Detection of nine respiratory RNA viruses using three multiplex RT-PCR assays incorporating a novel RNA internal control transcript

TY - JOUR

T1 - Detection of nine respiratory RNA viruses using three multiplex RT-PCR assays incorporating a novel RNA internal control transcript

AU - Auburn, Helen

AU - Zuckerman, Mark

AU - Broughton, Simon

AU - Greenough, Anne

AU - Smith, Melvyn

N1 - Copyright © 2011 Elsevier B.V. All rights reserved.

PY - 2011/9

Y1 - 2011/9

N2 - 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.

AB - 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.

KW - Sensitivity and Specificity

KW - Animals

KW - RNA Virus Infections

KW - Humans

KW - Reference Standards

KW - Infant, Newborn

KW - Infant, Premature, Diseases

KW - Nasopharynx

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Infant, Premature

KW - RNA, Viral

KW - Respiratory System

KW - Virus Cultivation

KW - DNA Probes

KW - Respiratory Tract Infections

KW - RNA Viruses

KW - Cell Line

U2 - 10.1016/j.jviromet.2011.05.017

DO - 10.1016/j.jviromet.2011.05.017

M3 - Article

C2 - 21620897

VL - 176

SP - 9

EP - 13

JO - Journal of Virological Methods

JF - Journal of Virological Methods

IS - 1-2

ER -