Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses

Abstract

Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1-4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.

Longitudinal Study on Respiratory Viral Co-Infections in the Presence or Absence of Clinical Manifestation in Infants Aged 0–2 Years

“…The primers used in this study are listed in Table 2. The detection limits of the in-house PCR were comparable to those obtained by the methods described previously (9,10). HAdV and HBoV were 10 copies/ml, respectively.…”

Development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses

“…Comparing analysis results with other diagnostic methods on clinical samples was considered the most relevant performance parameter in this case. The total positive diagnostic outcome of 57% of the 2004-2005 study is comparable to other prevalence studies of respiratory viruses in non-selected patient materials [Bellau-Pujol et al, 2005;Mahony et al, 2007]. Because the material is not clinically well defined, a large proportion of the diagnostic gap may be due to reasons such as patients sampled late in the course of the disease, patients tested for reasons other than viral respiratory tract disease, or inadequate sampling technique.…”

Molecular epidemiology of the attachment glycoprotein (G) gene in respiratory syncytial virus in children with acute respiratory infection in Japan in 2009/2010

“…We primarily isolated various viruses from the samples using cell-culture methods for Vero E6, RD18S and Madin-Derby canine kidney cells. Next, in the samples negative for isolation, we detected various respiratory viruses such as influenza A (H1N1) pdm09 virus, seasonal influenza virus (subtypes A, B and C), human parainfluenza virus (HPIV; types 1-4), adenovirus (AdV), human rhinovirus (HRV), human metapneumovirus (HMPV), enterovirus (EV) and human bocavirus (HBoV) by (RT)-PCR, as described previously (Allander et al, 2005;Bellau-Pujol et al, 2005;Echevarría et al, 1998;Miura-Ochiai et al, 2007;Nakauchi et al, 2011;Olive et al, 1990;Takao et al, 2004;Zhang & Evans, 1991). In addition, we attempted to isolate and/or detect various respiratory bacteria such as Haemophilus influenzae, Legionella pneumophila, Neisseria spp.…”