In 2005,the outbreak of avian influenza A(H5N1)occurred in 17 provinces in China,resulting in the death of 13 people.The outbreak of influenza A(H1N1)from Mexico in 2009 caused global panic.The outbreak of H7N9 avian influenza in eastern China in 2013 has once again attracted worldwide attention.In addition,the adenoviruses of type 7,type 14 and type 55 that have become popular in China in recent years has caused an outbreak of clustered outbreak.The shortage of on-site detection techniques for respiratory infectious diseases has severely affected the timeliness and pertinence of the emergency treatment of the epidemic.Objective and significance:The rapid detection of pathogens of respiratory infectious diseases is the key to detect infectious sourcesin a timely mannerand take timely measures to prevent the spread of the epidemic.It is an urgent need for scientific prevention and control of respiratory infectious diseases.This study intends to establish a rapid on-site detection technology for respiratory pathogens based on microfluidic chip and isothermal amplification technology,to solve the current lack of on-site detection technologies for respiratory infections and to meet the problem of screening for major pathogens of respiratory syndromes.It could provide technical support for timely and accurate treatment and isolation of the source of infection and the timely and effective scientific prevention and control of the epidemic.Methods:Firstly,we searched GenBank for the conserved sequences of influenza A H1,H3,H5,influenza B virus,adenovirus,respiratory syncytial virus,and parainfluenza viruses,to design its specific LAMP primers,and then optimized the best primers based on the amplification effect,and to establish and optimize the conventional fluorescent LAMP system.Secondly,we designed arrayed microfluidic chip,studied the immobilization methods and amplification performance of enzyme amplification,primer design,buffer and other amplification reagents in microfluidic chip,and established microfluidic isothermal amplification fluorescence detection method.We investigated and optimized the relevant parameters,focused on the anti-pollution techniques of nucleic acid amplification,reduced the false positives of isothermal amplification,and evaluated the specificity and sensitivity of established detection methods,and the ability to detect clinical samples.Results:Based on the conventional fluorescence LAMP detection method,we optimized to get the microfluidic isothermal amplification of fluorescence detection system.The amplification reaction was completed in 1.25 μL Eva Green at 65°C for 30 minutes,and a microfluidic isotherm was established.Specific evaluation results show that this method can achieve specific detection of influenza A virus H1,H3,H5,influenza B,adenovirus,respiratory syncytial virus and parainfluenza virus.Each reaction hole of the chip is independent,with only 3μL reaction system and no cross reaction;the typing of these 7 pathogens can be completed on a single chip.The microfluidic isothermal amplification fluorescence detection method has a sensitivity of 10 copies/μL for H1,H3,and adenovirus,and a detection sensitivity of 100 copies/μL for H5 subtype influenza virus,influenza B virus,and parainfluenza virus.Conclusion:The established microfluidic isothermal amplification fluorescence detection method in this study can realize the Real-time fluorescence response to the amplified results,and the operation is convenientand does not require thermal cycling;It could specificly regonise and amplify H1,H3,H5,influenza B virus,adenovirus,respiratory syncytial virus can be completed.The detection sensitivity is up to 10-100 copies/ L;the dosage of the reagent is small with low cost.The system is closed with plastic sealing and chip buckle to reduce the possibility of pollution.The microfluidic isothermal amplification fluorescence detection method is simple,rapid,specific and sensitive,to facilitate the on-site detection of respiratory viruses,and provide technical support for pathogen screening of respiratory infectious syndromes.