title: A sensitive, specific, and cost-effective multiplex reverse transcriptase-PCR assay for the detection of seven common respiratory viruses in respiratory samples.
abstract: Cell culture and direct fluorescent antibody (DFA) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections. Multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) is a sensitive, specific, and rapid method for detecting several DNA and RNA viruses in a single specimen. We developed a m-RT-PCR assay that utilizes multiple virus-specific primer pairs in a single reaction mix combined with an enzyme-linked amplicon hybridization assay (ELAHA) using virus-specific probes targeting unique gene sequences for each virus. Using this m-RT-PCR-ELAHA, we examined the presence of seven respiratory viruses in 598 nasopharyngeal aspirate (NPA) samples from patients with suspected respiratory infection. The specificity of each assay was 100%. The sensitivity of the DFA was 79.7% and the combined DFA/culture amplified-DFA (CA-DFA) was 88.6% when compared to the m-RT-PCR-ELAHA. Of the 598 NPA specimens screened by m-RT-PCR-ELAHA, 3% were positive for adenovirus (ADV), 2% for influenza A (Flu A) virus, 0.3% for influenza B (Flu B) virus, 1% for parainfluenza type 1 virus (PIV1), 1% for parainfluenza type 2 virus (PIV2), 5.5% for parainfluenza type 3 virus (PIV3), and 21% for respiratory syncytial virus (RSV). The enhanced sensitivity, specificity, rapid result turnaround time and reduced expense of the m-RT-PCR-ELAHA compared to DFA and CA-DFA, suggests that this assay would be a significant improvement over traditional assays for the detection of respiratory viruses in a clinical laboratory.
authors.affiliation: Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital and Health Service District, Queensland, Australia.
authors.forename: Melanie W
authors.initials: MW
authors.lastname: Syrmis
authors.forename: David M
authors.initials: DM
authors.lastname: Whiley
authors.forename: Marion
authors.initials: M
authors.lastname: Thomas
authors.forename: Ian M
authors.initials: IM
authors.lastname: Mackay
authors.forename: Jeanette
authors.initials: J
authors.lastname: Williamson
authors.forename: David J
authors.initials: DJ
authors.lastname: Siebert
authors.forename: Michael D
authors.initials: MD
authors.lastname: Nissen
authors.forename: Theo P
authors.initials: TP
authors.lastname: Sloots
mesh_terms: D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D019085:Fluorescent Antibody Technique, Direct; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D009305:Nasopharynx / Q000821:virology*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis*; D012141:Respiratory Tract Infections / Q000191:economics* / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral