High-Throughput, Sensitive, and Accurate Multiplex PCR-Microsphere Flow Cytometry System for Large-Scale Comprehensive Detection of Respiratory Viruses

Abstract

Human respiratory viruses are a diverse group of pathogens composed of hundreds of virus strains, and this presents a major challenge for diagnostic laboratories. To efficiently detect numerous viruses in a large epidemiologic study, we developed a fast, multitarget, sensitive, and specific assay named the Respiratory MultiCode-PLx Assay (RMA). The RMA utilizes improved multiplex PCR chemistry (EraGen MultiCode-PLx technology) coupled with high-throughput microsphere flow cytometry (Luminex). Eighteen sets of virusspecific multiplex PCR primers were developed based on the conserved sequences of all available respiratoryvirus sequences for eight distinct groups: human rhinovirus (HRV), respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus (InfV), metapneumovirus, adenovirus (Ad), coronavirus, and enterovirus. Each primer set detected 20 cDNA copies of the intended target per sample and had no reaction with 60,000 copies of human genomic DNA. The accuracy and sensitivity of the RMA for detecting respiratory viruses in human samples were tested with two sets of clinical specimens. First, 101 nasal-wash specimens that were positive for HRV, RSV, InfV, PIV, or Ad by traditional techniques were reanalyzed by RMA, and all target viruses were detected with an overall sensitivity of 94% and specificity of 99%. Second, 103 nasal-wash samples from 5-year-old children with asthma and respiratory symptoms were analyzed; RMA detected viruses in 74 specimens (71.8%) compared to only 24 (23.3%) by traditional culture and immunofluorescent-staining techniques. These results show that RMA is an accurate, sensitive, and practical test for respiratory-virus infections.

Increased H1N1 Infection Rate in Children with Asthma

“…These samples were analyzed for common respiratory viruses by multiplex polymerase chain reaction (respiratory MultiCode assay; EraGen Biosciences, Madison, WI) (12) and by seminested polymerase chain reaction specific for the hemagglutinin gene of the 2009 H1N1 influenza virus (see the online supplement for details). Partial genetic sequencing was used to identify HRV types (13).…”

Randomized Trial of Omalizumab (Anti-IgE) for Asthma in Inner-City Children

“…Dust from the participant's bed and bedroom floor was collected with the use of a validated self-collection procedure 12 Nasal-secretion samples were collected and frozen at four of the eight research sites at week 48 and within 7 days after the onset of an asthma exacerbation. Total RNA was extracted and analyzed by means of multiplex reverse-transcriptase-polymerase-chain-reaction assay 13,14 with the use of primers and probes specific for rhinoviruses, influenza virus, parainfluenza virus, coronavirus, respiratory syncytial virus, meta-pneumovirus, enterovirus, adenovirus, and boca-virus.…”

Development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses

“…High-throughput systems combining PCR with fluid microbead multiplex product detection have also been recently described [Lee et al, 2007;Mahony et al, 2007]. However, most published assays have not been adapted for large-scale diagnostic use, or lack one or more important virus targets.…”