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JMD 2004, Vol. 6, No. 2
Copyright © 2004 American Society for Investigative Pathology & Association for Molecular Pathology

A Sensitive, Specific, and Cost-Effective Multiplex Reverse Transcriptase-PCR Assay for the Detection of Seven Common Respiratory Viruses in Respiratory Samples

Melanie W. Syrmis*{dagger}{ddagger}, David M. Whiley*{dagger}, Marion Thomas{ddagger}, Ian M. Mackay*{dagger}{ddagger}, Jeanette Williamson§, David J. Siebert§, Michael D. Nissen{ddagger} and Theo P. Sloots*{dagger}{ddagger}§

From the Clinical Virology Research Unit, * Sir Albert Sakzewski Virus Research Centre, Royal Children’s Hospital and Health Service District; the Clinical Medical Virology Centre, {dagger} University of Queensland; the Microbiology Division, § Queensland Health Pathology Service, Royal Brisbane Hospital Campus; and the Department of Paediatrics and Child Health, {ddagger} University of Queensland, Brisbane, Queensland, Australia

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.




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