Detection Of Influenza Viruses And Identification Of Virulence Using A Microarray

Influenza virus has the characteristic of mutation and reassortment, so it is considered as a threat capable of rapid and lethal spread in the human population. Four historical influenza pandemics were result from reassortants or variant of influenza viruses. Due to the high mortality, H5N1 highly pathogenic avian influenza has caused severe public health problem and greater concern from the whole society. An increased awareness of a possible influenza pandemic has prompted worldwide efforts to actively pursue and institute influenza monitoring and pandemic preparedness measures. The study aims at establishing the technology platform of detecting common influenza viruses and avian influenza viruses using DNA microarray. The system is accurate, fast and high-throughput. This study provides the effective diagnosis tools for clinical diagnosis and protection of bioterrorism attack.In this study, the DNA microarray detected the presence of three seasonal influenza viruses H1N1, H3N2 and B and two human avian influenza viruses H5N1 and H9N2. The hemagglutinin, neuramidinase and nucleoprotein genes were chosen simultaneously as targets for designing and screening of subtype-specific probes and virulence-identification probes. At first, three methods of viral RNA amplification, i.e. Single Primer Amplification assay, Random Primer Amplification assay and In Vitro Transcription assay, were compared. On the basis of above experiments, the cutoff values were confirmed. Furthermore, specificity, sensitivity, reproducibility and the coincidence rate of the DNA microarray were determined respectively.The DNA microarray was able to specifically detect five target viruses. And there were no cross-reactions to each other. The limits for viruses of H1N1, H3N2, H5N1, H9N2 subtype and B type were 8HAU, 16HAU, 4HAU, 8HAU and 32HAU respectively, and the limit for high virulence was 32HAU. The microarray was of good repeatability. When DNA microarray was used to screen 56 specimens, the results compared with that of RT-PCR assay show that the coincidence rate was 83.9%(47/56), of which the positive coincidence was 82.7%(43/52).The DNA microarray for detection and typing five influenza viruses, and especially virulence identification, was specific, sensitive and reproducible. Because of its characteristics of high-throughput and parallelism, this array was suitable to simultaneously detect many target fragments in one test. The microarray would be sure to be helpful for copying with emergency.