Identification And Subtyping Of Influenza Viruses By DNA Microarray

Influenza is a highly contagious respiratory disease of humans, caused by negative-strand, segmented RNA viruses belonging to the family Orthomyxoviridae. There are three different genera of influenza virus; type A, type B, and type C. Seasonal outbreaks, caused by influenza A and B viruses, constitute a global health issue, leading to morbidity, mortality, and economic losses.IAV is classified into17HA and10NA subtypes, based on the antigenicity of two viral surface proteins-hemagglutinin (HA) and neuraminidase (NA). Almost all possible combinations of HA and NA have been isolated from aquatic birds, poultry, and other avian species. In humans and other mammals, limited subtypes of IAVs have been detected. IAV contains eight gene segments encoding the corresponding viral protein(s). Pandemic viruses are generated by the rearrangement (reassortment) of viral RNA segments in cells infected with two different strains of IAV. Thus, reassortment can theoretically result in256(28) different genotypes, and is a key source of pandemic viruses. Avian and human IAVs can generally infect swine, and generate pandemic IAVs by reassortment. Molecular diagnostic techniques for viral testing have undergone rapid development in recent years, because of its rapid, sensitive and accurate advantages, more and more appropriate detection techniques applied to the field of molecular detection of influenza viruses, a variety of molecular diagnostic techniques have been widely used.Most currently available methods for microbial detection and discovery using nucleic acid samples are based on three technologies. In order of increasing cost, these are the polymerase chain reaction (PCR), oligonucleotide microarrays and DNA sequencing. These platforms have different strengths and weaknesses. While sequencing provides the most in-depth, unbiased information, and is able to reveal completely novel organisms, it can be costly and time-consuming for some applications. Although multiplex sequencing of bar-coded samples reduces the cost per sample, it also decreases the coverage and thus the sensitivity of the analysis; this may be an issue when the organism of interest has low abundance and the sample has not been treated beforehand to remove host and/or background DNA.At the other end of the cost spectrum, PCR assays are very fast and sensitive, but have limited capacity for multiplexing.Microarrays occupy a middle ground with respect to cost, processing time, sensitivity, specificity and ability to detect novel organisms. Arrays can be designed with a combination of high-specificity probes and probes designed against conserved regions, so that they can be used in both detection and discovery modes.On this basis, the18pairs of PCR primers were designed to be divided into four groups, to establish a packet multiplex PCR amplified and labeled target genes. Prepared by in vitro transcription16kinds of HA gene of the virus RNA, dilution for susceptibility testing. After data analysis, and ultimately determine if there is a corresponding probe SNR532≥2or signal strength F532Mean-B532≥2000judged as positive results of this study the minimum detection limit of RNA copies at100-1000, and has good repeatability.To verify the DNA microarray method for the detection of a new influenza virus subtype performance, application reverse genetics rescued reassortant H1N1, H4N6, H8N4, H9N1subtype of influenza A virus, and its testing, the results show that the establishment of the detection method can be successfully identified H1, H4, H8, H9subtypes such as reverse genetics reassortant influenza virus strains.The application of the laboratory’s own strain of DNA microarray detection methods for sensitivity, specificity test, can specifically detect H1N1, H3N2, H9N2, H5N1subtype strains, etc., the minimum detection limit of1×103EIDso virus titers. Carried out DNA microarray genotyping influenza emergency detection research, the successful detection of a2004retrospective Baicheng City, Jilin caused by H5N1subtype avian influenza virus-positive clinical samples. And applied to the2009H1N1influenza virus detection and seasonal H1N1influenza virus to distinguish identification. The established method can be specifically identified2013H7N9subtype of avian influenza virus.The developed application microarray detection method can be very accurately on the A-type, B-type influenza virus subtypes and H1-16for genotyping. Furthermore, the test results demonstrated that our system displays higher sensitivity and specificity than other methods. Collectively, this study provides a rapid, convenient and accurate molecular biology diagnostic technique for routine monitoring as well as for emergency responses to influenza pandemics in the future.