High-Throughput, Sensitive, and Accurate Multiplex PCR-Microsphere Flow Cytometry System for Large-Scale Comprehensive Detection of Respiratory Viruses
- 1 August 2007
- journal article
- research article
- Published by American Society for Microbiology in Journal of Clinical Microbiology
- Vol. 45 (8), 2626-2634
- https://doi.org/10.1128/jcm.02501-06
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 virus-specific multiplex PCR primers were developed based on the conserved sequences of all available respiratory-virus 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.Keywords
This publication has 47 references indexed in Scilit:
- The alleles of PECAM-1Gene, 2006
- Human Bocavirus in ChildrenEmerging Infectious Diseases, 2006
- Human Bocavirus Infection, CanadaEmerging Infectious Diseases, 2006
- Coronavirus HKU1 Infection in the United StatesEmerging Infectious Diseases, 2006
- Multiplexed and microparticle‐based analyses: Quantitative tools for the large‐scale analysis of biological systemsCytometry Part A, 2006
- Multiplex Genotyping of Human Minor Histocompatibility AntigensHuman Immunology, 2005
- Characterization and Complete Genome Sequence of a Novel Coronavirus, Coronavirus HKU1, from Patients with PneumoniaJournal of Virology, 2005
- Viral infections and asthma inceptionJournal of Allergy and Clinical Immunology, 2004
- Identification of a new human coronavirusNature Medicine, 2004
- Advances in the laboratory diagnosis of viral respiratory diseaseThe Pediatric Infectious Disease Journal, 2004