Development of a multiplex one step RT-PCR that detects eighteen respiratory viruses in clinical specimens and comparison with real time RT-PCR

Abstract

Rapid and accurate diagnosis of viral respiratory infections is crucial for patient management. Multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) is used increasingly to diagnose respiratory infections and has shown to be more sensitive than viral culture and antigen detection. Objective of the present study was to develop a one-step mRT-PCR that could detect 18 respiratory viruses in three sets. The method was compared with real time RT-PCR (rRT-PCR) for its sensitivity and specificity. Clinical specimens from 843 pediatric patients with respiratory symptoms were used in the study. 503 (59.7%) samples were detected positive by mRT-PCR. Of these 462 (54.8%) exhibited presence of a single pathogen and 41 (4.9%) had multiple pathogens. rRT-PCR detected 439 (52.1%) positive samples, where 419 (49.7%) exhibited one virus and 20 (2.4%) showed co-infections. Concordance between mRT-PCR and rRT-PCR was 91.9% and kappa correlation 0.837. Sensitivity and specificity of mRT-PCR were 99.5% and 83.7% while that of rRT-PCR was 86.9% and 99.4% respectively. Rhinovirus (17.2%) was the most frequently detected virus followed by respiratory syncytial virus B (15.4%), H1N1pdm09 (8.54%), parainfluenza virus-3 (5.8%) and metapneumovirus (5.2%). In conclusion, mRT-PCR is a rapid, cost effective, specific and highly sensitive method for detection of respiratory viruses.

A new real-time RT-qPCR assay for the detection, subtyping and quantification of human respiratory syncytial viruses positive- and negative-sense RNAs

“…Real-time PCR is the most widely used method for RSV diagnostic, with proven sensitivity and specificity (Chi et al, 2007;Choudhary et al, 2013). Different RSV genes have been targeted in these assays, such as the matrix, the polymerase (Kuypers et al, 2004), or the fusion protein genes (Mentel et al, 2003;van Elden et al, 2003), but the N gene is the most widely used due to its high conservation and the number of N sequences available in Genbank.…”

“…Quality control samples from QCMD (Quality Control Molecular Diagnostics) with defined RSV subgroups were analyzed *According to QCMD. **CT obtained at the laboratory of diagnostic at the University Of Geneva Hospitals using Fast Track Diagnostics multiplex PCR (Choudhary et al, 2013). UD: undetermined.…”

“…These clinical samples were previously screened positive for RSV using a commercially available kit for multiplex real-time RT-PCR (Fast Track Diagnostics ® ; # FTD-2), a semi-quantitative method that cannot subtype RSV (Choudhary et al, 2013). Similarly, clinical samples screened positive for human parainfluenza virus (hPIV) or human metapneumovirus (hMPV) were analyzed as specificity controls.…”

“…The dilution factor between 1 ml of clinical specimen and the RNA input per reaction is 25.%pos: Percentage of detected samples. (Choudhary et al, 2013) are indicated as well as viral loads determined with the in-house assays. Viral loads above or below the validated LOQ (from Table 2) are shown in parenthesis.…”

“…Real-time quantitative PCR (RT-qPCR) is the gold standard for both accurate RSV diagnosis and quantification and several assays have been developed during the last decades (Chi et al, 2007;Choudhary et al, 2013;Dewhurst-Maridor et al, 2004;Do et al, 2012;Gueudin et al, 2003;Hu et al, 2003;Kuypers, http://dx.doi.org/10.1016/j.jviromet.2016.05.004 0166-0934/© 2016 Elsevier B.V. All rights reserved. Mentel et al, 2003;van Elden et al, 2003), although these have to be adapted constantly to take into account genetic variations among circulating strains.…”