Construction and application of microfluidics chip detection system for 9 respiratory viruses

ABSTRACT

In recent years, more and more new pathogenic respiratory viruses have been discovered, which has caused great problems for the prevention and control of port inspection and quarantine agencies. To establish detection systems for nine respiratory pathogens including influenza A virus (FluA) . influenza B virus (FluA) Human parainfluenza virus (HPIV-1 HPIV-2, HMV-3 HPTV-4), Corona virus (NL63, 0C43, 229E, HKUI, MERS), respiratory syncytial virus (RSV) . Human paratyphovirus (HMPV) human bocavirus (Hbov), Adenovirus(Adv) and Rhinovirus(Rhv). The human 3 actin gene was selected as the target gene to design the internal primer and probe. Primers and probes based on conservative regions of 9 respiratory pathogens were designed. Appropriate RT -PCR Mastermix based on the Ultra Fast Real-time PCR G2 -4 platform were selected. The primer and probe concentrations were optimized and the sensitivity and specificity of the primers and probes were verified by the optimized amplification system. Retrospective examination was carried out on 100 clinical specimens examined by Nantong International Travel Health Clinic in 2018. A rapid amplification RT-PCR Mastermix was selected with optimized upper primer, downstream primer and probe of 16 respiratory pathogens at concentrations of 500 nmol/L, 500 nmol/L and 250 nmol/L, respectively. The upstream primers, downstream primers, and probes of the internal gene were at concentrations of 300 nmol/L, 300rtmol/L. and 150 nmol/L. respectively. Under this amplification system, the minimum detection limit for 9 respiratory pathogens was 1.0 X 103copies/mL, without cross-reaction . The accuracy of 100 clinical samples was 10094. Thus, a microfluid chip amplification system for rapid detection of 9 respiratory viruses was established, which can he used to qualitatively detect nine kinds of viral nucleic acids within 25 min.