Development of High-throughput Liquid Chips for Respiratory Virus Detection

Abstract

Diagnosis of viral respiratory tract infections clinically is often nonspecific, and a rapid, high-throughput laboratory technique which can detect major respiratory viruses is desirable. Two liquid chip panels were developed which used a target-enriched multiplexed RT-PCR amplification followed by Luminex liquid chip hybridization based on xMAP technology. One was for the detection of 5 type/ subtypes of the influenza virus (called Influenza Panel), the other was for the identification of 14 major respiratory viruses, including emerging human metapneumovirus (HMPV), coronavirus (CoV NL63, HKU1), human bocavirus (HBoV), and WU polyomavirus (WUPyV) (called Respiratory Panel). The analytical sensitivity and specificity of these two panels were determined. 108 throat swabs from influenza-like-illness (ILI) cases and 88 nasopharyngeal aspirates (NPA) from hospitalized children with lower respiratory tract infections (LRTI) were used to evaluate the two panels. The Influenza Panel returned the analytical sensitivity of 4 - 10 copies vRNA/microL of sample for seasonal H1N1, H3N2, pandemic H1N1, and B, and 200 copies vRNA/microL of sample for H5. The analytical sensitivity of the Respiratory Panel was as follows: 10 - 150 copies vRNA/microL of sample for most of the viral targets tested in this panel except RSV (400 copies vRNA/microL of sample). No cross reactivity was observed for both panels. As determined by commercial multi-PCR kit and conventional PCR (for HBoV and WUPyV), the diagnostic sensitivity of the Respiratory Panel for each viral targets ranged from 50.0% to 100.0%, the diagnostic specificity was 96.2%-100.0%, and accordance rate was 93.2% - 100.0%, the kappa correlation of eight viral targets was > 0.75. Two liquid chip assay panels, developed based on xMAP technology, can detect 5 types/subtypes of influenza viruses and 14 respiratory viruses simultaneously in one reaction in 6 hours. They are potentially rapid, sensitive, specific, and high throughput diagnostic tools for major viral pathogens associated with respiratory tract infection.