A Multi-detection Method For Respiratory Virus Based On Suspension Chip

Respiratory viruses are the most common pathogens causing acute respiratory tract diseases. In the emergency of large scale epidemic diseases outbreak, or the needing of rapid clinical diagnostics in acute respiratory tract diseases, rapidly identifying the pathogens are most important. In this study, we describe a new method for detecting multiple respiratory viruses based on Bio-plex200liquichip system. All the repiratory virus specific primers and probes in this study were designed according to the pathogenic virus genomic regions from NCBI database. Using this multiplex asymmetric PCR associated with liquichip method, we can simultaneously detect10virus types or subtypes within5-6hours in order to save time, cut the cost and reduce the labour.All primers and probes in this study were designed and synthetised according to the highly conserved genomic regions of the pathogenic virus including adenovirus (AdV), human metapneumovirus (hMPV), influenza virus type A (IfA), influenza virus type B (IfB), respiratory syncytial virus (RSV), parainfluenza virus type1(PIV1), PIV2, and PIV3, severe acute respiratory syndrome coronavirus (SARS), Herpes simplex virus1(HSV1) from NCBI database. The target sequences of the virus nucleic acids were amplified by a PCR reaction directly or by a reverse transcript reaction followed with a PCR reaction. The amplifiction products were hybridized with the coupled nucleic acid probe beads set, and the mean fluorescence intensities were detected by the Bio-plex200liquichip system. After optimizing, the limits of detection for each virus can get to1×10-6ng DNAs (-30DNA copies) for IfA,1×10-7ng DNAs (-3DNA copies) for AdV, IfB, PIV3, PIV3, RSV, HSV1and SARS,1×10-8ng DNAs (-1DNA copies) for hMPV and PIV1.27of the29respiratory viral clinical samples were identified as positive, the detectable rate was93.1%. Part of the clinical samples were sequencing analyzed, and all of them were identified to be100%corresponding to that identified by liquid bead array assay.