Rapid Detection Method Of Influenza Virus Based On Microfluidic Isothermal Amplification

Influenza virus is a serious threat to human health.It has caused at least four pandemic in the world,and it has been a threat to public health and human health.Influenza viruses are divided into three kinds of typeinfluenza A,B,C.Influenza virus A and B can cause severe illness,including lower respiratory tract infections,pneumonia,meningitis and so on,which can cause death.Influenza viruses not only cause huge economic losses,but also may span the host barrier directly-infected with human.Influenza virus is a serious threat to human public health security.Therefore,the rapid and timely detection of influenza virus and targeted treatment for influenza epidemic is of great significance to prevent and control influenza virus.In recent years,molecular diagnostic methods such as polymerase chain reaction(PCR)is used to detect influenza virus,which can accurately identify the target sequence,but it requires a heat cycle system components,which usually rely on large and expensive equipment,so it is not conducive to rapid detection.Recently,Isothermal amplification techniques such as loop mediated isothermal amplification(LAMP).overcome the above shortcomings of PCR.The biggest difference between LAMP and PCR is that there is no thermal cycle.LAMPuse 4-6 primers to identify the target genes 6-8 specific sites.It is in constant temperature(60-65 ?)to complete chain replacement reactionunder 60min,with simple operation,high speed,high sensitivity and specificity,and has aroused widespread concern.Another microfluidic chip technology,In addition a microfluidic chip technology,it can be involved in areas such as chemical and biological sample preparation,reaction analysis,testing and other basic operating unit integrated into a chip,which has a few square centimeters.It can not only can achieve multi-channel parallel detection,but also help to integrate the detection system with the advantage of the reagent consumption,fast analysis speed,etc.As a new methodology platform,it has attracted widespread attention.Microfluidics and LAMP technology can be combined with the completion of parallel,multiplexed detection,and similar type of pathogen detection.It provides a new means of detection for quick and accurate distinction between common influenza virus.In this study,laboratory microfluidic microarray platform and real-time fluorescence LAMP instrument for the technical means to study the technical route is divided into three parts:1.LAMP primer screening.Through literature research to determine the target pathogen and target genes,design LAMP primersfor influenza virus.Collecting influenza virus strains to culture influenza virus.The sub-type influenza without virus samples to construct plasmid reference materials.Preparation of specific reference materials and reference material sensitivity,complete the respective sub-type of influenza virus LAMP primer preliminary screening.2.Optimization of experimental conditions.Optimize the reaction temperature,the reaction system,the establishment of detection methods.3.Evaluation of the performance of detection methods.Use specificity referenceproduct to assess the specificity detection of method,and use sensitivityreferenceproduct or other detection methods to comparemethods.Use clinical samples to evaluate the actual effect.The establishment of a microfluidic LAMP method for the detection of influenza A H1N1,influenza A H3N2,H5N1 avian influenza,H7N9 avian influenza and influenza B.Research method is mainly divided into two parts:1.LAMP method for detection of influenza viruses:Firstly,Collecting influenza virus strains to culture influenza virus.The sub-type influenza without virus samples to construct plasmid reference materials,and use reference materials viral plasmid as a template to prepared transcription of viral RNAin vitro.Secondly,designed and screened influenza A H1N1,influenza A H3N2 influenza,H5N1 avian influenza,H7N9 avian influenza HA gene and B influenza NEP gene for the purpose of designing LAMP primers.Thirdly,respectively optimized LAMP reaction system,and determined LAMP reaction system for the above influenza virus.Finally,specificity evaluation showed that,LAMP method for the detection of above types influenza type has good performance.Sensitivity evaluation showed that the detection limit of LAMP method was 10-100fg/reaction,the reaction entire process is completed within 30min.2.Microfluidic LAMP methods for detection of influenza viruses:Firstly,we used the above influenza virus LAMP primers optimized microfluidic LAMP reaction system.Secondly,the specificity results show that microfluidic LAMP methods established for the above type of influenza type have good performance,and no cross-reactivity with each other,80 clinical throat swab samples and cultured influenza virus samples test results also proved that for the above influenza have good subtype performance.The evaluation results indicate that the sensitivity of the microfluidic LAMP method was 10-100fg/reaction,10 to 100 times higher than the sensitivity of the RT-PCR detection method.In summary,microfluidic LAMP method reduces the LAMP method false positive problems,and has the advantage of the reagent consumption,analysis speed,etc.It provides a simple detection method for the detection of common type influenza virus,and improves the emergency responsecapability to the influenza outbreak,and makepatients with influenzahave a broader time frame to treat.At the same time,it lays the foundation for the establishment of integrated influenza virus detection platform.