title: Detection of nine respiratory RNA viruses using three multiplex RT-PCR assays incorporating a novel RNA internal control transcript.
abstract: Real-time PCR is a significant improvement over viral isolation and immunofluorescence for routinely detecting respiratory viruses. We developed three real-time internally controlled multiplex RT-PCR assays for detecting nine respiratory viruses. An internal control transcript consisting of a chimeric plasmid was synthesised and incorporated into each multiplex to monitor amplification efficiency, including inhibition. Each multiplex assay was developed on the Rotor-Gene 3000 and evaluated using RNA extracts from 126 nasopharyngeal aspirates from 112 pre-term infants. All 44/126 (35%) samples positive by immunofluorescence were confirmed by multiplex RT-PCR. Additionally, respiratory syncytial virus RNA was detected in 5 samples, influenza A virus RNA in 2 samples and thirteen (10%) dual infections by multiplex RT-PCR were noted. Inclusion of the RNA internal control did not affect the amplification efficiency of the target sequences and only 2 of 1256 (0.2%) samples tested over a 12 month period were inhibitory. Together with the improved sensitivity of the internally controlled multiplex RT-PCR assays over the older technology and the ability to detect co-infections, the internal control monitored the efficiency of both the RT and PCR steps and indicated inhibition, saving time and costs on running duplicate samples with a "spiked" inhibition control.
authors.affiliation: South London Specialist Virology Centre, King's College Hospital NHS Foundation Trust, Bessemer Wing, Denmark Hill, London SE5 9RS, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Auburn
authors.forename: Mark
authors.initials: M
authors.lastname: Zuckerman
authors.forename: Simon
authors.initials: S
authors.lastname: Broughton
authors.forename: Anne
authors.initials: A
authors.lastname: Greenough
authors.forename: Melvyn
authors.initials: M
authors.lastname: Smith
mesh_terms: D000818:Animals; D002460:Cell Line; D015342:DNA Probes; D006801:Humans; D007231:Infant, Newborn; D007234:Infant, Premature; D007235:Infant, Premature, Diseases / Q000821:virology*; D009305:Nasopharynx / Q000821:virology; D012327:RNA Virus Infections / Q000821:virology; D012328:RNA Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000592:standards*; D012015:Reference Standards; D012137:Respiratory System / Q000821:virology; D012141:Respiratory Tract Infections / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D014776:Virus Cultivation
chemical_list: D015342:DNA Probes; D012367:RNA, Viral