Development Of Visual DNA Microarray For Subtyping Influenza Virus

Influenza is a zoonotic infectious respiratory disease caused by influenza virus.In the nearly100years, different types of influenza virus have caused worldwideinfections, causing a great deal of threat to human life and property safety. Spanish fluin1918(H1N1subtype), resulted in4000-6000million deaths, Asian flu in1957(H2N2subtype), the death toll was more than1million,2009pandemic H1N1startedto spread quickly in many countries. The global epidemic quickly became ainternational public health events. In recent years, different types of influenza virusprevail alternately and mixed epidemics have become at every turn, clinical laboratorytest to determine the type of influenza virus is an important part of theepidemiological investigation and epidemic prevention.This study uses multiplex reverse transcription amplification, gene chiptechnology, and TSA-Quantum Dots-Silver enhancement technology to establish therapid, sensitive, specific, and visualization of microarray technology platform toprovide laboratory evidence for the diagnosis of important influenza viruses. The NAgene, M gene and HA gene of human influenza virus are selected as the target regionsto design subtype-specific probes and biotin-labeled primers for RT-PCRamplification, the amplified product take hybridization reaction with the microarraywhich is fixed with the probes, TSA-Quantum Dots-Silver enhance the color of thehybridization reaction result, making the probe showing visible signal to diagnoseinfluenza A viruses (2009pandemic H1N1,seasonal H1N1, seasonal H3N2), influenzaB virus genotyping. Five pairs of specific primers and five specific probes are selectedin the study, the RT-PCR system, the hybridization reaction and visual inspectionconditions are optimized and completed,18cases positive strains and10cases ofnegative strains are used to evaluate the specificity of the microarray. Test resultsprove that the microarray is extremely specific. The CV value of intra-chip was lessthan20%. The further comparison with real-time PCR determines that the detectionsensitivity of the microarray can be achieved as103PFU/mL.1247cases of clinicalsamples are tested by microarray analysis, the test results are consistent with thesequencing and real-time PCR. The microarray of good specificity, high sensitivity,low-cost can be used as a new technic for clinical diagnosis and prevention ofinfluenza virus.